A method for constructing a genetic linkage map of tobacco 8 parent magic population

Abstract

The present application relates to a method for constructing a genetic linkage map of a tobacco 8-parental MAGIC population. The genotypes, genotype frequency distribution and proportion of the 8-parental allo-tetraploid tobacco and its offspring are proposed for the first time, which makes up for the defects of the current MAGIC population containing only AA and aa two genotypes and 1:1 (0.5) genotype frequency distribution, increases the utilization amount of genotype data and improves the utilization efficiency; for the heterozygous genotype in the MAGIC population, the Aa genotype and its distribution frequency in each generation are proposed, which makes up for the default of the published method that the MAGIC population has no heterozygous genotype, reduces the false positive error rate of artificially missing heterozygous genotype and frequency; in addition, considering the wide genetic variation of the 8-parental allo-tetraploid plants, it is helpful to fully excavate the genetic variation sites and improve the quality of the genetic linkage map of the 8-parental MAGIC population.

Description

Technical Field

[0001] This invention belongs to the fields of computational biology and crop genetics, and relates to a method for constructing a genetic linkage map of a MAGIC population of 8 tobacco parents. Background Technology

[0002] Traditional biparental genetic populations are one of the most classic and widely used research materials in modern genetics, especially in quantitative trait locus (QTL) mapping studies. The parents in a traditional biparental genetic population are typically two homozygous representative individuals with significant differences in the target trait. However, the genetic variation based solely on two parents is very limited—it can only examine genes / QTL loci that differ between the two parents, and the allele frequency in biparental genetic populations is relatively high, making it unsuitable for studying rare alleles. Therefore, biparental genetic populations can only capture a small portion of the genetic factors influencing the target trait.

[0003] To overcome the limitations of traditional genetic populations, a novel and more complex genetic population—the Multi-Parent Advanced Generation Inter-Cross (MAGIC) population—was first proposed by Mott and Talbot and successfully constructed in mice (Mott R, Talbot CJ, Turri MG, et al. A method for fine mapping quantitative trait loci in outbred animal stocks [J]. Proceedings of the National Academy of Sciences, 2000, 97(23): 12649-12654. Mackay I, Powell W. Methods for linkage disequilibrium mapping in crops [J]. Trends in Plant Science, 2007, 12(2): 57-63.), and subsequently applied and popularized in plant and animal breeding. Compared with traditional biparental genetic populations, MAGIC populations contain rich genetic variation, which can not only make up for the shortcomings of traditional biparental genetic populations, but also discover more valuable functional genes, thus having higher breeding value. Therefore, the MAGIC population has been constructed and its genes / QTLs located in crops such as wheat, rice, barley, tomato, corn, cowpea, sorghum, tobacco, and cotton, and many new functional genes / QTLs with breeding value have been discovered.

[0004] Constructing high-quality genetic linkage maps is a crucial preliminary step for gene / QTL localization and marker-assisted selection. While the core idea and algorithm for constructing linkage maps of MAGIC populations are largely the same as those for traditional biparental genetic populations, the computational complexity increases significantly as more genotype combinations are considered for each locus. Furthermore, the size of the transition probability matrix between adjacent markers increases exponentially with the number of parents, posing a considerable challenge to computational time complexity.

[0005] Mott et al. developed the first genetic linkage map construction software, R / happy, specifically for MAGIC populations. Subsequently, software such as R / qtl and R / mpMap emerged, but these programs could only handle molecular markers in the thousands. However, with the development of sequencing technology, the number of high-quality molecular markers has gradually increased to tens of thousands, hundreds of thousands, or even higher levels, and the sample size of genetic populations has become increasingly massive. Therefore, there is an urgent need for software capable of constructing linkage maps in large-sample, high-throughput MAGIC populations. The software R / mpMap2, co-authored by Shah and Huang, meets this need. This software uses a hybrid programming approach of R and C++, balancing ease of use and high efficiency, and can construct linkage maps in MAGIC populations with sample sizes in the thousands and over hundreds of thousands of molecular markers.

