A method for parentage identification in tetraploid oysters based on SNP loci

Abstract

This invention discloses a method for identifying the parentage of tetraploid Pacific oysters based on SNP loci, belonging to the field of molecular-assisted breeding technology for tetraploid Pacific oysters. The method includes the following steps: (1) sequencing the adductor muscle of the tetraploid Pacific oyster to be tested; (2) comparing the genotype of the designated SNP locus in the tetraploid Pacific oyster with the reference genome; (3) calculating the IBS and LOD of all tested parents and offspring based on the SNP typing results; and (4) combining the IBS and LOD to identify the parentage of the tested parents and offspring. The advantages of this invention are: it discloses for the first time 1000 SNP loci for identifying the parentage of tetraploid Pacific oysters, providing data support for the identification of parentage in tetraploid Pacific oysters and laying the foundation for the development of liquid-phase chips for identifying the parentage of tetraploid Pacific oysters, showing good application prospects.

Description

TECHNICAL FIELD

[0001] The application relates to a parent-offspring relationship identification method, in particular to a tetraploid Crassostrea gigas parent-offspring relationship identification method based on SNP sites, and belongs to the technical field of tetraploid Crassostrea gigas molecular assisted breeding. BACKGROUND

[0002] Crassostrea gigas is the largest and highest-yield marine economic shellfish in China and the world, has the advantages of high plumpness, fast growth, rich nutrition and wide adaptability. Triploid Crassostrea gigas has more abundant nutrition, and its poor fertility ensures that the gonad is plump all year round, which can meet the market demand. Tetraploid Crassostrea gigas is the core germplasm for producing full triploid Crassostrea gigas. With the progress of breeding technology, the main method for obtaining tetraploid Crassostrea gigas has been changed from traditional drug-induced transformation to self-population breeding, and through selective breeding, the breeding speed of tetraploid Crassostrea gigas has been greatly accelerated. At present, family selection is an important method for breeding tetraploid Crassostrea gigas, and through breeding, families with stable ploidy and excellent traits can be obtained to promote the research and development of new varieties. However, family selection requires each family to be bred separately, which greatly increases the manpower and material resources required for breeding. At the same time, family selection requires accurate recording of pedigree information of each family, and incorrect pedigree will reduce the accuracy of genetic evaluation and affect germplasm improvement. In addition, incorrect pedigree information will also increase the risk of inbreeding depression and lead to degeneration of germplasm. However, in large-scale breeding, incorrect pedigree information recording often occurs. Therefore, a method for efficiently and accurately recording pedigree relationships is needed to promote the stable development of tetraploid Crassostrea gigas core germplasm breeding.

[0003] Parent-offspring relationship identification is a scientific method for determining biological genetic relationship by comparing the similarity of genetic material between individuals and using genetic rules. DNA molecular marker identification technology has become the preferred technology for parent-offspring relationship identification due to its high accuracy and specificity. This method determines parent-offspring relationship by detecting whether the genetic pattern of DNA molecular markers between individuals conforms to Mendelian inheritance. With the development of high-throughput sequencing, DNA molecular markers used for parent-offspring relationship identification have evolved from restriction fragment length polymorphism (RFLP) to simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP). Compared to RFLP and SSR, SNP, as the third generation of genetic markers, has the following advantages: (1) widely distributed in the genome with a large number; (2) low mutation and recombination rate, high reproducibility; (3) high polymorphism; (4) high typing efficiency and low error rate. At the same time, studies have shown that a relatively small number of SNPs can accurately determine the parent-offspring relationship between individuals. Based on the above advantages, SNP has become the main molecular marker for parent-offspring identification.

[0004] Currently, Cervus, PaternityT, PediHaplotyper and R package have been widely used in livestock and marine organisms due to their high accuracy and high identification efficiency. Among them, Cervus series software calculates the likelihood ratio logarithm (LOD) between samples by simulating the non-parent probability, thereby accurately identifying the parent-offspring relationship between individuals; PaternityT calculates the cumulative non-parent exclusion probability by constructing the cumulative paternity index and paternity probability matrix, thereby identifying the parent-offspring relationship. The above two methods have been widely used in marine organisms. For example, Cervus has realized accurate identification of the genetic relationship between Megalobrama punctatus populations based on 116 SNPs; PaternityT identifies the parent-offspring relationship between individuals of Pseudosciaena crocea by calculating the cumulative non-parent exclusion probability of mixed samples of each family of Pseudosciaena crocea, and accurately assigns them to each family. However, the above software only supports the analysis of diploid organisms, and is not suitable for tetraploid organisms with higher genetic complexity, and there is no software and R package for tetraploid parent identification. Therefore, it is necessary to develop a parent-offspring relationship identification method for tetraploids by combining the genetic rules of tetraploids, and improve the universality of the parent-offspring relationship identification method.

[0005] For the important aquaculture species Crassostrea talajana, a SNP site combination specifically used for efficient and accurate identification of its parent-offspring relationship has not been systematically screened and identified, and there is currently no method for identifying its parent-offspring relationship. SUMMARY

[0006] To solve the problems of the prior art, the purpose of the present application is to screen SNP sites that can efficiently and accurately identify the parent-offspring relationship of tetraploid Crassostrea talajana, and to develop a method suitable for identifying the parent-offspring relationship of tetraploid Crassostrea talajana based on this, so as to solve the problem that the parent-offspring relationship cannot be accurately recorded in the breeding process of tetraploid Crassostrea talajana.

[0007] To achieve the above goal, the technical scheme adopted by the present application is as follows:

[0008] A SNP site-based tetraploid Crassostrea talajana parent-offspring relationship identification method, comprising the following steps:

[0009] (1) Take the adductor muscle of the tetraploid Crassostrea talajana to be tested for sequencing;

[0010] (2) Align the Crassostrea talajana reference genome to obtain the genotype of the specified SNP site of the tetraploid Crassostrea talajana, wherein the GenBank accession number of the Crassostrea talajana reference genome is GCA_011032805.1, and the genotype of the specified SNP site is as follows:

[0011] 1 chromosome: G / A mutation at position 121890 bp, A / G mutation at position 1057743 bp, T / G mutation at position 1282310 bp, G / T mutation at position 1672864 bp, T / C mutation at position 2377246 bp, T / A mutation at position 3067043 bp, T / C mutation at position 3586786 bp, A / T mutation at position 3999280 bp, T / C mutation at position 4728772 bp, T / C mutation at position 5044925 bp, T / C mutation at position 5300940 bp, A / G mutation at position 5594188 bp, C / T mutation at position 5903109 bp, T / G mutation at position 6330374 bp, T / C mutation at position 6833762 bp, A / G mutation at position 7943920 bp, A / T mutation at position 8487382 bp, A / G mutation at position 9704220 bp, T / C mutation at position 10224929 bp, T / C mutation at position 10862004 bp, T / G mutation at position 11750498 bp, T / C mutation at position 11990399 bp, T / C mutation at position 12218512 bp, G / A mutation at position 12483376 bp, G / A mutation at position 13361474 bp, G / T mutation at position 13765448 bp, T / A mutation at position 14300439 bp, T / A mutation at position 15205025 bp, A / G mutation at position 15645080 bp, G / T mutation at position 15940394 bp, T / C mutation at position 17045095 bp, T / C mutation at position 17268995 bp, T / A mutation at position 17538044 bp, A / T mutation at position 17899956 bp, G / T mutation at position 18451368 bp, C / T mutation at position 18760584 bp, C / G mutation at position 19501089 bp, G / T mutation at position 21531956 bp, T / A mutation at position 22401721 bp, T / A mutation at position 24526392 bp, A / T mutation at position 25189059 bp, G / A mutation at position 25603252 bp, A / T mutation at position 26161161 bp, C / T mutation at position 26611917 bp, A / C mutation at position 27004404 bp, A / G mutation at position 27425048 bp, C / T mutation at position 27805924 bp, A / T mutation at position 28632425 bp, A / T mutation at position 29021720 bp, C / T mutation at position 33066462 bp, A / T mutation at position 33718201 bp, G / A mutation at position 34717118 bp, A / C mutation at position 35189356 bp, C / T mutation at position 36608599 bp, C / T mutation at position 36886136 bp, C / A mutation at position 37186110 bp,G / C mutation at position 37483058 bp, C / A mutation at position 38812679 bp, A / G mutation at position 39546717 bp, C / T mutation at position 40317002 bp, A / G mutation at position 41392051 bp, G / A mutation at position 43046392 bp, C / T mutation at position 43389852 bp, T / A mutation at position 45510791 bp, A / G mutation at position 46464535 bp, A / G mutation at position 47037306 bp, T / G mutation at position 47275653 bp, A / T mutation at position 47543454 bp, A / T mutation at position 48596129 bp, A / G mutation at position 49583230 bp, T / C mutation at position 49868879 bp, T / G mutation at position 50086266 bp, T / G mutation at position 50302873 bp, A / C mutation at position 51214188 bp, C / T mutation at position 51485365 bp, G / A mutation at position 52121167 bp, A / T mutation at position 52424658 bp, C / T mutation at position 52758029 bp, A / G mutation at position 53158981 bp, C / T mutation at position 53542635 bp, T / C mutation at position 53910234 bp, C / T mutation at position 54359105 bp, T / C mutation at position 55808097 bp, T / A mutation at position 56281482 bp, C / T mutation at position 57138543 bp, G / A mutation at position 58299726 bp, C / T mutation at position 59081390 bp, G / T mutation at position 59774992 bp, T / G mutation at position 60482812 bp, C / T mutation at position 60889634 bp, G / A mutation at position 61245346 bp, C / G mutation at position 61505117 bp, C / A mutation at position 61817752 bp, A / T mutation at position 62517352 bp, T / C mutation at position 62882914 bp, A / G mutation at position 63105443 bp, C / T mutation at position 64259558 bp, C / A mutation at position 65252225 bp, T / A mutation at position 66625596 bp, A / G mutation at position 66835219 bp, C / T mutation at position 67248469 bp, T / A mutation at position 67503687 bp, A / G mutation at position 67906236 bp, C / T mutation at position 68247170 bp, C / T mutation at position 68945233 bp, T / C mutation at position 69565892 bp, G / A mutation at position 70058662 bp, A / G mutation at position 70314116 bp, A / T mutation at position 70765241 bp, A / G mutation at position 72411555 bp, T / C mutation at position 73822739 bp,A / G mutation at position 74137180 bp, A / G mutation at position 75053096 bp, A / C mutation at position 75443458 bp, A / G mutation at position 76271402 bp, T / A mutation at position 76958049 bp, C / G mutation at position 77190736 bp, T / A mutation at position 77491425 bp, A / T mutation at position 77966387 bp, C / T mutation at position 78744623 bp,

[0012] 2: T / C at position 126208, G / A at position 530724, A / T at position 1030962, C / G at position 1883338, T / A at position 2385548, A / G at position 2612768, T / C at position 2905649, T / C at position 3408267, A / G at position 4209622, T / C at position 4735213, C / G at position 5034762, C / A at position 5605969, A / G at position 5935587, T / G at position 6189866, A / T at position 6451484, A / G at position 7186787, A / G at position 7919353, T / A at position 8150271, T / C at position 8400126, A / G at position 8695230, G / T at position 9416767, C / T at position 9823074, T / C at position 10397975, A / T at position 10863126, A / G at position 11074140, A / G at position 11296287, A / G at position 11502471, C / G at position 12364482, T / G at position 12574976, T / C at position 12794499, C / A at position 13083777, T / C at position 13511367, T / C at position 13877414, C / A at position 14299527, G / A at position 14524308, A / G at position 14736903, G / A at position 15063383, A / G at position 15597292, T / A at position 15867029, G / A at position 16101395, C / A at position 16326428, T / C at position 16905050, A / T at position 17266489, A / T at position 17579365, G / T at position 18124593, G / C at position 18474645, A / G at position 18705311, C / A at position 19267782, A / G at position 21377238, T / A at position 22776786, A / C at position 23709017, A / G at position 24156723, A / C at position 24530341, G / A at position 25094735, G / C at position 25421687, A / G at position 25763249,C / T mutation at position 25980517 bp, T / A mutation at position 27508872 bp, T / C mutation at position 28212532 bp, C / T mutation at position 29113539 bp, G / C mutation at position 29485500 bp, T / C mutation at position 29809495 bp, A / T mutation at position 30098613 bp, C / T mutation at position 30391267 bp, A / G mutation at position 31955766 bp, G / A mutation at position 32672496 bp, C / T mutation at position 33448952 bp, C / T mutation at position 34565522 bp, G / T mutation at position 34854460 bp, T / G mutation at position 35956877 bp, G / A mutation at position 37299837 bp, T / A mutation at position 37768791 bp, A / G mutation at position 38364999 bp, C / T mutation at position 38772687 bp, A / T mutation at position 39119217 bp, G / A mutation at position 39791727 bp, G / A mutation at position 40956496 bp, A / T mutation at position 42059421 bp, C / T mutation at position 42389769 bp, A / G mutation at position 42831401 bp, T / C mutation at position 43232251 bp, T / A mutation at position 43943244 bp, A / T mutation at position 44224577 bp, A / T mutation at position 44910891 bp, T / G mutation at position 46742409 bp, A / G mutation at position 47231207 bp, A / T mutation at position 47919343 bp, G / A mutation at position 48360872 bp, G / T mutation at position 49359594 bp, G / A mutation at position 49898952 bp, G / A mutation at position 50319548 bp, T / C mutation at position 51049661 bp, A / C mutation at position 51306897 bp, A / G mutation at position 51516277 bp, A / G mutation at position 52191511 bp, G / A mutation at position 52579004 bp, C / G mutation at position 53640899 bp, A / T mutation at position 53976693 bp, T / C mutation at position 54891372 bp, C / T mutation at position 56410343 bp, G / C mutation at position 56902918 bp, A / G mutation at position 57143044 bp, G / A mutation at position 57422435 bp, G / A mutation at position 58005227 bp, T / C mutation at position 58329882 bp, T / C mutation at position 58802812 bp, T / C mutation at position 59036884 bp, G / T mutation at position 60176975 bp, G / A mutation at position 60593132 bp, A / G mutation at position 60929419 bp, A / G mutation at position 61642762 bp,