[0006] There are many research reports on the construction methods of genetic linkage maps of MAGIC populations, and there are also many mapping software programs that match them. However, there are certain deficiencies in the quality (accuracy) of the constructed genetic linkage maps: (1) The currently available methods or software for constructing genetic maps of MAGIC populations are all for MAGIC populations with 4 parents and a small number of 8 parents. However, all 8 parents are diploid animals and plants. To date, no mapping method for 8-parent MAGIC populations based on allotetraploid plants has been developed; (2) The publicly available methods based on MAGIC populations The genetic linkage map construction method (software) only has two genotypes, AA and aa, for the offspring and parents produced by multiple parents, and the genotype frequency (ratio) is only 1:1. It does not take into account the multiple genotypes and their frequencies of the offspring produced by crosses between all numbers (4 or 8) of parents; (3) When using the published mapping method (software) to analyze the genotypes of 4 or 8 parent MAGIC populations, it assumes that there are no heterozygous genotypes (Aa) in the MAGIC population. This assumption deviates significantly from the actual genotypes in the MAGIC population. In view of this, developing a new genetic linkage map method applicable to the 8-parent MAGIC population of allotetraploid tobacco to solve the genotypes and genotype frequencies, heterozygous genotypes and their frequencies of the 8 parents and their offspring will help to effectively solve the shortcomings of the published MAGIC population mapping method and can also significantly improve the accuracy of mapping the 8-parent MAGIC population of allotetraploid tobacco. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a method for constructing a genetic linkage map of an 8-parental MAGIC population in tobacco, developing a new method applicable to the construction of genetic linkage maps of 8-parental MAGIC populations in allotetraploid tobacco, with the aim of promoting the development of the field of 8-parental MAGIC population genetic linkage map construction.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing a genetic linkage map of a MAGIC population of eight tobacco parents, the method comprising the following steps: (1) Obtain genotype data of the MAGIC population of tobacco parental lines 8, wherein the genotype data includes SNPs and Indels; (2) Screening polymorphic loci between parents that meet the requirements of the 8-parent MAGIC population type and typing offspring individuals. The 8-parent MAGIC population has four MAF distributions of 0.125, 0.25, 0.375 and 0.5, and each MAF corresponds to one or more offspring genotype distributions. Based on the percentage of each genotype in the offspring before self-fertilization, the frequency of each genotype in the nth generation is calculated using the following formula: , , ; in, p aa , p Aa , p AA This represents the percentage of each genotype in the offspring before self-fertilization. A chi-square test with 2 degrees of freedom can also be performed on the genotype distribution of offspring corresponding to each marker, with a threshold adjusted for Bonferroni: 0.05 divided by the number of markers in that class. m ,Right now ; (3) Perform linkage disequilibrium pruning on chromosomes, and group genotype data and construct genetic linkage maps.

[0009] Optionally, the method for obtaining genotype data in step (1) includes performing genome resequencing and detecting SNP and Indel variant information based on the genome resequencing data.

[0010] Optionally, the depth of the genome resequencing is 5× or 10×.

[0011] Optionally, the genome resequencing data undergoes preprocessing, which includes filtering quality control and alignment with a reference genome.

[0012] Optionally, the distribution of one or more progeny genotypes for each MAF is shown in Table 1, where P1-P8 are eight allotetraploid tobacco varieties.

[0013] Table 1. Genetic linkage map of 8 parental MAGIC populations and statistics of offspring genotypes and parental genotype frequencies. Optionally, the linkage disequilibrium pruning in step (3) includes filtering genotype data within 100 kb on the chromosome.

[0014] Optionally, the filtering method includes retaining only one genotype with the lowest deletion rate and the highest minimum allele frequency per 100 kb.

[0015] Optionally, the linkage imbalance pruning parameters in step (3) are set to an interval size of 100 kb, a step size of 1 mark, and LD( r 2 The threshold is 0.99.

[0016] Optionally, the method of grouping the genotypes in step (3) includes: dividing the genotypes into 24 chromosomes (the somatic cells of cultivated tobacco of allotetraploid species contain 48 chromosomes, i.e., 2n=48, n=24).