[0013] 3: T / C at position 167511, G / T at position 444560, T / G at position 1740957, A / C at position 2247068, A / T at position 2663410, A / G at position 3153958, A / G at position 3443363, T / A at position 3918303, T / A at position 4164736, T / G at position 4395261, G / A at position 5114758, A / T at position 5385938, C / T at position 5778988, G / T at position 6150113, A / T at position 6576463, T / A at position 7403440, C / G at position 8943900, T / A at position 9256892, T / C at position 9919466, A / C at position 10174343, A / T at position 11633388, G / A at position 12017984, A / G at position 12915914, C / T at position 14184322, A / T at position 14610187, C / T at position 15112027, A / C at position 15742955, T / C at position 18055450, T / C at position 20548205, A / T at position 20806332, C / T at position 21239659, A / T at position 21451371, A / G at position 21908751, C / T at position 22900014, T / G at position 23437899, C / G at position 24068761, C / T at position 24297401, A / G at position 25467666, A / T at position 26854208, G / A at position 27503027, C / T at position 27774729, T / C at position 28167758, A / G at position 29499237, G / A at position 29844353, A / G at position 30081511, G / T at position 30294248, G / A at position 31271920, C / T at position 31611643, C / T at position 31900387, A / T at position 32420847, A / G at position 33314059, C / G at position 33562476, A / G at position 34537410, A / C at position 35525002, G / A at position 36168038, G / A at position 37291953,G / C mutation at 37938922bp, T / C mutation at 38158633bp, G / T mutation at 38470830bp, G / A mutation at 39158628bp, G / A mutation at 39719498bp, A / C mutation at 40603131bp, A / C mutation at 40865370bp, T / G mutation at 41259567bp, T / C mutation at 41597965bp, G / A mutation at 41910605bp, T / A mutation at 42938999bp, A / T mutation at 43500081bp, C / T mutation at 44213296bp. The following mutations were observed: A / G at 44459501 bp, C / A at 45077111 bp, A / T at 46562102 bp, T / A at 47015472 bp, A / C at 47248636 bp, A / G at 47755545 bp, G / A at 48068295 bp, G / A at 48403665 bp, C / A at 48859386 bp, A / G at 49155040 bp, A / T at 49514429 bp, T / A at 49801952 bp, and G / C at 50508624 bp. T / C mutation at position 50867456 bp, C / T mutation at position 51172288 bp, G / A mutation at position 51555200 bp, T / A mutation at position 51897141 bp, C / G mutation at position 53046160 bp, T / A mutation at position 53359071 bp, T / A mutation at position 53636779 bp, C / T mutation at position 53869457 bp, T / C mutation at position 54594267 bp, T / C mutation at position 55269130 bp, A / G mutation at position 55572008 bp, A / C mutation at position 55896201 bp, C / T mutation at position 56783882 bp The following mutations were observed: A / G at 57104330 bp, C / G at 57532214 bp, T / C at 57807917 bp, T / A at 58451281 bp, C / G at 58805029 bp, A / C at 59273801 bp, C / A at 59815549 bp, A / G at 60128400 bp, G / A at 60681360 bp, C / T at 61275765 bp, C / T at 61522130 bp, C / A at 61801828 bp, and T / C at 62069187 bp.

[0014] Chromosome 4: C / A mutation at 489947 bp, G / C mutation at 886286 bp, A / G mutation at 2582998 bp, G / A mutation at 3139977 bp, G / A mutation at 3595215 bp, T / A mutation at 3859888 bp, A / G mutation at 4344155 bp, C / A mutation at 4566835 bp, A / G mutation at 5145569 bp, T / C mutation at 6456871 bp, G / C mutation at 7321215 bp, T / A mutation at 7817588 bp, T / G mutation at 8179181 bp, G / T mutation at 8910032 bp, ... G / T mutation at 6 bp, T / G mutation at 9552736 bp, G / A mutation at 10022246 bp, T / G mutation at 10354612 bp, G / A mutation at 10907817 bp, C / T mutation at 11734493 bp, G / A mutation at 12580554 bp, T / C mutation at 13260828 bp, T / C mutation at 13765169 bp, C / T mutation at 14310601 bp, T / C mutation at 16355043 bp, G / A mutation at 17630046 bp, T / A mutation at 19429489 bp, A / T mutation at 19679728 bp, 21079 A / G mutation at 213bp, T / C mutation at 22164490bp, A / C mutation at 22482582bp, T / A mutation at 22780082bp, C / T mutation at 23190102bp, A / G mutation at 23587372bp, A / G mutation at 24228102bp, A / G mutation at 24961164bp, G / T mutation at 25252848bp, A / T mutation at 25844871bp, G / T mutation at 26112318bp, G / A mutation at 27430034bp, C / A mutation at 27889194bp, T / C mutation at 28186129bp, 28 G / T mutation at 862600bp, T / A mutation at 29613029bp, A / T mutation at 29849521bp, C / T mutation at 30074147bp, G / T mutation at 30551177bp, A / C mutation at 31624189bp, T / C mutation at 31868549bp, T / C mutation at 32085476bp, T / C mutation at 33390588bp, T / A mutation at 33885908bp, T / C mutation at 34321828bp, C / A mutation at 34763173bp, A / G mutation at 36691587bp, A / G mutation at 36995284bp.The following mutations were observed at the following locations: A / C at 37364904 bp, C / T at 39303404 bp, G / C at 40040754 bp, T / C at 40300785 bp, T / C at 41007997 bp, T / C at 41213469 bp, A / G at 41586776 bp, A / G at 42194520 bp, G / T at 42525010 bp, C / T at 42726376 bp, T / G at 43723088 bp, A / T at 44141447 bp, and T / C at 44798262 bp. A / G mutation at 45230255bp, A / G mutation at 45691471bp, T / A mutation at 45924080bp, A / G mutation at 46335423bp, G / A mutation at 46609807bp, C / T mutation at 47649738bp, C / A mutation at 48054473bp, G / A mutation at 49037444bp, T / C mutation at 49356447bp, T / A mutation at 49823141bp, C / T mutation at 50253140bp, T / A mutation at 50495698bp, G / A mutation at 50782734bp. G / A mutation at 51328829bp, T / C mutation at 51912827bp, T / C mutation at 52524776bp, C / T mutation at 52849059bp, C / T mutation at 53628954bp, C / T mutation at 54121390bp, C / T mutation at 54500655bp, A / T mutation at 54780363bp, C / A mutation at 55043635bp, T / C mutation at 55269248bp, C / T mutation at 56009969bp, T / C mutation at 56360644bp, T / A mutation at 56621645bp. The following mutations were observed: A / G mutation at 57033970 bp, T / A mutation at 57487468 bp, C / T mutation at 58248810 bp, A / G mutation at 58569969 bp, C / A mutation at 58804428 bp, G / A mutation at 59177752 bp, A / G mutation at 59780097 bp, C / A mutation at 60118503 bp, G / T mutation at 60642124 bp, T / A mutation at 60872702 bp, T / G mutation at 61335564 bp, G / A mutation at 61988945 bp, and G / T mutation at 62219348 bp.

[0015] Chromosome 5: C / G mutation at 879645bp, G / A mutation at 1415586bp, C / T mutation at 2111293bp, C / A mutation at 2314335bp, A / T mutation at 2635565bp, G / A mutation at 2920345bp, T / C mutation at 3467779bp, T / A mutation at 3884050bp, C / G mutation at 4393916bp, A / C mutation at 4660080bp, T / C mutation at 5064983bp, G / A mutation at 5762044bp, G / T mutation at 6544275bp, G / T mutation at 6749408bp, G / T mutation at 699... A / G mutation at 7717bp, T / G mutation at 7345402bp, A / G mutation at 7654532bp, A / G mutation at 7932212bp, A / G mutation at 8303258bp, A / C mutation at 8661151bp, T / C mutation at 9176468bp, T / C mutation at 9464239bp, C / A mutation at 9931584bp, T / C mutation at 10539507bp, A / G mutation at 10875599bp, A / T mutation at 11217368bp, G / A mutation at 11702291bp, C / T mutation at 12003330bp, T / G mutation at 12366955bp A / T mutation at 12808603bp, T / C mutation at 13875823bp, T / C mutation at 14157737bp, A / G mutation at 15150248bp, G / A mutation at 15517737bp, A / G mutation at 15749958bp, A / G mutation at 16257325bp, A / G mutation at 16594460bp, T / C mutation at 17783174bp, T / A mutation at 18355754bp, C / A mutation at 18594492bp, G / A mutation at 18998718bp, A / T mutation at 19767072bp, C / T mutation at 202... T / C mutation at 53708bp, A / C mutation at 20548723bp, G / A mutation at 22378076bp, A / G mutation at 23213346bp, A / C mutation at 23730799bp, T / C mutation at 23996432bp, C / T mutation at 24241420bp, G / A mutation at 24721774bp, T / C mutation at 26586598bp, A / T mutation at 26893151bp, C / T mutation at 27345167bp, T / C mutation at 27679094bp, C / G mutation at 28185342bp, T / A mutation at 29149844bp.C / A mutation at 29649330bp, G / A mutation at 30587198bp, C / A mutation at 30997742bp, G / T mutation at 31256011bp, T / C mutation at 31705376bp, T / C mutation at 32770700bp, G / C mutation at 33339763bp, A / G mutation at 33573910bp, C / T mutation at 34514253bp, A / G mutation at 35112239bp, T / C mutation at 35669268bp, T / A mutation at 36705272bp, C / T mutation at 36922640bp, A mutation at 37131668bp / G mutation, C / T mutation at 37581075bp, C / T mutation at 38542724bp, G / A mutation at 39044859bp, G / C mutation at 39688041bp, G / A mutation at 40000656bp, T / C mutation at 40395464bp, G / A mutation at 41001494bp, T / C mutation at 41681731bp, T / C mutation at 42174184bp, A / T mutation at 42576737bp, A / G mutation at 43098562bp, A / G mutation at 43487720bp, A / G mutation at 43692812bp, A / G mutation at 44169923bp A / C mutation at 44373192bp, A / C mutation at 44895351bp, C / T mutation at 45190288bp, A / G mutation at 46681737bp, T / A mutation at 47488756bp, T / C mutation at 49326052bp, T / C mutation at 49639111bp, G / T mutation at 50108002bp, C / G mutation at 50436257bp, T / A mutation at 50696264bp, A / T mutation at 50903386bp, T / C mutation at 51419745bp, A / G mutation at 51669577bp, C / T mutation at 524bp T / A mutation at 14271bp, A / C mutation at 53368505bp, A / G mutation at 53605312bp, T / C mutation at 53894100bp, T / A mutation at 54916254bp, A / G mutation at 55477238bp, A / C mutation at 55999091bp, G / C mutation at 56214132bp, G / A mutation at 57070120bp, T / A mutation at 57313644bp, A / C mutation at 57666549bp, G / T mutation at 57877313bp, C / T mutation at 58133420bp, G / A mutation at 58891833bp.T / C mutation at 59245611bp, A / T mutation at 59751484bp, and A / G mutation at 59976681bp;

[0016] Chromosome 6: G / A mutation at 587350 bp, A / G mutation at 1075497 bp, T / A mutation at 1679222 bp, G / T mutation at 2172358 bp, T / C mutation at 2739350 bp, G / A mutation at 3206148 bp, C / A mutation at 3421291 bp, G / A mutation at 3843416 bp, A / T mutation at 4064437 bp, G / T mutation at 4469234 bp, T / C mutation at 4760643 bp, T / A mutation at 4992337 bp, A / T mutation at 5778802 bp, A / C mutation at 6114864 bp, ... T / C mutation at 355bp, G / A mutation at 6932249bp, G / A mutation at 8026496bp, C / T mutation at 8885122bp, T / C mutation at 9620041bp, G / T mutation at 10908040bp, C / T mutation at 11117547bp, A / G mutation at 12753363bp, C / T mutation at 14268050bp, T / G mutation at 14586618bp, G / C mutation at 14879882bp, G / A mutation at 15232542bp, A / C mutation at 15807647bp, C / T mutation at 16152642bp, 164216 A / G mutation at 43bp, A / T mutation at 16738467bp, C / A mutation at 17154757bp, G / A mutation at 17669050bp, T / A mutation at 17995298bp, A / T mutation at 18653957bp, T / C mutation at 18914180bp, T / A mutation at 19545790bp, C / T mutation at 20589164bp, A / G mutation at 21516044bp, T / A mutation at 22028166bp, A / T mutation at 22624328bp, T / A mutation at 23340429bp, C / T mutation at 23832772bp, 24 A / G mutation at 266242bp, A / C mutation at 25073811bp, A / G mutation at 26222500bp, A / C mutation at 26774119bp, T / C mutation at 27210621bp, C / T mutation at 27689291bp, G / A mutation at 28235309bp, T / C mutation at 28437764bp, C / T mutation at 28668444bp, T / A mutation at 29329850bp, A / G mutation at 29645718bp, G / T mutation at 30524553bp, G / A mutation at 31121205bp, C / T mutation at 31359984bp.The following mutations were observed: A / G at 32106045 bp, G / A at 32457595 bp, T / C at 32753400 bp, T / A at 33117124 bp, T / C at 33570502 bp, T / G at 34176629 bp, C / T at 34667060 bp, A / G at 38181718 bp, T / A at 39546371 bp, and G / C at 39866670 bp. G / T mutation at 40351063bp, A / G mutation at 40681703bp, T / C mutation at 41023800bp, G / T mutation at 41459997bp, T / C mutation at 42016811bp, T / C mutation at 42364688bp, T / A mutation at 42605575bp, C / A mutation at 43099589bp, C / T mutation at 43383766bp, C / T mutation at 43791635bp. The mutations are: A / T at 44792472 bp, C / T at 45098188 bp, A / G at 45791769 bp, A / G at 46506334 bp, T / C at 47036051 bp, G / C at 47496058 bp, C / T at 47812848 bp, A / C at 48271374 bp, T / A at 49169061 bp, and A / T at 49429004 bp. C / T mutation at position 49743523 bp, T / C mutation at position 50128359 bp, A / G mutation at position 50887722 bp, A / T mutation at position 51813382 bp, C / T mutation at position 52554583 bp, T / C mutation at position 53230845 bp, C / T mutation at position 54083112 bp, C / T mutation at position 54380581 bp, C / T mutation at position 55428291 bp, A / C mutation at position 56179611 bp;