[0017] Optionally, the method for constructing the genetic linkage map in step (3) includes using genetic analysis mapping software, such as GAPL and mpMap2.

[0018] Optionally, the method for constructing a genetic linkage map in step (3) specifically includes the following steps: (3-1) Generate a pedigree corresponding to the 8-parent MAGIC population. Before self-pollination, there are n offspring, each offspring has m F1 plants. Then, self-pollinate continuously for x generations, x>3. (3-2) The recombination rate was calculated using the estimateRFSingleDesign function; (3-3) Use the clusterOrderCross and orderCross functions to perform repeated sorting of the graph and set the parameter randomStart = T so that the function sorts the graph starting from a random starting point. (3-4) Use the impute function to fill in the missing part of the recombination rate, and use estimateMap to construct a linkage map based on the recombination rate, and set maxOffset=200 (this parameter can be used to select the number of adjacent markers used in nonlinear least squares regression, which can reduce unreasonable extension of the map caused by recombination rate fluctuations). (3-5) The linkage map was constructed using the GAPL tool. The filtering criteria for each chromosome were set as follows: deletion rate less than 0.05 and deviation P value less than 0.05. At the same time, the optimal marker within the same Bin was selected according to the deletion rate to remove redundancy.

[0019] Optionally, the method further includes a step of quality assessment of the constructed genetic linkage map, including plotting a collinearity graph between the constructed genetic linkage map and the reference genome (using R packages such as ggplot2, plyr, and reshape2).

[0020] In a second aspect, the present invention provides a genetic linkage map of a tobacco 8-parent MAGIC population, wherein the genetic linkage map is obtained by the method described in the first aspect for constructing a genetic linkage map of a tobacco 8-parent MAGIC population.

[0021] Thirdly, the present invention provides the application of the genetic linkage map of the 8-parental MAGIC population of tobacco described in the second aspect in the mapping of quantitative trait loci (QTLs) in tobacco.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: This invention is the first to propose the distribution and proportion of various genotypes and genotype frequencies for eight parents and their progeny in allotetraploid tobacco, effectively compensating for the current deficiency of only having two genotypes, AA and aa, and a genotype frequency distribution of 1:1 (0.5), in the MAGIC population. This increases the amount of genotype data available and improves the efficiency of using various genotype data. For the heterozygous genotypes that actually exist in the MAGIC population, the invention proposes the Aa genotype and its distribution frequency in each generation. This effectively compensates for the deficiency of the assumption that there are no heterozygous genotypes in the MAGIC population in the currently published methods, and greatly reduces the false positive error rate of artificially omitting heterozygous genotypes and frequencies. In addition, it fully considers the extensive genetic variation of up to eight parents in allotetraploid plants, which helps to more comprehensively explore and utilize the rich genetic variation sites and improves the quality of the genetic linkage map of the eight-parent MAGIC population. Attached Figure Description

[0023] Figure 1A The parental MAF distribution map of the MAGIC population of tobacco parent line 8 (without quality control).

[0024] Figure 1BMAF distribution map of offspring from the MAGIC population of tobacco parent 8 (without quality control).

[0025] Figure 1C SNP distribution map of the MAGIC population of 8 parental tobacco plants (without quality control).

[0026] Figure 2A The parental MAF distribution map of the MAGIC population of 8 parental tobacco lines (quality control of only 8 parental materials).

[0027] Figure 2B MAF distribution map of offspring from the MAGIC population of 8 parent tobacco lines (quality control of only 8 parent materials).

[0028] Figure 2C SNP distribution map of the MAGIC population of 8 parental tobacco plants (quality control of only 8 parental materials).

[0029] Figure 3A The parental MAF distribution map of the MAGIC population of tobacco parent line 8 (quality control of parents and all progeny materials).

[0030] Figure 3B MAF distribution map of the progeny of the MAGIC population of tobacco parent 8 (quality control of parents and all progeny materials).

[0031] Figure 3C SNP distribution map of the MAGIC population of tobacco parent line 8 (quality control of parents and all progeny materials).

[0032] Figure 4 Genetic map of SNP markers for the MAGIC population of 8 parental tobacco plants.

[0033] Figure 5 This is a genetic linkage map of the MAGIC population of tobacco parental lineage 8 and a collinearity map of SNP markers between the K326 reference genome. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0035] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0036] Unless otherwise defined, scientific and technical terms and their abbreviations used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Some of the terms and abbreviations used in this invention are listed below.

[0037] SNP: Single nucleotide polymorphism.

[0038] Indel: Insertion-deletion variant.

[0039] Distance (DST): refers to the genetic distance of allele similarity between individuals.

[0040] MAF (Minor Allele Frequency): refers to the frequency of alleles that occur less frequently in a specific population / group.

[0041] GAPL (Genetic Analysis Software of Pure-Line Populations) is a tool developed by the Institute of Crop Science, Chinese Academy of Agricultural Sciences, specifically for genetic analysis of pure-line progeny (such as DH and RIL populations) derived from four or eight pure-line parents.

[0042] mpMap2 (R Package for Multi-Parent Recombinant Inbred Lines) is a flexible multi-parent analysis package in the R environment.

[0043] Pedigree: refers to structured information describing the kinship between samples.

[0044] LD pruning: refers to a key step in genetic data analysis that aims to remove a set of closely related markers (SNPs) to obtain a statistically approximately independent subset of SNPs.

[0045] Constructing a genetic linkage map directly from the MAGIC parental population of tobacco using currently published software or mapping methods can significantly reduce the accuracy of the constructed genetic map due to factors such as severe omissions of genotypes and genotype frequencies in the parents and offspring, and the artificial selective omission of heterozygous genotypes in the offspring population. Consequently, the accuracy of QTL analysis results for one or more phenotypic traits based on this map is extremely low, making it impossible to effectively apply the rich variations in QTL analysis results within the MAGIC population to actual production.

[0046] To further optimize the quality of genetic linkage maps based on the 8-parental MAGIC population of tobacco and improve the accuracy of the constructed genetic linkage maps, this invention proposes a method for constructing genetic linkage maps based on the 8-parental MAGIC population of tobacco. This method first performs quality control on the offspring genotype and parental genotype frequency distributions of the 8-parental MAGIC population of tobacco; secondly, it proposes strategies for constructing genetic linkage maps of the 8-parental MAGIC population of tobacco; and finally, it evaluates the quality of the constructed genetic linkage maps.

[0047] The experimental materials and data analysis in a specific embodiment of the present invention are shown below.

[0048] I. Experimental Materials The F4 generation MAGIC population was constructed using tobacco varieties K326, NC82, Y3, RBST, CF965, NC95, Cuibi No. 1, and K346 as parents.

[0049] II. Data Analysis The data processing and analysis methods for genome resequencing data from 8 parental lines and 1541 F4 progeny lines are as follows: Filtering raw data The raw data is filtered to remove connector contamination and low-quality reads, resulting in high-quality CleanData for subsequent comparative analysis. The specific filtering conditions are as follows: (1) Remove the connectors from the reads; (2) Remove low-quality reads (40% of the bases have a quality value of 20 or less). (3) Remove reads containing more than 5% N bases.

[0050] b Reference genome The genome of the superior flue-cured tobacco variety K326, originating from the United States, is based on the allotetraploid cultivar published in the 2017 article "A reference genome for Nicotiana tabacum enables map-based cloning of homologous lociimplicated in nitrogen utilization efficiency". The download URL is: ftp: / / ftp.sgn.cornell.edu / genomes / Nicotiana_tabacum / edwards_et_al_2017 / assembly / Nitab-v4.5_genome_Scf_Edwards2017.fasta.gz. This reference genome contains a total of 1,084,432 scaffolds, with a genome size of 4,694,948,798 bp and an effective genome size of 4,049,118,047 bp. During the analysis process of this project, after further quality control and filtering of the genome data, the scaffolds were linked into 24 pseudochromosomes for analysis (1000 N were added between the scaffolds for connection). Subsequent variant detection and analysis will be performed according to the pseudochromosomes. The total length of the K326 genome after linking the pseudochromosomes is 5,779,360,798 bp, and the effective genome size is 4,049,118,047 bp.