[0017] Chromosome 7: A / G mutation at 603788 bp, G / T mutation at 896391 bp, T / C mutation at 1282724 bp, G / T mutation at 1535834 bp, G / A mutation at 1795922 bp, T / A mutation at 2124131 bp, G / T mutation at 2694802 bp, G / A mutation at 3023240 bp, T / C mutation at 3227288 bp, T / C mutation at 3539581 bp, T / C mutation at 3946542 bp, T / A mutation at 4409015 bp, G / A mutation at 4610418 bp, A / G mutation at 5099145 bp, 5726... T / C mutation at 774bp, T / C mutation at 6090181bp, T / A mutation at 7174959bp, G / T mutation at 7732660bp, T / A mutation at 7943436bp, C / T mutation at 8179975bp, A / T mutation at 9080480bp, T / A mutation at 9322949bp, A / G mutation at 9569794bp, T / C mutation at 9911214bp, A / C mutation at 10663904bp, T / G mutation at 10877221bp, G / A mutation at 11467271bp, T / A mutation at 11736967bp, 12053878b T / C mutation at p, C / T mutation at 13007410bp, A / G mutation at 13768058bp, G / A mutation at 15198787bp, A / T mutation at 15771972bp, T / G mutation at 16027470bp, G / A mutation at 16465374bp, T / G mutation at 16680508bp, A / G mutation at 17192258bp, T / C mutation at 17558072bp, C / T mutation at 17969940bp, C / T mutation at 18480038bp, C / T mutation at 18732421bp, A / C mutation at 19150831bp, 1935 A / T mutation at 8092 bp, C / T mutation at 19653197 bp, T / C mutation at 20077315 bp, T / G mutation at 20839591 bp, A / T mutation at 21779850 bp, A / G mutation at 23771667 bp, C / A mutation at 24586945 bp, A / G mutation at 25311728 bp, A / C mutation at 25544379 bp, T / C mutation at 26417838 bp, G / C mutation at 26642847 bp, A / G mutation at 26965429 bp, A / T mutation at 29758485 bp, T / A mutation at 30141591 bp.C / T mutation at 30468300bp, T / C mutation at 31400778bp, G / T mutation at 31778180bp, A / G mutation at 32075251bp, C / T mutation at 33230067bp, C / T mutation at 33744108bp, T / A mutation at 36023112bp, T / A mutation at 36396469bp, A / G mutation at 36693769bp, and so on. A / T mutation at 37760599bp, C / T mutation at 38161672bp, T / A mutation at 38702757bp, G / T mutation at 39210903bp, A / G mutation at 39699534bp, A / T mutation at 40385742bp, G / C mutation at 41026442bp, T / C mutation at 41307086bp, C / A mutation at 42969755bp, G / A mutation. T / C mutation at 43571847bp, A / G mutation at 43993986bp, T / C mutation at 44394370bp, G / A mutation at 44703340bp, A / G mutation at 45524469bp, C / T mutation at 46045824bp, A / G mutation at 47359820bp, C / A mutation at 47598126bp, A / G mutation at 49292205bp, and at 50492560bp. T / C mutation at 50866106bp, T / C mutation at 51554060bp, C / T mutation at 51867069bp, T / G mutation at 52186344bp, T / C mutation at 52386979bp, A / G mutation at 52719201bp, A / G mutation at 53780380bp, C / T mutation at 54228211bp, G / A mutation at 54901003bp;

[0018] Chromosome 8: G / A mutation at 412562bp, T / A mutation at 985611bp, T / C mutation at 1303653bp, C / T mutation at 1544349bp, T / C mutation at 1922320bp, T / C mutation at 2361255bp, T / A mutation at 2719405bp, A / G mutation at 3000604bp, T / C mutation at 3288220bp, G / C mutation at 3518544bp, T / C mutation at 3933031bp, T / C mutation at 4572621bp, T / C mutation at 5346224bp, A / T mutation at 5602024bp, 59030... T / C mutation at 95bp, C / T mutation at 6172964bp, T / A mutation at 6581392bp, T / A mutation at 7427921bp, T / C mutation at 7653299bp, G / A mutation at 10833172bp, C / T mutation at 11143937bp, C / T mutation at 11563545bp, T / C mutation at 12240151bp, A / G mutation at 12664163bp, C / A mutation at 12976720bp, C / G mutation at 13428217bp, T / A mutation at 13690299bp, C / A mutation at 14184965bp, 145407 A / G mutation at 37bp, A / C mutation at 14927534bp, T / A mutation at 15164050bp, A / C mutation at 16228337bp, C / G mutation at 16839194bp, T / A mutation at 17073823bp, A / G mutation at 17560361bp, C / T mutation at 17805117bp, C / T mutation at 18668691bp, C / T mutation at 19077073bp, A / T mutation at 19321381bp, A / G mutation at 20212862bp, A / G mutation at 20498190bp, T / C mutation at 20748906bp, 21 A / G mutation at 088463bp, A / G mutation at 21591018bp, A / G mutation at 22165379bp, A / T mutation at 23067147bp, T / C mutation at 23844408bp, A / G mutation at 24104507bp, T / A mutation at 24665627bp, G / A mutation at 25079866bp, T / G mutation at 25958988bp, A / G mutation at 27292396bp, G / A mutation at 27511452bp, C / T mutation at 27712853bp, G / A mutation at 27981923bp, G / A mutation at 28485338bp.G / A mutation at 28816819bp, C / A mutation at 29401272bp, C / T mutation at 29733986bp, C / T mutation at 30150021bp, A / T mutation at 30361015bp, A / C mutation at 30665469bp, T / G mutation at 30927726bp, C / G mutation at 31612011bp, and at 31949923bp... C / G mutation at 32350947bp, T / C mutation at 32829805bp, T / C mutation at 33830538bp, T / C mutation at 34428621bp, G / A mutation at 34631910bp, C / A mutation at 34878333bp, T / C mutation at 35167435bp, G / A mutation at 35384611bp C / T mutation at 36295451bp, A / G mutation at 36758686bp, T / C mutation at 38408273bp, T / C mutation at 39084417bp, T / C mutation at 40515577bp, A / G mutation at 40784564bp, C / T mutation at 41770053bp, G / T mutation at 42205041bp, and so on. G / A mutation at 43574397bp, G / A mutation at 44913196bp, T / G mutation at 46413323bp, C / A mutation at 47254739bp, C / T mutation at 47569565bp, T / C mutation at 48443108bp, A / T mutation at 48947202bp, T / C mutation at 50071965bp;

[0019] Chromosome 9: T / C mutation at 217853 bp, A / G mutation at 583042 bp, A / C mutation at 796208 bp, T / C mutation at 1046351 bp, T / G mutation at 1327845 bp, G / A mutation at 2838975 bp, A / G mutation at 3240786 bp, T / C mutation at 3675421 bp, G / T mutation at 4651246 bp, C / T mutation at 4959856 bp, C / T mutation at 5227751 bp, C / T mutation at 5933068 bp, G / A mutation at 6728623 bp, C / T mutation at 7235075 bp, ... C / G mutation at 85bp, A / T mutation at 7915378bp, A / G mutation at 8155620bp, A / C mutation at 8437140bp, C / A mutation at 9041049bp, T / G mutation at 9320125bp, G / C mutation at 9523788bp, A / G mutation at 10256842bp, A / T mutation at 10516635bp, C / G mutation at 11295005bp, C / G mutation at 11568023bp, T / A mutation at 12159413bp, C / T mutation at 12529731bp, T / G mutation at 12735255bp, 13042605 C / A mutation at 13493638bp, A / G mutation at 13853217bp, A / T mutation at 14058546bp, G / A mutation at 15219775bp, A / T mutation at 15704220bp, G / T mutation at 16140181bp, G / A mutation at 16412881bp, T / C mutation at 17010061bp, T / C mutation at 17928904bp, C / T mutation at 18193136bp, T / C mutation at 18452448bp, T / C mutation at 18653747bp, A / T mutation at 18878597bp, 196 C / T mutation at 85478bp, G / A mutation at 20221044bp, T / C mutation at 20993779bp, G / T mutation at 21586001bp, A / G mutation at 22002234bp, C / T mutation at 22394787bp, T / A mutation at 22969695bp, A / T mutation at 23876847bp, C / T mutation at 25194999bp, A / G mutation at 25462264bp, A / T mutation at 25741402bp, A / G mutation at 26959004bp, A / G mutation at 27955752bp, C / A mutation at 28258725bp, A / G mutation.A / T mutation at 29236123bp, A / G mutation at 29908750bp, C / T mutation at 30127537bp, T / C mutation at 31200772bp, C / T mutation at 31445189bp, T / C mutation at 31759757bp, C / A mutation at 32418276bp, T / C mutation at 33430899bp, G / A mutation at 33857260bp, and so on. A / G mutation at 35910999bp, A / G mutation at 36657698bp, T / C mutation at 37111454bp, A / T mutation at 38333366bp, A / T mutation at 38956659bp, G / T mutation at 41437696bp, T / G mutation at 41655725bp, G / T mutation at 42066006bp, T / C mutation at 43778385bp. T / C mutation at 44072139bp, C / T mutation at 44341586bp, T / C mutation at 46347906bp, C / A mutation at 46681275bp, A / G mutation at 47089848bp, T / C mutation at 47332573bp, G / A mutation at 47545232bp, T / C mutation at 47787454bp, T / A mutation at 48000048bp, and so on. T / C mutation at 49478480bp, T / C mutation at 50204065bp, G / A mutation at 50425019bp, C / T mutation at 52132614bp, T / G mutation at 52785787bp, C / T mutation at 53663195bp, C / T mutation at 54046362bp, T / G mutation at 54605490bp, T / C mutation at 54826243bp;

[0020] Chromosome 10: T / C mutation at 1534163bp, T / G mutation at 2455069bp, C / T mutation at 3038564bp, T / C mutation at 4197932bp, A / T mutation at 4800809bp, A / T mutation at 5676331bp, T / C mutation at 6642222bp, A / G mutation at 7400941bp, T / C mutation at 7668457bp, T / C mutation at 7897420bp, A / C mutation at 8180708bp, C / T mutation at 8755317bp, G / A mutation at 9215005bp, C / A mutation at 9779335bp, ... A / G mutation at 5131bp, A / G mutation at 10801950bp, T / C mutation at 11089097bp, C / A mutation at 11732224bp, T / C mutation at 11959146bp, C / T mutation at 12182036bp, A / G mutation at 12384483bp, T / A mutation at 12977779bp, C / G mutation at 13228019bp, G / A mutation at 13725920bp, A / G mutation at 14297949bp, C / T mutation at 14599462bp, A / C mutation at 15316648bp, C / T mutation at 16226209bp, 17 T / A mutation at 125566bp, G / T mutation at 19323146bp, C / A mutation at 20329041bp, C / A mutation at 20545842bp, A / G mutation at 20831667bp, A / G mutation at 21077840bp, T / C mutation at 21855677bp, C / T mutation at 22076843bp, T / G mutation at 22285822bp, C / A mutation at 22573506bp, T / C mutation at 23139968bp, A / T mutation at 23927295bp, C / T mutation at 24217188bp, T / A mutation at 24958349bp, ... A / T mutation at 26265143bp, A / C mutation at 26789789bp, A / G mutation at 27013959bp, C / T mutation at 27270677bp, T / A mutation at 27522828bp, A / T mutation at 28230594bp, A / T mutation at 28621223bp, T / C mutation at 28976931bp, G / C mutation at 30161642bp, G / T mutation at 30602940bp, A / G mutation at 30978727bp, T / A mutation at 31649003bp, G / A mutation at 32503776bp, T / C mutation at 33082095bp.G / A mutation at 33398327bp, A / T mutation at 34352656bp, C / T mutation at 34825626bp, A / G mutation at 35322952bp, A / T mutation at 35627155bp, C / T mutation at 35967237bp, C / G mutation at 36252688bp, G / A mutation at 36722144bp, T / C mutation at 37789019bp;

[0021] (3) Calculate the IBS and LOD of all tested parents and offspring based on the SNP typing results. The calculation formula is as follows:

[0022] ,

[0023] Where n represents the number of SNP loci successfully genotyped, k represents the k-th SNP locus, and G ik For the genotype of individual i at the k-th SNP locus, G jk For the genotype of individual j at the k-th SNP locus, I k (G) ik G jk () represents the genotype matching score between individual i and individual j at the k-th SNP locus. I is the score when the genotypes of individuals i and j at the k-th SNP locus match as parent-child. k (G) ik G jk If ) is 1, otherwise I k (G) ik G jk P1 is 0, representing the probability that the candidate parent and the simulated parent will produce offspring, and P2 represents the background probability.

[0024] (4) Combine IBS and LOD to identify the parent-child relationship between the parent and offspring;

[0025] Preferably, in step (3), the probability P1 of offspring produced by the candidate parent and the simulated parent is calculated using the following formula:

[0026]

[0027] in, This represents the probability of the appearance of candidate parent gametes. λ represents the probability of the occurrence of simulated parental gametes, w represents the frequency of simulated parental genotypes in the population, m represents the number of mismatched genotypes between parents and offspring, and λ represents the mismatch penalty weight, which is 0.1.

[0028] Preferably, in step (3), when calculating the background probability P2, the genotypes of the simulated parents are all based on the population allele frequencies.

[0029] Preferably, in step (4), the method for identifying the parent-child relationship between the parent and offspring using IBS and LOD is as follows:

[0030] (1) When LOD is greater than 0, the candidate parent may be the real parent, and at least one parent with the highest LOD is most likely to be the real parent of the offspring. In this case, the IBS between the candidate parent and the offspring should be higher than the preset threshold of 0.98.

[0031] (2) When LOD is less than 0, the candidate parent cannot be the real parent.

[0032] The advantages of this invention are:

[0033] (1) This invention discloses for the first time 1,000 SNP loci for the identification of parentage in tetraploid oysters, providing data support for the identification of parentage in tetraploid oysters and laying the foundation for the development of liquid-phase chips for the identification of parentage in tetraploid oysters, which has good application prospects.

[0034] (2) Based on the genetic laws of tetraploids, this invention has developed a method for identifying the parentage of tetraploid oysters. Based on the two developed indicators IBS and LOD, the parentage can be accurately identified through low-density SNP locus information, which makes up for the current lack of molecular means to identify the parentage of tetraploid oysters. It also provides a guarantee for identifying and repairing potential pedigree errors, thereby improving the accuracy of molecular breeding of tetraploid oysters.

[0035] (3) The parentage identification method provided by the present invention is low in cost and can be used for large-scale parentage identification and family tree construction.

[0036] (4) The parentage identification method provided by the present invention is also applicable to the parentage identification of other tetraploid organisms and has high universality. Attached Figure Description

[0037] Figure 1 This is a distribution diagram of the Level of Displacement (LOD) among samples with parent-child relationships;

[0038] Figure 2 This is a partial LOD and IBS heatmap between samples. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0040] I. Screening for SNP loci that can efficiently and accurately identify parentage in tetraploid oysters.

[0041] The method for screening SNP loci that can efficiently and accurately identify parentage in tetraploid oysters according to this invention is as follows:

[0042] 1. Adductor muscles of tetraploid oysters with a clear parent-child relationship were used for DNA purification, sequencing library construction, and high-throughput sequencing.

[0043] Five individuals were collected from each of four tetraploid oyster families, with each family consisting of two parents and three offspring.

[0044] Adductor muscles of tetraploid oysters were collected, and DNA from each sample was purified using the GenoPrep Polysaccharide-Polyphenol DNA Extraction Kit (Shijiazhuang Borui Biotechnology Co., Ltd.). DNA concentration was determined using a Qubit 2.0 real-time fluorescence analyzer, and DNA quality was assessed using 1% agarose gel electrophoresis.

[0045] Sequencing libraries were constructed using the GenoBaits® DNA Library Prep Kit for ILM.