[0051] c Comparison The filtered data is aligned to the tobacco reference genome K326 to generate an alignment result file (sam file), which is then converted into a sort.bam file.

[0052] d. Processing of comparison files The alignment results were further processed using multiple software programs (e.g., using samtools to convert the alignment file format, using the samtools merge tool to merge alignment files of different libraries / lanes of the same sample (merging parameter: samtools merge -@ 8), and using the MarkDuplicates tool in GATK 4.2.0 to mark duplicates generated during PCR amplification when preparing the library). Considering that tobacco is an allotetraploid species, only reads with a mapQ value greater than 10 and properly paired alignments were selected for subsequent variant detection analysis (filtering parameter: samtools view -f 2 -q 10).

[0053] e. Detection of variant information SNP and Indel variant detection was performed. First, individual SNP variant detection was performed on the filtered alignment results file to obtain a gVCF file for each chromosome of each sample. Second, the gVCF files of all samples were merged. Finally, the variant information of each chromosome of all individuals in the population was integrated to generate a vcf format file of the population variant (including SNP and Indel variant) results.

[0054] Example 1 This embodiment provides a method for constructing a genetic linkage map of a MAGIC population of 8 tobacco parents.

[0055] The original population SNPs and Indels were filtered and selected to obtain high-quality population SNPs and Indels. Then, inter-parental polymorphic loci that meet the requirements of the population type were selected for genotyping of offspring individuals, linkage group division, and map construction. The details are as follows: First, since markers within a physical distance of 100kb have nearly the same genetic distance, the case of multiple markers within 100kb was filtered before constructing the genetic map. The specific filtering method is: only one marker with the lowest deletion rate and the highest minimum allele frequency is retained within a physical distance of 100k (because the closer the physical distance between markers, the lower the probability of recombination and the higher the possibility of marker redundancy).

[0056] Secondly, linkage maps were constructed for the aforementioned SNPs and Indel markers using GAPL or mpMap2 software. Since the markers had already been clustered at the chromosome level according to the K326 reference genome, the markers were divided into 24 chromosomes, and each chromosome was mapped using mpMap2. (1) Generate a pedigree corresponding to the 8-parent MAGIC population in the software. There are 16 offspring (including reciprocal cross offspring) before self-pollination. Each offspring has 100 F1 plants. Then, self-pollinate for 4 generations. (2) The estimateRFSingleDesign function is used to calculate the recombination rate. Compared with the estimateRF function, this function performs better in MAGIC populations with known pedigree single funnels. (3) Combine the clusterOrderCross and orderCross functions to implement repeated sorting of the map and set the parameter randomStart = T so that the function starts the sorting from a random starting point; (4) Use the impute function to fill in the missing part of the recombination rate calculated above, and use estimateMap to construct the linkage map based on the recombination rate, and set maxOffset=200 (this parameter can be used to select the number of adjacent markers used in nonlinear least squares regression, which can reduce the unreasonable extension of the map caused by the recombination rate fluctuation). (5) Finally, the linkage map was constructed using GAPL software. The filtering criteria for each chromosome were set as follows: the deletion rate was less than 0.05 and the deviation P value was less than 0.05. At the same time, the optimal marker within the same Bin was selected according to the deletion rate to remove redundancy.

[0057] The collinearity between the genetic linkage map constructed in Example 1 and the K326 reference genome was plotted for quality assessment.

[0058] The results are shown below: (1) SNP variation analysis of the MAGIC population with K326 as the reference genome Based on the genome of the allotetraploid tobacco variety K326, SNP variation was detected in the population using GATK (GATK 4.1.2) bioinformatics analysis software. A total of 6,962,820 SNP loci were obtained from 1,549 samples (8 parents K326, NC82, Y3, RBST, CF965, NC95, Cuibi No. 1 CB1 and K346; and 1,541 progeny samples). The distribution of these SNP loci on each chromosome of the K326 reference genome is shown in Table 2.