[0046] High-throughput sequencing of DNA fragments in the sequencing library was performed using an MGISEQ-2000 sequencer (Shenzhen BGI Genomics Co., Ltd.) in PE150 mode.

[0047] 2. Genotyping the sequencing data.

[0048] The raw data were aligned to the reference genome of Pacific oyster (GenBank accession number: GCA_011032805.1), and genotyping was performed on the aligned data using GATK.

[0049] 3. Quality control of SNP sites

[0050] Quality control was performed on the genotyped sites. First, Indel sites and multi-allelic SNP sites were filtered out. Then, the remaining diallelic SNP sites were filtered according to the following criteria: (1) minor allele frequency (MAF) < 0.35; (2) site detection rate < 100%; (3) sequencing depth (DP) < 100.

[0051] 4. Calculate the MAF, expected heterozygosity (Hexp), and polymorphism information content (PIC) of SNP loci that meet the quality control standards. Select SNP loci with high MAF, PIC, and Hepx from each chromosome. The number of SNP loci on each chromosome is positively correlated with the length of each chromosome, and the distance between adjacent SNP loci exceeds 200K.

[0052] This invention ultimately screened 1000 SNP loci on 10 chromosomes for parentage identification in tetraploid oysters. The information of these 1000 SNP loci is as follows:

[0053] Chromosome 1: G / A mutation at 121890 bp, A / G mutation at 1057743 bp, T / G mutation at 1282310 bp, G / T mutation at 1672864 bp, T / C mutation at 2377246 bp, T / A mutation at 3067043 bp, T / C mutation at 3586786 bp, A / T mutation at 3999280 bp, T / C mutation at 4728772 bp, T / C mutation at 5044925 bp, T / C mutation at 5300940 bp, A / G mutation at 5594188 bp, C / T mutation at 5903109 bp, T / G mutation at 6330374 bp, ... T / C mutation at 762bp, A / G mutation at 7943920bp, A / T mutation at 8487382bp, A / G mutation at 9704220bp, T / C mutation at 10224929bp, T / C mutation at 10862004bp, T / G mutation at 11750498bp, T / C mutation at 11990399bp, T / C mutation at 12218512bp, G / A mutation at 12483376bp, G / A mutation at 13361474bp, G / T mutation at 13765448bp, T / A mutation at 14300439bp, T / A mutation at 15205025bp, T / A mutation at 15645bp A / G mutation at 080bp, G / T mutation at 15940394bp, T / C mutation at 17045095bp, T / C mutation at 17268995bp, T / A mutation at 17538044bp, A / T mutation at 17899956bp, G / T mutation at 18451368bp, C / T mutation at 18760584bp, C / G mutation at 19501089bp, G / T mutation at 21531956bp, T / A mutation at 22401721bp, T / A mutation at 24526392bp, A / T mutation at 25189059bp, G / A mutation at 25603252bp, 26 A / T mutation at 161161bp, C / T mutation at 26611917bp, A / C mutation at 27004404bp, A / G mutation at 27425048bp, C / T mutation at 27805924bp, A / T mutation at 28632425bp, A / T mutation at 29021720bp, A / T mutation at 33066462bp, C / T mutation at 33718201bp, A / T mutation at 34717118bp, G / A mutation at 35189356bp, A / C mutation at 36608599bp, C / T mutation at 36886136bp, C / A mutation at 37186110bp.G / C mutation at 37483058bp, C / A mutation at 38812679bp, A / G mutation at 39546717bp, C / T mutation at 40317002bp, A / G mutation at 41392051bp, G / A mutation at 43046392bp, C / T mutation at 43389852bp, T / A mutation at 45510791bp, A / G mutation at 46464535bp, A / G mutation at 47037306bp, A / G mutation at 47275653bp, T / G mutation at 47543454bp, A / T mutation at 48596129bp, A / T mutation at 49583230bp. / G mutation, T / C mutation at 49868879bp, T / G mutation at 50086266bp, T / G mutation at 50302873bp, A / C mutation at 51214188bp, C / T mutation at 51485365bp, G / A mutation at 52121167bp, A / T mutation at 52424658bp, C / T mutation at 52758029bp, A / G mutation at 53158981bp, C / T mutation at 53542635bp, T / C mutation at 53910234bp, C / T mutation at 54359105bp, T / C mutation at 55808097bp, 56281482 T / A mutation at 57138543bp, C / T mutation at 58299726bp, G / A mutation at 59081390bp, C / T mutation at 59774992bp, G / T mutation at 60482812bp, T / G mutation at 60889634bp, G / A mutation at 61245346bp, C / G mutation at 61505117bp, C / A mutation at 61817752bp, A / T mutation at 62517352bp, T / C mutation at 62882914bp, A / G mutation at 63105443bp, C / T mutation at 64259558bp, and so on. C / A mutation at 52225bp, T / A mutation at 66625596bp, A / G mutation at 66835219bp, C / T mutation at 67248469bp, T / A mutation at 67503687bp, A / G mutation at 67906236bp, C / T mutation at 68247170bp, C / T mutation at 68945233bp, T / C mutation at 69565892bp, G / A mutation at 70058662bp, A / G mutation at 70314116bp, A / T mutation at 70765241bp, A / G mutation at 72411555bp, T / C mutation at 73822739bp.The following mutations were observed: A / G at 74137180 bp, A / G at 75053096 bp, A / C at 75443458 bp, A / G at 76271402 bp, T / A at 76958049 bp, C / G at 77190736 bp, T / A at 77491425 bp, A / T at 77966387 bp, and C / T at 78744623 bp.

[0054] Chromosome 2: T / C mutation at 126208 bp, G / A mutation at 530724 bp, A / T mutation at 1030962 bp, C / G mutation at 1883338 bp, T / A mutation at 2385548 bp, A / G mutation at 2612768 bp, T / C mutation at 2905649 bp, T / C mutation at 3408267 bp, A / G mutation at 4209622 bp, T / C mutation at 4735213 bp, C / G mutation at 5034762 bp, C / A mutation at 5605969 bp, A / G mutation at 5935587 bp, T / G mutation at 6189866 bp, ... A / T mutation at 484bp, A / G mutation at 7186787bp, A / G mutation at 7919353bp, T / A mutation at 8150271bp, T / C mutation at 8400126bp, A / G mutation at 8695230bp, G / T mutation at 9416767bp, C / T mutation at 9823074bp, T / C mutation at 10397975bp, A / T mutation at 10863126bp, A / G mutation at 11074140bp, A / G mutation at 11296287bp, A / G mutation at 11502471bp, C / G mutation at 12364482bp, and so on. T / G mutation at 172 bp, T / C mutation at 12794499 bp, C / A mutation at 13083777 bp, T / C mutation at 13511367 bp, T / C mutation at 13877414 bp, C / A mutation at 14299527 bp, G / A mutation at 14524308 bp, A / G mutation at 14736903 bp, G / A mutation at 15063383 bp, A / G mutation at 15597292 bp, T / A mutation at 15867029 bp, G / A mutation at 16101395 bp, C / A mutation at 16326428 bp, T / C mutation at 16905050 bp, T / C mutation at 172 bp A / T mutation at 66489bp, A / T mutation at 17579365bp, G / T mutation at 18124593bp, G / C mutation at 18474645bp, A / G mutation at 18705311bp, C / A mutation at 19267782bp, A / G mutation at 21377238bp, T / A mutation at 22776786bp, A / C mutation at 23709017bp, A / G mutation at 24156723bp, A / C mutation at 24530341bp, G / A mutation at 25094735bp, G / C mutation at 25421687bp, A / G mutation at 25763249bp.C / T mutation at 25980517bp, T / A mutation at 27508872bp, T / C mutation at 28212532bp, C / T mutation at 29113539bp, G / C mutation at 29485500bp, T / C mutation at 29809495bp, A / T mutation at 30098613bp, C / T mutation at 30391267bp, A / G mutation at 31955766bp, G / A mutation at 32672496bp, C / T mutation at 33448952bp, C / T mutation at 34565522bp, G / T mutation at 34854460bp, T mutation at 35956877bp / G mutation, G / A mutation at 37299837bp, T / A mutation at 37768791bp, A / G mutation at 38364999bp, C / T mutation at 38772687bp, A / T mutation at 39119217bp, G / A mutation at 39791727bp, G / A mutation at 40956496bp, A / T mutation at 42059421bp, C / T mutation at 42389769bp, A / G mutation at 42831401bp, T / C mutation at 43232251bp, T / A mutation at 43943244bp, A / T mutation at 44224577bp, 44910891 A / T mutation at 46742409bp, T / G mutation at 47231207bp, A / G mutation at 47919343bp, A / T mutation at 48360872bp, G / A mutation at 49359594bp, G / A mutation at 49898952bp, G / A mutation at 50319548bp, G / A mutation at 51049661bp, T / C mutation at 51306897bp, A / C mutation at 51516277bp, A / G mutation at 52191511bp, A / G mutation at 52579004bp, G / A mutation at 53640899bp, C / G mutation at 5397bp, ... A / T mutation at 6693bp, T / C mutation at 54891372bp, C / T mutation at 56410343bp, G / C mutation at 56902918bp, A / G mutation at 57143044bp, G / A mutation at 57422435bp, G / A mutation at 58005227bp, T / C mutation at 58329882bp, T / C mutation at 58802812bp, T / C mutation at 59036884bp, G / T mutation at 60176975bp, G / A mutation at 60593132bp, A / G mutation at 60929419bp, A / G mutation at 61642762bp;

[0055] Chromosome 3: T / C mutation at 167511 bp, G / T mutation at 444560 bp, T / G mutation at 1740957 bp, A / C mutation at 2247068 bp, A / T mutation at 2663410 bp, A / G mutation at 3153958 bp, A / G mutation at 3443363 bp, T / A mutation at 3918303 bp, T / A mutation at 4164736 bp, T / G mutation at 4395261 bp, G / A mutation at 5114758 bp, A / T mutation at 5385938 bp, C / T mutation at 5778988 bp, G / T mutation at 6150113 bp, ... A / T mutation at 63bp, T / A mutation at 7403440bp, C / G mutation at 8943900bp, T / A mutation at 9256892bp, T / C mutation at 9919466bp, A / C mutation at 10174343bp, A / T mutation at 11633388bp, G / A mutation at 12017984bp, A / G mutation at 12915914bp, C / T mutation at 14184322bp, A / T mutation at 14610187bp, C / T mutation at 15112027bp, A / C mutation at 15742955bp, T / C mutation at 18055450bp, and so on. T / C mutation at 0.05 bp, A / T mutation at 20806332 bp, C / T mutation at 21239659 bp, A / T mutation at 21451371 bp, A / G mutation at 21908751 bp, C / T mutation at 22900014 bp, T / G mutation at 23437899 bp, C / G mutation at 24068761 bp, C / T mutation at 24297401 bp, A / G mutation at 25467666 bp, A / T mutation at 26854208 bp, G / A mutation at 27503027 bp, C / T mutation at 27774729 bp, T / C mutation at 28167758 bp, 29 A / G mutation at 499237bp, G / A mutation at 29844353bp, A / G mutation at 30081511bp, G / T mutation at 30294248bp, G / A mutation at 31271920bp, C / T mutation at 31611643bp, C / T mutation at 31900387bp, A / T mutation at 32420847bp, A / G mutation at 33314059bp, A / G mutation at 33562476bp, C / G mutation at 34537410bp, A / G mutation at 35525002bp, A / C mutation at 36168038bp, G / A mutation at 37291953bp.G / C mutation at 37938922bp, T / C mutation at 38158633bp, G / T mutation at 38470830bp, G / A mutation at 39158628bp, G / A mutation at 39719498bp, A / C mutation at 40603131bp, A / C mutation at 40865370bp, T / G mutation at 41259567bp, T / C mutation at 41597965bp, G / A mutation at 41910605bp, T / A mutation at 42938999bp, A / T mutation at 43500081bp, C / T mutation at 44213296bp. The following mutations were observed: A / G at 44459501 bp, C / A at 45077111 bp, A / T at 46562102 bp, T / A at 47015472 bp, A / C at 47248636 bp, A / G at 47755545 bp, G / A at 48068295 bp, G / A at 48403665 bp, C / A at 48859386 bp, A / G at 49155040 bp, A / T at 49514429 bp, T / A at 49801952 bp, and G / C at 50508624 bp. T / C mutation at position 50867456 bp, C / T mutation at position 51172288 bp, G / A mutation at position 51555200 bp, T / A mutation at position 51897141 bp, C / G mutation at position 53046160 bp, T / A mutation at position 53359071 bp, T / A mutation at position 53636779 bp, C / T mutation at position 53869457 bp, T / C mutation at position 54594267 bp, T / C mutation at position 55269130 bp, A / G mutation at position 55572008 bp, A / C mutation at position 55896201 bp, C / T mutation at position 56783882 bp The following mutations were observed: A / G at 57104330 bp, C / G at 57532214 bp, T / C at 57807917 bp, T / A at 58451281 bp, C / G at 58805029 bp, A / C at 59273801 bp, C / A at 59815549 bp, A / G at 60128400 bp, G / A at 60681360 bp, C / T at 61275765 bp, C / T at 61522130 bp, C / A at 61801828 bp, and T / C at 62069187 bp.

[0056] Chromosome 4: C / A mutation at 489947 bp, G / C mutation at 886286 bp, A / G mutation at 2582998 bp, G / A mutation at 3139977 bp, G / A mutation at 3595215 bp, T / A mutation at 3859888 bp, A / G mutation at 4344155 bp, C / A mutation at 4566835 bp, A / G mutation at 5145569 bp, T / C mutation at 6456871 bp, G / C mutation at 7321215 bp, T / A mutation at 7817588 bp, T / G mutation at 8179181 bp, G / T mutation at 8910032 bp, ... G / T mutation at 6 bp, T / G mutation at 9552736 bp, G / A mutation at 10022246 bp, T / G mutation at 10354612 bp, G / A mutation at 10907817 bp, C / T mutation at 11734493 bp, G / A mutation at 12580554 bp, T / C mutation at 13260828 bp, T / C mutation at 13765169 bp, C / T mutation at 14310601 bp, T / C mutation at 16355043 bp, G / A mutation at 17630046 bp, T / A mutation at 19429489 bp, A / T mutation at 19679728 bp, 21079 A / G mutation at 213bp, T / C mutation at 22164490bp, A / C mutation at 22482582bp, T / A mutation at 22780082bp, C / T mutation at 23190102bp, A / G mutation at 23587372bp, A / G mutation at 24228102bp, A / G mutation at 24961164bp, G / T mutation at 25252848bp, A / T mutation at 25844871bp, G / T mutation at 26112318bp, G / A mutation at 27430034bp, C / A mutation at 27889194bp, T / C mutation at 28186129bp, 28 G / T mutation at 862600bp, T / A mutation at 29613029bp, A / T mutation at 29849521bp, C / T mutation at 30074147bp, G / T mutation at 30551177bp, A / C mutation at 31624189bp, T / C mutation at 31868549bp, T / C mutation at 32085476bp, T / C mutation at 33390588bp, T / A mutation at 33885908bp, T / C mutation at 34321828bp, C / A mutation at 34763173bp, A / G mutation at 36691587bp, A / G mutation at 36995284bp.The following mutations were observed at the following locations: A / C at 37364904 bp, C / T at 39303404 bp, G / C at 40040754 bp, T / C at 40300785 bp, T / C at 41007997 bp, T / C at 41213469 bp, A / G at 41586776 bp, A / G at 42194520 bp, G / T at 42525010 bp, C / T at 42726376 bp, T / G at 43723088 bp, A / T at 44141447 bp, and T / C at 44798262 bp. A / G mutation at 45230255bp, A / G mutation at 45691471bp, T / A mutation at 45924080bp, A / G mutation at 46335423bp, G / A mutation at 46609807bp, C / T mutation at 47649738bp, C / A mutation at 48054473bp, G / A mutation at 49037444bp, T / C mutation at 49356447bp, T / A mutation at 49823141bp, C / T mutation at 50253140bp, T / A mutation at 50495698bp, G / A mutation at 50782734bp. G / A mutation at 51328829bp, T / C mutation at 51912827bp, T / C mutation at 52524776bp, C / T mutation at 52849059bp, C / T mutation at 53628954bp, C / T mutation at 54121390bp, C / T mutation at 54500655bp, A / T mutation at 54780363bp, C / A mutation at 55043635bp, T / C mutation at 55269248bp, C / T mutation at 56009969bp, T / C mutation at 56360644bp, T / A mutation at 56621645bp. The following mutations were observed: A / G mutation at 57033970 bp, T / A mutation at 57487468 bp, C / T mutation at 58248810 bp, A / G mutation at 58569969 bp, C / A mutation at 58804428 bp, G / A mutation at 59177752 bp, A / G mutation at 59780097 bp, C / A mutation at 60118503 bp, G / T mutation at 60642124 bp, T / A mutation at 60872702 bp, T / G mutation at 61335564 bp, G / A mutation at 61988945 bp, and G / T mutation at 62219348 bp.

[0057] Chromosome 5: C / G mutation at 879645bp, G / A mutation at 1415586bp, C / T mutation at 2111293bp, C / A mutation at 2314335bp, A / T mutation at 2635565bp, G / A mutation at 2920345bp, T / C mutation at 3467779bp, T / A mutation at 3884050bp, C / G mutation at 4393916bp, A / C mutation at 4660080bp, T / C mutation at 5064983bp, G / A mutation at 5762044bp, G / T mutation at 6544275bp, G / T mutation at 6749408bp, G / T mutation at 699... A / G mutation at 7717bp, T / G mutation at 7345402bp, A / G mutation at 7654532bp, A / G mutation at 7932212bp, A / G mutation at 8303258bp, A / C mutation at 8661151bp, T / C mutation at 9176468bp, T / C mutation at 9464239bp, C / A mutation at 9931584bp, T / C mutation at 10539507bp, A / G mutation at 10875599bp, A / T mutation at 11217368bp, G / A mutation at 11702291bp, C / T mutation at 12003330bp, T / G mutation at 12366955bp A / T mutation at 12808603bp, T / C mutation at 13875823bp, T / C mutation at 14157737bp, A / G mutation at 15150248bp, G / A mutation at 15517737bp, A / G mutation at 15749958bp, A / G mutation at 16257325bp, A / G mutation at 16594460bp, T / C mutation at 17783174bp, T / A mutation at 18355754bp, C / A mutation at 18594492bp, G / A mutation at 18998718bp, A / T mutation at 19767072bp, C / T mutation at 202... T / C mutation at 53708bp, A / C mutation at 20548723bp, G / A mutation at 22378076bp, A / G mutation at 23213346bp, A / C mutation at 23730799bp, T / C mutation at 23996432bp, C / T mutation at 24241420bp, G / A mutation at 24721774bp, T / C mutation at 26586598bp, A / T mutation at 26893151bp, C / T mutation at 27345167bp, T / C mutation at 27679094bp, C / G mutation at 28185342bp, T / A mutation at 29149844bp.C / A mutation at 29649330bp, G / A mutation at 30587198bp, C / A mutation at 30997742bp, G / T mutation at 31256011bp, T / C mutation at 31705376bp, T / C mutation at 32770700bp, G / C mutation at 33339763bp, A / G mutation at 33573910bp, C / T mutation at 34514253bp, A / G mutation at 35112239bp, T / C mutation at 35669268bp, T / A mutation at 36705272bp, C / T mutation at 36922640bp, A mutation at 37131668bp / G mutation, C / T mutation at 37581075bp, C / T mutation at 38542724bp, G / A mutation at 39044859bp, G / C mutation at 39688041bp, G / A mutation at 40000656bp, T / C mutation at 40395464bp, G / A mutation at 41001494bp, T / C mutation at 41681731bp, T / C mutation at 42174184bp, A / T mutation at 42576737bp, A / G mutation at 43098562bp, A / G mutation at 43487720bp, A / G mutation at 43692812bp, A / G mutation at 44169923bp A / C mutation at 44373192bp, A / C mutation at 44895351bp, C / T mutation at 45190288bp, A / G mutation at 46681737bp, T / A mutation at 47488756bp, T / C mutation at 49326052bp, T / C mutation at 49639111bp, G / T mutation at 50108002bp, C / G mutation at 50436257bp, T / A mutation at 50696264bp, A / T mutation at 50903386bp, T / C mutation at 51419745bp, A / G mutation at 51669577bp, C / T mutation at 524bp T / A mutation at 14271bp, A / C mutation at 53368505bp, A / G mutation at 53605312bp, T / C mutation at 53894100bp, T / A mutation at 54916254bp, A / G mutation at 55477238bp, A / C mutation at 55999091bp, G / C mutation at 56214132bp, G / A mutation at 57070120bp, T / A mutation at 57313644bp, A / C mutation at 57666549bp, G / T mutation at 57877313bp, C / T mutation at 58133420bp, G / A mutation at 58891833bp.T / C mutation at 59245611bp, A / T mutation at 59751484bp, and A / G mutation at 59976681bp;

[0058] Chromosome 6: G / A mutation at 587350 bp, A / G mutation at 1075497 bp, T / A mutation at 1679222 bp, G / T mutation at 2172358 bp, T / C mutation at 2739350 bp, G / A mutation at 3206148 bp, C / A mutation at 3421291 bp, G / A mutation at 3843416 bp, A / T mutation at 4064437 bp, G / T mutation at 4469234 bp, T / C mutation at 4760643 bp, T / A mutation at 4992337 bp, A / T mutation at 5778802 bp, A / C mutation at 6114864 bp, ... T / C mutation at 355bp, G / A mutation at 6932249bp, G / A mutation at 8026496bp, C / T mutation at 8885122bp, T / C mutation at 9620041bp, G / T mutation at 10908040bp, C / T mutation at 11117547bp, A / G mutation at 12753363bp, C / T mutation at 14268050bp, T / G mutation at 14586618bp, G / C mutation at 14879882bp, G / A mutation at 15232542bp, A / C mutation at 15807647bp, C / T mutation at 16152642bp, 164216 A / G mutation at 43bp, A / T mutation at 16738467bp, C / A mutation at 17154757bp, G / A mutation at 17669050bp, T / A mutation at 17995298bp, A / T mutation at 18653957bp, T / C mutation at 18914180bp, T / A mutation at 19545790bp, C / T mutation at 20589164bp, A / G mutation at 21516044bp, T / A mutation at 22028166bp, A / T mutation at 22624328bp, T / A mutation at 23340429bp, C / T mutation at 23832772bp, 24 A / G mutation at 266242bp, A / C mutation at 25073811bp, A / G mutation at 26222500bp, A / C mutation at 26774119bp, T / C mutation at 27210621bp, C / T mutation at 27689291bp, G / A mutation at 28235309bp, T / C mutation at 28437764bp, C / T mutation at 28668444bp, T / A mutation at 29329850bp, A / G mutation at 29645718bp, G / T mutation at 30524553bp, G / A mutation at 31121205bp, C / T mutation at 31359984bp.The following mutations were observed: A / G at 32106045 bp, G / A at 32457595 bp, T / C at 32753400 bp, T / A at 33117124 bp, T / C at 33570502 bp, T / G at 34176629 bp, C / T at 34667060 bp, A / G at 38181718 bp, T / A at 39546371 bp, and G / C at 39866670 bp. G / T mutation at 40351063bp, A / G mutation at 40681703bp, T / C mutation at 41023800bp, G / T mutation at 41459997bp, T / C mutation at 42016811bp, T / C mutation at 42364688bp, T / A mutation at 42605575bp, C / A mutation at 43099589bp, C / T mutation at 43383766bp, C / T mutation at 43791635bp. The mutations are: A / T at 44792472 bp, C / T at 45098188 bp, A / G at 45791769 bp, A / G at 46506334 bp, T / C at 47036051 bp, G / C at 47496058 bp, C / T at 47812848 bp, A / C at 48271374 bp, T / A at 49169061 bp, and A / T at 49429004 bp. C / T mutation at position 49743523 bp, T / C mutation at position 50128359 bp, A / G mutation at position 50887722 bp, A / T mutation at position 51813382 bp, C / T mutation at position 52554583 bp, T / C mutation at position 53230845 bp, C / T mutation at position 54083112 bp, C / T mutation at position 54380581 bp, C / T mutation at position 55428291 bp, A / C mutation at position 56179611 bp;

[0059] Chromosome 7: A / G mutation at 603788 bp, G / T mutation at 896391 bp, T / C mutation at 1282724 bp, G / T mutation at 1535834 bp, G / A mutation at 1795922 bp, T / A mutation at 2124131 bp, G / T mutation at 2694802 bp, G / A mutation at 3023240 bp, T / C mutation at 3227288 bp, T / C mutation at 3539581 bp, T / C mutation at 3946542 bp, T / A mutation at 4409015 bp, G / A mutation at 4610418 bp, A / G mutation at 5099145 bp, 5726... T / C mutation at 774bp, T / C mutation at 6090181bp, T / A mutation at 7174959bp, G / T mutation at 7732660bp, T / A mutation at 7943436bp, C / T mutation at 8179975bp, A / T mutation at 9080480bp, T / A mutation at 9322949bp, A / G mutation at 9569794bp, T / C mutation at 9911214bp, A / C mutation at 10663904bp, T / G mutation at 10877221bp, G / A mutation at 11467271bp, T / A mutation at 11736967bp, 12053878b T / C mutation at p, C / T mutation at 13007410bp, A / G mutation at 13768058bp, G / A mutation at 15198787bp, A / T mutation at 15771972bp, T / G mutation at 16027470bp, G / A mutation at 16465374bp, T / G mutation at 16680508bp, A / G mutation at 17192258bp, T / C mutation at 17558072bp, C / T mutation at 17969940bp, C / T mutation at 18480038bp, C / T mutation at 18732421bp, A / C mutation at 19150831bp, 1935 A / T mutation at 8092 bp, C / T mutation at 19653197 bp, T / C mutation at 20077315 bp, T / G mutation at 20839591 bp, A / T mutation at 21779850 bp, A / G mutation at 23771667 bp, C / A mutation at 24586945 bp, A / G mutation at 25311728 bp, A / C mutation at 25544379 bp, T / C mutation at 26417838 bp, G / C mutation at 26642847 bp, A / G mutation at 26965429 bp, A / T mutation at 29758485 bp, T / A mutation at 30141591 bp.C / T mutation at 30468300bp, T / C mutation at 31400778bp, G / T mutation at 31778180bp, A / G mutation at 32075251bp, C / T mutation at 33230067bp, C / T mutation at 33744108bp, T / A mutation at 36023112bp, T / A mutation at 36396469bp, A / G mutation at 36693769bp, and so on. A / T mutation at 37760599bp, C / T mutation at 38161672bp, T / A mutation at 38702757bp, G / T mutation at 39210903bp, A / G mutation at 39699534bp, A / T mutation at 40385742bp, G / C mutation at 41026442bp, T / C mutation at 41307086bp, C / A mutation at 42969755bp, G / A mutation. T / C mutation at 43571847bp, A / G mutation at 43993986bp, T / C mutation at 44394370bp, G / A mutation at 44703340bp, A / G mutation at 45524469bp, C / T mutation at 46045824bp, A / G mutation at 47359820bp, C / A mutation at 47598126bp, A / G mutation at 49292205bp, and at 50492560bp. T / C mutation at 50866106bp, T / C mutation at 51554060bp, C / T mutation at 51867069bp, T / G mutation at 52186344bp, T / C mutation at 52386979bp, A / G mutation at 52719201bp, A / G mutation at 53780380bp, C / T mutation at 54228211bp, G / A mutation at 54901003bp;

[0060] Chromosome 8: G / A mutation at 412562bp, T / A mutation at 985611bp, T / C mutation at 1303653bp, C / T mutation at 1544349bp, T / C mutation at 1922320bp, T / C mutation at 2361255bp, T / A mutation at 2719405bp, A / G mutation at 3000604bp, T / C mutation at 3288220bp, G / C mutation at 3518544bp, T / C mutation at 3933031bp, T / C mutation at 4572621bp, T / C mutation at 5346224bp, A / T mutation at 5602024bp, 59030... T / C mutation at 95bp, C / T mutation at 6172964bp, T / A mutation at 6581392bp, T / A mutation at 7427921bp, T / C mutation at 7653299bp, G / A mutation at 10833172bp, C / T mutation at 11143937bp, C / T mutation at 11563545bp, T / C mutation at 12240151bp, A / G mutation at 12664163bp, C / A mutation at 12976720bp, C / G mutation at 13428217bp, T / A mutation at 13690299bp, C / A mutation at 14184965bp, 145407 A / G mutation at 37bp, A / C mutation at 14927534bp, T / A mutation at 15164050bp, A / C mutation at 16228337bp, C / G mutation at 16839194bp, T / A mutation at 17073823bp, A / G mutation at 17560361bp, C / T mutation at 17805117bp, C / T mutation at 18668691bp, C / T mutation at 19077073bp, A / T mutation at 19321381bp, A / G mutation at 20212862bp, A / G mutation at 20498190bp, T / C mutation at 20748906bp, 21 A / G mutation at 088463bp, A / G mutation at 21591018bp, A / G mutation at 22165379bp, A / T mutation at 23067147bp, T / C mutation at 23844408bp, A / G mutation at 24104507bp, T / A mutation at 24665627bp, G / A mutation at 25079866bp, T / G mutation at 25958988bp, A / G mutation at 27292396bp, G / A mutation at 27511452bp, C / T mutation at 27712853bp, G / A mutation at 27981923bp, G / A mutation at 28485338bp.G / A mutation at 28816819bp, C / A mutation at 29401272bp, C / T mutation at 29733986bp, C / T mutation at 30150021bp, A / T mutation at 30361015bp, A / C mutation at 30665469bp, T / G mutation at 30927726bp, C / G mutation at 31612011bp, and at 31949923bp... C / G mutation at 32350947bp, T / C mutation at 32829805bp, T / C mutation at 33830538bp, T / C mutation at 34428621bp, G / A mutation at 34631910bp, C / A mutation at 34878333bp, T / C mutation at 35167435bp, G / A mutation at 35384611bp C / T mutation at 36295451bp, A / G mutation at 36758686bp, T / C mutation at 38408273bp, T / C mutation at 39084417bp, T / C mutation at 40515577bp, A / G mutation at 40784564bp, C / T mutation at 41770053bp, G / T mutation at 42205041bp, and so on. G / A mutation at 43574397bp, G / A mutation at 44913196bp, T / G mutation at 46413323bp, C / A mutation at 47254739bp, C / T mutation at 47569565bp, T / C mutation at 48443108bp, A / T mutation at 48947202bp, T / C mutation at 50071965bp;

[0061] Chromosome 9: T / C mutation at 217853 bp, A / G mutation at 583042 bp, A / C mutation at 796208 bp, T / C mutation at 1046351 bp, T / G mutation at 1327845 bp, G / A mutation at 2838975 bp, A / G mutation at 3240786 bp, T / C mutation at 3675421 bp, G / T mutation at 4651246 bp, C / T mutation at 4959856 bp, C / T mutation at 5227751 bp, C / T mutation at 5933068 bp, G / A mutation at 6728623 bp, C / T mutation at 7235075 bp, ... C / G mutation at 85bp, A / T mutation at 7915378bp, A / G mutation at 8155620bp, A / C mutation at 8437140bp, C / A mutation at 9041049bp, T / G mutation at 9320125bp, G / C mutation at 9523788bp, A / G mutation at 10256842bp, A / T mutation at 10516635bp, C / G mutation at 11295005bp, C / G mutation at 11568023bp, T / A mutation at 12159413bp, C / T mutation at 12529731bp, T / G mutation at 12735255bp, 13042605 C / A mutation at 13493638bp, A / G mutation at 13853217bp, A / T mutation at 14058546bp, G / A mutation at 15219775bp, A / T mutation at 15704220bp, G / T mutation at 16140181bp, G / A mutation at 16412881bp, T / C mutation at 17010061bp, T / C mutation at 17928904bp, C / T mutation at 18193136bp, T / C mutation at 18452448bp, T / C mutation at 18653747bp, A / T mutation at 18878597bp, 196 C / T mutation at 85478bp, G / A mutation at 20221044bp, T / C mutation at 20993779bp, G / T mutation at 21586001bp, A / G mutation at 22002234bp, C / T mutation at 22394787bp, T / A mutation at 22969695bp, A / T mutation at 23876847bp, C / T mutation at 25194999bp, A / G mutation at 25462264bp, A / T mutation at 25741402bp, A / G mutation at 26959004bp, A / G mutation at 27955752bp, C / A mutation at 28258725bp, A / G mutation.A / T mutation at 29236123bp, A / G mutation at 29908750bp, C / T mutation at 30127537bp, T / C mutation at 31200772bp, C / T mutation at 31445189bp, T / C mutation at 31759757bp, C / A mutation at 32418276bp, T / C mutation at 33430899bp, G / A mutation at 33857260bp, and so on. A / G mutation at 35910999bp, A / G mutation at 36657698bp, T / C mutation at 37111454bp, A / T mutation at 38333366bp, A / T mutation at 38956659bp, G / T mutation at 41437696bp, T / G mutation at 41655725bp, G / T mutation at 42066006bp, T / C mutation at 43778385bp. T / C mutation at 44072139bp, C / T mutation at 44341586bp, T / C mutation at 46347906bp, C / A mutation at 46681275bp, A / G mutation at 47089848bp, T / C mutation at 47332573bp, G / A mutation at 47545232bp, T / C mutation at 47787454bp, T / A mutation at 48000048bp, and so on. T / C mutation at 49478480bp, T / C mutation at 50204065bp, G / A mutation at 50425019bp, C / T mutation at 52132614bp, T / G mutation at 52785787bp, C / T mutation at 53663195bp, C / T mutation at 54046362bp, T / G mutation at 54605490bp, T / C mutation at 54826243bp;

[0062] Chromosome 10: T / C mutation at 1534163bp, T / G mutation at 2455069bp, C / T mutation at 3038564bp, T / C mutation at 4197932bp, A / T mutation at 4800809bp, A / T mutation at 5676331bp, T / C mutation at 6642222bp, A / G mutation at 7400941bp, T / C mutation at 7668457bp, T / C mutation at 7897420bp, A / C mutation at 8180708bp, C / T mutation at 8755317bp, G / A mutation at 9215005bp, C / A mutation at 9779335bp, ... A / G mutation at 5131bp, A / G mutation at 10801950bp, T / C mutation at 11089097bp, C / A mutation at 11732224bp, T / C mutation at 11959146bp, C / T mutation at 12182036bp, A / G mutation at 12384483bp, T / A mutation at 12977779bp, C / G mutation at 13228019bp, G / A mutation at 13725920bp, A / G mutation at 14297949bp, C / T mutation at 14599462bp, A / C mutation at 15316648bp, C / T mutation at 16226209bp, 17 T / A mutation at 125566bp, G / T mutation at 19323146bp, C / A mutation at 20329041bp, C / A mutation at 20545842bp, A / G mutation at 20831667bp, A / G mutation at 21077840bp, T / C mutation at 21855677bp, C / T mutation at 22076843bp, T / G mutation at 22285822bp, C / A mutation at 22573506bp, T / C mutation at 23139968bp, A / T mutation at 23927295bp, C / T mutation at 24217188bp, T / A mutation at 24958349bp, ... A / T mutation at 26265143bp, A / C mutation at 26789789bp, A / G mutation at 27013959bp, C / T mutation at 27270677bp, T / A mutation at 27522828bp, A / T mutation at 28230594bp, A / T mutation at 28621223bp, T / C mutation at 28976931bp, G / C mutation at 30161642bp, G / T mutation at 30602940bp, A / G mutation at 30978727bp, T / A mutation at 31649003bp, G / A mutation at 32503776bp, T / C mutation at 33082095bp.G / A mutation at 33398327bp, A / T mutation at 34352656bp, C / T mutation at 34825626bp, A / G mutation at 35322952bp, A / T mutation at 35627155bp, C / T mutation at 35967237bp, C / G mutation at 36252688bp, G / A mutation at 36722144bp, T / C mutation at 37789019bp.

[0063] II. Identification of parentage in tetraploid Crassula ovata based on the aforementioned 1000 SNP loci

[0064] 1. Sequencing of tetraploid long oysters

[0065] We collected data from 66 offspring and 2 parents with clear parent-child relationships. We also collected data from 10 offspring and 6 parents with unknown kinship relationships.

[0066] Adductor muscles of tetraploid oysters were collected, and DNA from each sample was purified using the GenoPrep Polysaccharide-Polyphenol DNA Extraction Kit (Shijiazhuang Borui Biotechnology Co., Ltd.). DNA concentration was determined using a Qubit 2.0 real-time fluorescence analyzer, and DNA quality was assessed using 1% agarose gel electrophoresis.

[0067] Sequencing libraries were constructed using the GenoBaits® DNA Library Prep Kit for ILM.

[0068] High-throughput sequencing of DNA fragments in the sequencing library was performed using an MGISEQ-2000 sequencer (Shenzhen BGI Genomics Co., Ltd.) in PE150 mode.

[0069] 2. Obtain the genotypes of the above 1000 SNP loci in tetraploid oysters.

[0070] The raw data was aligned to the reference genome of the Pacific oyster, and GATK was used to perform genotyping on the aligned data to obtain the genotypes of the above 1000 SNP loci in the tetraploid Pacific oyster to be tested.

[0071] 3. Calculate the IBS and LOD of all tested parents and offspring based on the SNP genotyping results.

[0072] (1) Calculate the individual consistency (IBS) of all tested parents and offspring.

[0073] Based on the laws of tetraploid inheritance, the IBS calculation formula developed in this invention for identifying parentage in tetraploid oysters is as follows:

[0074]

[0075] Where n represents the number of SNP loci successfully genotyped, k represents the k-th SNP locus, and G ik For the genotype of individual i at the k-th SNP locus, G jk For the genotype of individual j at the k-th SNP locus, I k (G) ik G jk () represents the genotype matching score between individual i and individual j at the k-th SNP locus. If the genotypes of individuals i and j at the k-th SNP locus match as parent and child, then I k (G) ik G jk If ) is 1, otherwise I k (G) ik G jk The value is 0.

[0076] (2) Calculate the log likelihood ratio (LOD) for all tested parents and offspring.

[0077] Based on the genetic principles of tetraploidy, the LOD calculation formula developed in this invention for identifying parentage in tetraploid oysters is as follows:

[0078]

[0079] Where n represents the number of SNP loci successfully genotyped, k represents the kth SNP locus, P1 represents the probability of offspring produced by the candidate parent and the simulated parent, and P2 represents the background probability (i.e. the probability of producing offspring when the genotypes of the two parents are completely random).

[0080] The formula for calculating P1 is as follows:

[0081]

[0082] in, This represents the probability of the appearance of candidate parent gametes. λ represents the probability of the occurrence of simulated parental gametes, w represents the frequency of simulated parental genotypes in the population, m represents the number of mismatched genotypes between parents and offspring, which is 0 or 1, and λ represents the mismatch penalty weight, which is 0.1.

[0083] When calculating P2, the genotypes of the simulated parents are determined based on the population allele frequencies.

[0084] 4. Combine IBS and LOD to determine the parentage between the tested parents and offspring.

[0085] When LOD is greater than 0, the candidate parent may be the real parent, and at least one parent with the highest LOD is most likely to be the real parent of the offspring. In this case, the IBS between the candidate parent and the offspring should be higher than the preset threshold of 0.98.

[0086] When LOD is less than 0, the candidate parent cannot be the real parent.

[0087] In this specific embodiment, the paternity and maternity identification results of the tetraploid *Crassostrea gigas* are shown in Tables 1-1 and 1-2. The parentage identification results among these samples fully conformed to pedigree relationships; all offspring with clear parentage were identified as having a true parent, while none of the 10 offspring with unknown parentage were identified as having a parent. The distribution diagram of the level of distance (LOD) among parent-related samples is shown below. Figure 1 See the LOD and IBS heatmaps for some samples. Figure 2 .

[0088] Table 1-1 Results of Paternity and Maternity Identification in Tetraploid Crassula ovata

[0089]

[0090] Table 1-2 Results of Paternity and Maternity Identification in Tetraploid Crassula ovata

[0091]

[0092] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A tetraploid long oyster parentage identification method based on SNP sites, characterized by, The method comprises the following steps: (1) taking the adductor muscle of the tetraploid Crassostrea gigas to be tested for sequencing; (2) comparing the Crassostrea gigas reference genome to obtain the genotype of the specified SNP site of the tetraploid Crassostrea gigas, wherein the GenBank accession number of the Crassostrea gigas reference genome is GCA_011032805.1, and the genotype of the specified SNP site is as follows: 1 chromosome: G / A mutation at position 121890 bp, A / G mutation at position 1057743 bp, T / G mutation at position 1282310 bp, G / T mutation at position 1672864 bp, T / C mutation at position 2377246 bp, T / A mutation at position 3067043 bp, T / C mutation at position 3586786 bp, A / T mutation at position 3999280 bp, T / C mutation at position 4728772 bp, T / C mutation at position 5044925 bp, T / C mutation at position 5300940 bp, A / G mutation at position 5594188 bp, C / T mutation at position 5903109 bp, T / G mutation at position 6330374 bp, T / C mutation at position 6833762 bp, A / G mutation at position 7943920 bp, A / T mutation at position 8487382 bp, A / G mutation at position 9704220 bp, T / C mutation at position 10224929 bp, T / C mutation at position 10862004 bp, T / G mutation at position 11750498 bp, T / C mutation at position 11990399 bp, T / C mutation at position 12218512 bp, G / A mutation at position 12483376 bp, G / A mutation at position 13361474 bp, G / T mutation at position 13765448 bp, T / A mutation at position 14300439 bp, T / A mutation at position 15205025 bp, A / G mutation at position 15645080 bp, G / T mutation at position 15940394 bp, T / C mutation at position 17045095 bp, T / C mutation at position 17268995 bp, T / A mutation at position 17538044 bp, A / T mutation at position 17899956 bp, G / T mutation at position 18451368 bp, C / T mutation at position 18760584 bp, C / G mutation at position 19501089 bp, G / T mutation at position 21531956 bp, T / A mutation at position 22401721 bp, T / A mutation at position 24526392 bp, A / T mutation at position 25189059 bp, G / A mutation at position 25603252 bp, A / T mutation at position 26161161 bp, C / T mutation at position 26611917 bp, A / C mutation at position 27004404 bp, A / G mutation at position 27425048 bp, C / T mutation at position 27805924 bp, A / T mutation at position 28632425 bp, A / T mutation at position 29021720 bp, C / T mutation at position 33066462 bp, A / T mutation at position 33718201 bp, G / A mutation at position 34717118 bp, A / C mutation at position 35189356 bp, C / T mutation at position 36608599 bp, C / T mutation at position 36886136 bp, C / A mutation at position 37186110 bp,G / C mutation at position 37483058 bp, C / A mutation at position 38812679 bp, A / G mutation at position 39546717 bp, C / T mutation at position 40317002 bp, A / G mutation at position 41392051 bp, G / A mutation at position 43046392 bp, C / T mutation at position 43389852 bp, T / A mutation at position 45510791 bp, A / G mutation at position 46464535 bp, A / G mutation at position 47037306 bp, T / G mutation at position 47275653 bp, A / T mutation at position 47543454 bp, A / T mutation at position 48596129 bp, A / G mutation at position 49583230 bp, T / C mutation at position 49868879 bp, T / G mutation at position 50086266 bp, T / G mutation at position 50302873 bp, A / C mutation at position 51214188 bp, C / T mutation at position 51485365 bp, G / A mutation at position 52121167 bp, A / T mutation at position 52424658 bp, C / T mutation at position 52758029 bp, A / G mutation at position 53158981 bp, C / T mutation at position 53542635 bp, T / C mutation at position 53910234 bp, C / T mutation at position 54359105 bp, T / C mutation at position 55808097 bp, T / A mutation at position 56281482 bp, C / T mutation at position 57138543 bp, G / A mutation at position 58299726 bp, C / T mutation at position 59081390 bp, G / T mutation at position 59774992 bp, T / G mutation at position 60482812 bp, C / T mutation at position 60889634 bp, G / A mutation at position 61245346 bp, C / G mutation at position 61505117 bp, C / A mutation at position 61817752 bp, A / T mutation at position 62517352 bp, T / C mutation at position 62882914 bp, A / G mutation at position 63105443 bp, C / T mutation at position 64259558 bp, C / A mutation at position 65252225 bp, T / A mutation at position 66625596 bp, A / G mutation at position 66835219 bp, C / T mutation at position 67248469 bp, T / A mutation at position 67503687 bp, A / G mutation at position 67906236 bp, C / T mutation at position 68247170 bp, C / T mutation at position 68945233 bp, T / C mutation at position 69565892 bp, G / A mutation at position 70058662 bp, A / G mutation at position 70314116 bp, A / T mutation at position 70765241 bp, A / G mutation at position 72411555 bp, T / C mutation at position 73822739 bp,A / G mutation at position 74137180 bp, A / G mutation at position 75053096 bp, A / C mutation at position 75443458 bp, A / G mutation at position 76271402 bp, T / A mutation at position 76958049 bp, C / G mutation at position 77190736 bp, T / A mutation at position 77491425 bp, A / T mutation at position 77966387 bp, C / T mutation at position 78744623 bp; 2: T / C at position 126208, G / A at position 530724, A / T at position 1030962, C / G at position 1883338, T / A at position 2385548, A / G at position 2612768, T / C at position 2905649, T / C at position 3408267, A / G at position 4209622, T / C at position 4735213, C / G at position 5034762, C / A at position 5605969, A / G at position 5935587, T / G at position 6189866, A / T at position 6451484, A / G at position 7186787, A / G at position 7919353, T / A at position 8150271, T / C at position 8400126, A / G at position 8695230, G / T at position 9416767, C / T at position 9823074, T / C at position 10397975, A / T at position 10863126, A / G at position 11074140, A / G at position 11296287, A / G at position 11502471, C / G at position 12364482, T / G at position 12574976, T / C at position 12794499, C / A at position 13083777, T / C at position 13511367, T / C at position 13877414, C / A at position 14299527, G / A at position 14524308, A / G at position 14736903, G / A at position 15063383, A / G at position 15597292, T / A at position 15867029, G / A at position 16101395, C / A at position 16326428, T / C at position 16905050, A / T at position 17266489, A / T at position 17579365, G / T at position 18124593, G / C at position 18474645, A / G at position 18705311, C / A at position 19267782, A / G at position 21377238, T / A at position 22776786, A / C at position 23709017, A / G at position 24156723, A / C at position 24530341, G / A at position 25094735, G / C at position 25421687, A / G at position 25763249,C / T mutation at position 25980517 bp, T / A mutation at position 27508872 bp, T / C mutation at position 28212532 bp, C / T mutation at position 29113539 bp, G / C mutation at position 29485500 bp, T / C mutation at position 29809495 bp, A / T mutation at position 30098613 bp, C / T mutation at position 30391267 bp, A / G mutation at position 31955766 bp, G / A mutation at position 32672496 bp, C / T mutation at position 33448952 bp, C / T mutation at position 34565522 bp, G / T mutation at position 34854460 bp, T / G mutation at position 35956877 bp, G / A mutation at position 37299837 bp, T / A mutation at position 37768791 bp, A / G mutation at position 38364999 bp, C / T mutation at position 38772687 bp, A / T mutation at position 39119217 bp, G / A mutation at position 39791727 bp, G / A mutation at position 40956496 bp, A / T mutation at position 42059421 bp, C / T mutation at position 42389769 bp, A / G mutation at position 42831401 bp, T / C mutation at position 43232251 bp, T / A mutation at position 43943244 bp, A / T mutation at position 44224577 bp, A / T mutation at position 44910891 bp, T / G mutation at position 46742409 bp, A / G mutation at position 47231207 bp, A / T mutation at position 47919343 bp, G / A mutation at position 48360872 bp, G / T mutation at position 49359594 bp, G / A mutation at position 49898952 bp, G / A mutation at position 50319548 bp, T / C mutation at position 51049661 bp, A / C mutation at position 51306897 bp, A / G mutation at position 51516277 bp, A / G mutation at position 52191511 bp, G / A mutation at position 52579004 bp, C / G mutation at position 53640899 bp, A / T mutation at position 53976693 bp, T / C mutation at position 54891372 bp, C / T mutation at position 56410343 bp, G / C mutation at position 56902918 bp, A / G mutation at position 57143044 bp, G / A mutation at position 57422435 bp, G / A mutation at position 58005227 bp, T / C mutation at position 58329882 bp, T / C mutation at position 58802812 bp, T / C mutation at position 59036884 bp, G / T mutation at position 60176975 bp, G / A mutation at position 60593132 bp, A / G mutation at position 60929419 bp, A / G mutation at position 61642762 bp, 3: T / C at position 167511, G / T at position 444560, T / G at position 1740957, A / C at position 2247068, A / T at position 2663410, A / G at position 3153958, A / G at position 3443363, T / A at position 3918303, T / A at position 4164736, T / G at position 4395261, G / A at position 5114758, A / T at position 5385938, C / T at position 5778988, G / T at position 6150113, A / T at position 6576463, T / A at position 7403440, C / G at position 8943900, T / A at position 9256892, T / C at position 9919466, A / C at position 10174343, A / T at position 11633388, G / A at position 12017984, A / G at position 12915914, C / T at position 14184322, A / T at position 14610187, C / T at position 15112027, A / C at position 15742955, T / C at position 18055450, T / C at position 20548205, A / T at position 20806332, C / T at position 21239659, A / T at position 21451371, A / G at position 21908751, C / T at position 22900014, T / G at position 23437899, C / G at position 24068761, C / T at position 24297401, A / G at position 25467666, A / T at position 26854208, G / A at position 27503027, C / T at position 27774729, T / C at position 28167758, A / G at position 29499237, G / A at position 29844353, A / G at position 30081511, G / T at position 30294248, G / A at position 31271920, C / T at position 31611643, C / T at position 31900387, A / T at position 32420847, A / G at position 33314059, C / G at position 33562476, A / G at position 34537410, A / C at position 35525002, G / A at position 36168038, G / A at position 37291953,G / C mutation at position 37938922 bp, T / C mutation at position 38158633 bp, G / T mutation at position 38470830 bp, G / A mutation at position 39158628 bp, G / A mutation at position 39719498 bp, A / C mutation at position 40603131 bp, A / C mutation at position 40865370 bp, T / G mutation at position 41259567 bp, T / C mutation at position 41597965 bp, G / A mutation at position 41910605 bp, T / A mutation at position 42938999 bp, A / T mutation at position 43500081 bp, C / T mutation at position 44213296 bp, A / G mutation at position 44459501 bp, C / A mutation at position 45077111 bp, A / T mutation at position 46562102 bp, T / A mutation at position 47015472 bp, A / C mutation at position 47248636 bp, A / G mutation at position 47755545 bp, G / A mutation at position 48068295 bp, G / A mutation at position 48403665 bp, C / A mutation at position 48859386 bp, A / G mutation at position 49155040 bp, A / T mutation at position 49514429 bp, T / A mutation at position 49801952 bp, G / C mutation at position 50508624 bp, T / C mutation at position 50867456 bp, C / T mutation at position 51172288 bp, G / A mutation at position 51555200 bp, T / A mutation at position 51897141 bp, C / G mutation at position 53046160 bp, T / A mutation at position 53359071 bp, T / A mutation at position 53636779 bp, C / T mutation at position 53869457 bp, T / C mutation at position 54594267 bp, T / C mutation at position 55269130 bp, A / G mutation at position 55572008 bp, A / C mutation at position 55896201 bp, C / T mutation at position 56783882 bp, A / G mutation at position 57104330 bp, C / G mutation at position 57532214 bp, T / C mutation at position 57807917 bp, T / A mutation at position 58451281 bp, C / G mutation at position 58805029 bp, A / C mutation at position 59273801 bp, C / A mutation at position 59815549 bp, A / G mutation at position 60128400 bp, G / A mutation at position 60681360 bp, C / T mutation at position 61275765 bp, C / T mutation at position 61522130 bp, C / A mutation at position 61801828 bp, T / C mutation at position 62069187 bp; 4: C / A at position 489947, G / C at position 886286, A / G at position 2582998, G / A at position 3139977, G / A at position 3595215, T / A at position 3859888, A / G at position 4344155, C / A at position 4566835, A / G at position 5145569, T / C at position 6456871, G / C at position 7321215, T / A at position 7817588, T / G at position 8179181, G / T at position 8910032, G / T at position 9218976, T / G at position 9552736, G / A at position 10022246, T / G at position 10354612, G / A at position 10907817, C / T at position 11734493, G / A at position 12580554, T / C at position 13260828, T / C at position 13765169, C / T at position 14310601, T / C at position 16355043, G / A at position 17630046, T / A at position 19429489, A / T at position 19679728, A / G at position 21079213, T / C at position 22164490, A / C at position 22482582, T / A at position 22780082, C / T at position 23190102, A / G at position 23587372, A / G at position 24228102, A / G at position 24961164, G / T at position 25252848, A / T at position 25844871, G / T at position 26112318, G / A at position 27430034, C / A at position 27889194, T / C at position 28186129, G / T at position 28862600, T / A at position 29613029, A / T at position 29849521, C / T at position 30074147, G / T at position 30551177, A / C at position 31624189, T / C at position 31868549, T / C at position 32085476, T / C at position 33390588, T / A at position 33885908, T / C at position 34321828, C / A at position 34763173, A / G at position 36691587, A / G at position 36995284,A / C mutation at position 37364904 bp, C / T mutation at position 39303404 bp, G / C mutation at position 40040754 bp, T / C mutation at position 40300785 bp, T / C mutation at position 41007997 bp, T / C mutation at position 41213469 bp, A / G mutation at position 41586776 bp, A / G mutation at position 42194520 bp, G / T mutation at position 42525010 bp, C / T mutation at position 42726376 bp, T / G mutation at position 43723088 bp, A / T mutation at position 44141447 bp, T / C mutation at position 44798262 bp, A / G mutation at position 45230255 bp, A / G mutation at position 45691471 bp, T / A mutation at position 45924080 bp, A / G mutation at position 46335423 bp, G / A mutation at position 46609807 bp, C / T mutation at position 47649738 bp, C / A mutation at position 48054473 bp, G / A mutation at position 49037444 bp, T / C mutation at position 49356447 bp, T / A mutation at position 49823141 bp, C / T mutation at position 50253140 bp, T / A mutation at position 50495698 bp, G / A mutation at position 50782734 bp, G / A mutation at position 51328829 bp, T / C mutation at position 51912827 bp, T / C mutation at position 52524776 bp, C / T mutation at position 52849059 bp, C / T mutation at position 53628954 bp, C / T mutation at position 54121390 bp, C / T mutation at position 54500655 bp, A / T mutation at position 54780363 bp, C / A mutation at position 55043635 bp, T / C mutation at position 55269248 bp, C / T mutation at position 56009969 bp, T / C mutation at position 56360644 bp, T / A mutation at position 56621645 bp, A / G mutation at position 57033970 bp, T / A mutation at position 57487468 bp, C / T mutation at position 58248810 bp, A / G mutation at position 58569969 bp, C / A mutation at position 58804428 bp, G / A mutation at position 59177752 bp, A / G mutation at position 59780097 bp, C / A mutation at position 60118503 bp, G / T mutation at position 60642124 bp, T / A mutation at position 60872702 bp, T / G mutation at position 61335564 bp, G / A mutation at position 61988945 bp, G / T mutation at position 62219348 bp; 5: C / G at position 879645, G / A at position 1415586, C / T at position 2111293, C / A at position 2314335, A / T at position 2635565, G / A at position 2920345, T / C at position 3467779, T / A at position 3884050, C / G at position 4393916, A / C at position 4660080, T / C at position 5064983, G / A at position 5762044, G / T at position 6544275, G / T at position 6749408, A / G at position 6997717, T / G at position 7345402, A / G at position 7654532, A / G at position 7932212, A / C at position 8303258, T / C at position 8661151, T / C at position 9176468, C / A at position 9464239, T / C at position 9931584, A / G at position 10539507, A / T at position 10875599, G / A at position 11217368, C / T at position 11702291, T / G at position 12003330, A / T at position 12366955, T / C at position 12808603, T / C at position 13875823, A / G at position 14157737, G / A at position 15150248, A / G at position 15517737, A / G at position 15749958, A / G at position 16257325, T / C at position 16594460, T / A at position 17783174, C / A at position 18355754, G / A at position 18594492, A / T at position 18998718, C / T at position 19767072, T / C at position 20253708, A / C at position 20548723, G / A at position 22378076, A / G at position 23213346, A / C at position 23730799, T / C at position 23996432, C / T at position 24241420, G / A at position 24721774, T / C at position 26586598, A / T at position 26893151, C / T at position 27345167, T / C at position 27679094, C / G at position 28185342, T / A at position 29149844,C / A mutation at position 2964933, G / A mutation at position 30587198, C / A mutation at position 30997742, G / T mutation at position 31256011, T / C mutation at position 31705376, T / C mutation at position 32770700, G / C mutation at position 33339763, A / G mutation at position 33573910, C / T mutation at position 34514253, A / G mutation at position 35112239, T / C mutation at position 35669268, T / A mutation at position 36705272, C / T mutation at position 36922640, A / G mutation at position 37131668, C / T mutation at position 37581075, C / T mutation at position 38542724, G / A mutation at position 39044859, G / C mutation at position 39688041, G / A mutation at position 40000656, T / C mutation at position 40395464, G / A mutation at position 41001494, T / C mutation at position 41681731, T / C mutation at position 42174184, A / T mutation at position 42576737, A / G mutation at position 43098562, A / G mutation at position 43487720, A / G mutation at position 43692812, A / C mutation at position 44169923, A / C mutation at position 44373192, C / T mutation at position 44895351, A / G mutation at position 45190288, T / A mutation at position 46681737, T / C mutation at position 47488756, T / C mutation at position 49326052, G / T mutation at position 49639111, C / G mutation at position 50108002, T / A mutation at position 50436257, A / T mutation at position 50696264, T / C mutation at position 50903386, A / G mutation at position 51419745, C / T mutation at position 51669577, T / A mutation at position 52414271, A / C mutation at position 53368505, A / G mutation at position 53605312, T / C mutation at position 53894100, T / A mutation at position 54916254, A / G mutation at position 55477238, A / C mutation at position 55999091, G / C mutation at position 56214132, G / A mutation at position 57070120, T / A mutation at position 57313644, A / C mutation at position 57666549, G / T mutation at position 57877313, C / T mutation at position 58133420, G / A mutation at position 58891833,T / C mutation at position 59245611 bp, A / T mutation at position 59751484 bp, A / G mutation at position 59976681 bp; Chromosome 6: G / A mutation at position 587350, A / G mutation at position 1075497, T / A mutation at position 1679222, G / T mutation at position 2172358, T / C mutation at position 2739350, G / A mutation at position 3206148, C / A mutation at position 3421291, G / A mutation at position 3843416, A / T mutation at position 4064437, G / T mutation at position 4469234, T / C mutation at position 4760643, T / A mutation at position 4992337, A / T mutation at position 5778802, A / C mutation at position 6114864, T / C mutation at position 6400355, G / A mutation at position 6932249, G / A mutation at position 8026496, C / T mutation at position 8885122, T / C mutation at position 9620041, G / T mutation at position 10908040, C / T mutation at position 11117547, A / G mutation at position 12753363, C / T mutation at position 14268050, T / G mutation at position 14586618, G / C mutation at position 14879882, G / A mutation at position 15232542, A / C mutation at position 15807647, C / T mutation at position 16152642, A / G mutation at position 16421643, A / T mutation at position 16738467, C / A mutation at position 17154757, G / A mutation at position 17669050, T / A mutation at position 17995298, A / T mutation at position 18653957, T / C mutation at position 18914180, T / A mutation at position 19545790, C / T mutation at position 20589164, A / G mutation at position 21516044, T / A mutation at position 22028166, A / T mutation at position 22624328, T / A mutation at position 23340429, C / T mutation at position 23832772, A / G mutation at position 24266242, A / C mutation at position 25073811, A / G mutation at position 26222500, A / C mutation at position 26774119, T / C mutation at position 27210621, C / T mutation at position 27689291, G / A mutation at position 28235309, T / C mutation at position 28437764, C / T mutation at position 28668444, T / A mutation at position 29329850, A / G mutation at position 29645718, G / T mutation at position 30524553, G / A mutation at position 31121205, C / T mutation at position 31359984,A / G mutation at position 32106045 bp, G / A mutation at position 32457595 bp, T / C mutation at position 32753400 bp, T / A mutation at position 33117124 bp, T / C mutation at position 33570502 bp, T / G mutation at position 34176629 bp, C / T mutation at position 34667060 bp, A / G mutation at position 38181718 bp, T / A mutation at position 39546371 bp, G / C mutation at position 39866670 bp, G / T mutation at position 40351063 bp, A / G mutation at position 40681703 bp, T / C mutation at position 41023800 bp, G / T mutation at position 41459997 bp, T / C mutation at position 42016811 bp, T / C mutation at position 42364688 bp, T / A mutation at position 42605575 bp, C / A mutation at position 43099589 bp, C / T mutation at position 43383766 bp, C / T mutation at position 43791635 bp, A / T mutation at position 44792472 bp, C / T mutation at position 45098188 bp, A / G mutation at position 45791769 bp, A / G mutation at position 46506334 bp, T / C mutation at position 47036051 bp, G / C mutation at position 47496058 bp, C / T mutation at position 47812848 bp, A / C mutation at position 48271374 bp, T / A mutation at position 49169061 bp, A / T mutation at position 49429004 bp, C / T mutation at position 49743523 bp, T / C mutation at position 50128359 bp, A / G mutation at position 50887722 bp, A / T mutation at position 51813382 bp, C / T mutation at position 52554583 bp, T / C mutation at position 53230845 bp, C / T mutation at position 54083112 bp, C / T mutation at position 54380581 bp, C / T mutation at position 55428291 bp, A / C mutation at position 56179611 bp; 7: A / G at position 603788, G / T at position 896391, T / C at position 1282724, G / T at position 1535834, G / A at position 1795922, T / A at position 2124131, G / T at position 2694802, G / A at position 3023240, T / C at position 3227288, T / C at position 3539581, T / C at position 3946542, T / A at position 4409015, G / A at position 4610418, A / G at position 5099145, T / C at position 5726774, T / C at position 6090181, T / A at position 7174959, G / T at position 7732660, T / A at position 7943436, C / T at position 8179975, A / T at position 9080480, T / A at position 9322949, A / G at position 9569794, T / C at position 9911214, A / C at position 10663904, T / G at position 10877221, G / A at position 11467271, T / A at position 11736967, T / C at position 12053878, C / T at position 13007410, A / G at position 13768058, G / A at position 15198787, A / T at position 15771972, T / G at position 16027470, G / A at position 16465374, T / G at position 16680508, A / G at position 17192258, T / C at position 17558072, C / T at position 17969940, C / T at position 18480038, C / T at position 18732421, A / C at position 19150831, A / T at position 19358092, C / T at position 19653197, T / C at position 20077315, T / G at position 20839591, A / T at position 21779850, A / G at position 23771667, C / A at position 24586945, A / G at position 25311728, A / C at position 25544379, T / C at position 26417838, G / C at position 26642847, A / G at position 26965429, A / T at position 29758485, T / A at position 30141591,C / T mutation at position 30468300 bp, T / C mutation at position 31400778 bp, G / T mutation at position 31778180 bp, A / G mutation at position 32075251 bp, C / T mutation at position 33230067 bp, C / T mutation at position 33744108 bp, T / A mutation at position 36023112 bp, T / A mutation at position 36396469 bp, A / G mutation at position 36693769 bp, A / T mutation at position 36913195 bp, C / T mutation at position 37760599 bp, T / A mutation at position 38161672 bp, G / T mutation at position 38702757 bp, A / G mutation at position 39210903 bp, A / T mutation at position 39699534 bp, G / C mutation at position 40385742 bp, T / C mutation at position 41026442 bp, C / A mutation at position 41307086 bp, G / A mutation at position 42969755 bp, T / C mutation at position 43571847 bp, A / G mutation at position 43993986 bp, T / C mutation at position 44394370 bp, G / A mutation at position 44703340 bp, A / G mutation at position 45524469 bp, C / T mutation at position 46045824 bp, A / G mutation at position 47359820 bp, C / A mutation at position 47598126 bp, A / G mutation at position 49292205 bp, T / C mutation at position 50492560 bp, T / C mutation at position 50866106 bp, C / T mutation at position 51554060 bp, T / G mutation at position 51867069 bp, T / C mutation at position 52186344 bp, A / G mutation at position 52386979 bp, A / G mutation at position 52719201 bp, C / T mutation at position 53780380 bp, G / A mutation at position 54228211 bp, A / C mutation at position 54901003 bp, 8: G / A at position 412562, T / A at position 985611, T / C at position 1303653, C / T at position 1544349, T / C at position 1922320, T / C at position 2361255, C / A at position 2719405, A / G at position 3000604, T / C at position 3288220, G / C at position 3518544, T / C at position 3933031, T / C at position 4572621, T / C at position 5346224, A / T at position 5602024, T / C at position 5903095, C / T at position 6172964, T / A at position 6581392, T / A at position 7427921, T / C at position 7653299, G / A at position 10833172, C / T at position 11143937, C / T at position 11563545, T / C at position 12240151, A / G at position 12664163, C / A at position 12976720, C / G at position 13428217, T / A at position 13690299, C / A at position 14184965, A / G at position 14540737, A / C at position 14927534, T / A at position 15164050, A / C at position 16228337, C / G at position 16839194, T / A at position 17073823, A / G at position 17560361, C / T at position 17805117, C / T at position 18668691, C / T at position 19077073, A / T at position 19321381, A / G at position 20212862, A / G at position 20498190, T / C at position 20748906, A / G at position 21088463, A / G at position 21591018, A / G at position 22165379, A / T at position 23067147, T / C at position 23844408, A / G at position 24104507, T / A at position 24665627, G / A at position 25079866, T / G at position 25958988, A / G at position 27292396, G / A at position 27511452, C / T at position 27712853, G / A at position 27981923, G / A at position 28485338,G / A mutation at position 28816819 bp, C / A mutation at position 29401272 bp, C / T mutation at position 29733986 bp, C / T mutation at position 30150021 bp, A / T mutation at position 30361015 bp, A / C mutation at position 30665469 bp, T / G mutation at position 30927726 bp, C / G mutation at position 31612011 bp, C / G mutation at position 31949923 bp, T / C mutation at position 32350947 bp, T / C mutation at position 32829805 bp, T / C mutation at position 33830538 bp, T / C mutation at position 34428621 bp, G / A mutation at position 34631910 bp, C / A mutation at position 34878333 bp, T / C mutation at position 35167435 bp, G / A mutation at position 35384611 bp, C / T mutation at position 36295451 bp, A / G mutation at position 36758686 bp, T / C mutation at position 38408273 bp, T / C mutation at position 39084417 bp, T / C mutation at position 40515577 bp, A / G mutation at position 40784564 bp, C / T mutation at position 41770053 bp, G / T mutation at position 42205041 bp, G / A mutation at position 42605472 bp, G / A mutation at position 43574397 bp, T / G mutation at position 44913196 bp, C / A mutation at position 46413323 bp, C / T mutation at position 47254739 bp, T / C mutation at position 47569565 bp, C / T mutation at position 48443108 bp, A / T mutation at position 48947202 bp, T / C mutation at position 50071965 bp; 9: T / C at position 217853, A / G at position 583042, A / C at position 796208, T / C at position 1046351, T / G at position 1327845, G / A at position 2838975, A / G at position 3240786, T / C at position 3675421, G / T at position 4651246, C / T at position 4959856, C / T at position 5227751, C / T at position 5933068, G / A at position 6728623, C / T at position 7235075, C / G at position 7492985, A / T at position 7915378, A / G at position 8155620, A / C at position 8437140, C / A at position 9041049, T / G at position 9320125, G / C at position 9523788, A / G at position 10256842, A / T at position 10516635, C / G at position 11295005, C / G at position 11568023, T / A at position 12159413, C / T at position 12529731, T / G at position 12735255, C / A at position 13042605, A / G at position 13493638, A / T at position 13853217, G / A at position 14058546, A / T at position 15219775, G / T at position 15704220, G / A at position 16140181, T / C at position 16412881, T / C at position 17010061, T / C at position 17928904, C / T at position 18193136, T / C at position 18452448, T / C at position 18653747, A / T at position 18878597, C / T at position 19685478, G / A at position 20221044, T / C at position 20993779, G / T at position 21586001, A / G at position 22002234, C / T at position 22394787, T / A at position 22969695, A / T at position 23876847, C / T at position 25194999, A / G at position 25462264, A / T at position 25741402, A / G at position 26959004, C / A at position 27955752, A / G at position 28258725,A / T mutation at position 29236123 bp, A / G mutation at position 29908750 bp, C / T mutation at position 30127537 bp, T / C mutation at position 31200772 bp, C / T mutation at position 31445189 bp, T / C mutation at position 31759757 bp, C / A mutation at position 32418276 bp, T / C mutation at position 33430899 bp, G / A mutation at position 33857260 bp, A / G mutation at position 34836159 bp, A / G mutation at position 35910999 bp, T / C mutation at position 36657698 bp, A / T mutation at position 37111454 bp, A / T mutation at position 38333366 bp, G / T mutation at position 38956659 bp, T / G mutation at position 41437696 bp, G / T mutation at position 41655725 bp, T / C mutation at position 42066006 bp, G / C mutation at position 43778385 bp, T / C mutation at position 44072139 bp, C / T mutation at position 44341586 bp, T / C mutation at position 46347906 bp, C / A mutation at position 46681275 bp, A / G mutation at position 47089848 bp, T / C mutation at position 47332573 bp, G / A mutation at position 47545232 bp, T / C mutation at position 47787454 bp, T / A mutation at position 48000048 bp, T / C mutation at position 48209798 bp, T / C mutation at position 49478480 bp, G / A mutation at position 50204065 bp, C / T mutation at position 50425019 bp, T / G mutation at position 52132614 bp, T / C mutation at position 52785787 bp, C / T mutation at position 53663195 bp, C / T mutation at position 54046362 bp, T / G mutation at position 54605490 bp, T / C mutation at position 54826243 bp; 10: T / C at position 1534163, T / G at position 2455069, C / T at position 3038564, T / C at position 4197932, A / T at position 4800809, A / T at position 5676331, T / C at position 6642222, A / G at position 7400941, T / C at position 7668457, T / C at position 7897420, A / C at position 8180708, C / T at position 8755317, G / A at position 9215005, C / A at position 9779335, A / G at position 10405131, A / G at position 10801950, T / C at position 11089097, C / A at position 11732224, T / C at position 11959146, C / T at position 12182036, A / G at position 12384483, T / A at position 12977779, C / G at position 13228019, G / A at position 13725920, A / G at position 14297949, C / T at position 14599462, A / C at position 15316648, C / T at position 16226209, T / A at position 17125566, G / T at position 19323146, C / A at position 20329041, C / A at position 20545842, A / G at position 20831667, A / G at position 21077840, T / C at position 21855677, C / T at position 22076843, T / G at position 22285822, C / A at position 22573506, T / C at position 23139968, A / T at position 23927295, C / T at position 24217188, T / A at position 24958349, A / T at position 26265143, A / C at position 26789789, A / G at position 27013959, C / T at position 27270677, T / A at position 27522828, A / T at position 28230594, A / T at position 28621223, T / C at position 28976931, G / C at position 30161642, G / T at position 30602940, A / G at position 30978727, T / A at position 31649003, G / A at position 32503776, T / C at position 33082095,G / A mutation at position 33398327 bp, A / T mutation at position 34352656 bp, C / T mutation at position 34825626 bp, A / G mutation at position 35322952 bp, A / T mutation at position 35627155 bp, C / T mutation at position 35967237 bp, C / G mutation at position 36252688 bp, G / A mutation at position 36722144 bp, T / C mutation at position 37789019 bp; (3) calculating the IBS and the LOD of all the tested parents and offspring according to the SNP typing result, and the calculation formula is as follows: ， ， ； wherein n represents the number of SNP loci successfully typed, k represents the kth SNP locus, G ik represents the genotype of individual i at the kth SNP locus, G jk represents the genotype of individual j at the kth SNP locus, I k (G ik , G jk ) represents the genotype matching score of individual i and individual j at the kth SNP locus, I k (G ik , G jk ) is 1 when the genotype of individual i and individual j at the kth SNP locus can be matched as parent-offspring, otherwise I k (G ik , G jk ) is 0, P1 represents the probability of the candidate parent and the simulated parent producing offspring, represents the probability of the candidate parent gamete appearing, represents the probability of the simulated parent gamete appearing, w represents the frequency of the simulated parent genotype in the population, m represents the number of mismatched genotypes between the parent and the offspring, λ represents the mismatch penalty weight, which is 0.1, P2 represents the background probability, and when calculating the background probability P2, the genotypes of the simulated parents are based on the population allele frequency. (4) identifying the parent-offspring relationship of the tested parents and offspring in combination with the IBS and the LOD: When the LOD is greater than 0, and the IBS between the candidate parent and the offspring is higher than the preset threshold value 0.98, the candidate parent is most likely to be the real parent of the offspring; When the LOD is less than 0, the candidate parent is not likely to be the real parent.

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