[0059] Table 2. SNP statistics of the MAGIC population based on the K326 reference genome. The distribution maps of the least allele frequency (MAF) of SNPs in 8 parents and 1,541 offspring, as well as their distribution maps on each chromosome, are shown below. Figures 1A-1C As shown.

[0060] The MAF distribution map reveals some heterozygous loci and non-polymorphic loci in the parents. Quality control of these loci and deletion of deleted loci in the parents yields 2,569,085 SNPs for quality control based solely on the parents (pre-QC). Their distribution on each chromosome and the MAF distribution are shown below. Figures 2A-2C As shown.

[0061] (2) Quality control based on the frequency distribution of offspring genotypes and parental genotypes Genotypic quality control in multi-parental genetic populations cannot be performed using the minimum allele frequency (MAF) threshold (e.g., 0.05) found in biparental genetic populations due to the unique characteristics of their allele frequency distribution. In a MAGIC population, the allele frequency at a specific locus depends on the frequency of that locus in the parents. For an eight-parent population, there are four minimum allele frequencies (MAFs): 0.125, 0.250, 0.375, and 0.5. Each MAF corresponds to one or more genotype distributions in the offspring, as shown in Table 1.

[0062] After obtaining the proportions of each genotype in the offspring before self-fertilization, the genotype distribution of the offspring in any generation can be obtained, denoted as... p aa , p Aa , p AA Let be the percentage of each genotype in the offspring before self-fertilization. Then, the... n The genotype frequencies of each phase in each generation are as follows: , , Therefore, a chi-square test with 2 degrees of freedom can be performed on the genotype distribution of the offspring corresponding to each marker, with a Bonferroni corrected threshold: 0.05 divided by the number of markers in that class. m ,Right now .

[0063] (3) Quality control results of SNP markers Quality control was performed on the genotypes of the MAGIC population progeny and the genotype frequencies of the eight parental isolates (i.e., 1,541 MAGIC population progeny individuals were selected for quality control, primarily based on a deletion rate of less than 0.05 and the chi-square test shown in Table 1). A total of 621,019 high-quality SNP markers were obtained after quality control. The specific distribution of the SNP markers is shown in Table 1. Figures 3A-3C As shown.

[0064] Although the minimum allele frequency (e.g., 0.015, 1 / 64) was not included in the data quality control process, the chi-square test results based on genotype distribution are equivalent to performing MAF quality control at 0.03 (1 / 32). The MAF plot from this quality control study shows that parental allele frequencies are predominantly distributed at low frequencies (0.125). A particularly extreme example is that almost all variations on chromosome 21 originate from the parental Y3, resulting in the minimum allele frequency on chromosome 21 being almost entirely around 0.125. However, this result does not affect subsequent map construction and association analysis.

[0065] (4) Construction of genetic linkage map of the MAGIC population of 8 tobacco parents After filtering, a total of 32,489 high-quality SNP markers were obtained, which can be used for subsequent construction and association analysis of the genetic linkage map of the MAGIC population. Using the genetic linkage map construction method based on an 8-parental MAGIC population of tobacco provided in this invention, the filtered SNP markers were used to construct the linkage map of the 8-parental MAGIC population of tobacco. Finally, a genetic linkage map of the tobacco MAGIC population based on SNP markers was obtained. This genetic map contains 32,489 SNP markers, covering a total genome length of 5,797.50 cM, with an average SNP marker density of 0.181 cM. The average number of SNP markers per linkage group is 1,354, with the highest number of SNP markers (1,895) on LG24 and the lowest (891) on LG16. The average length of each linkage group was 241.563 cM, with LG18 being the longest at 367.59 cM and LG17 the shortest at 164.94 cM. The average maximum gap on each linkage group was 6.01 cM, with a maximum gap of 19.44 cM located on LG07 (corresponding to chromosome chr.7 of the K326 genome). Detailed statistical results are shown in Table 3 and... Figure 4 As shown.

[0066] Table 3. Statistical information on the genetic map of the MAGIC population based on SNP markers. (5) Quality assessment of genetic linkage maps of the MAGIC population of 8 tobacco parents To evaluate the quality of the constructed SNP marker genetic linkage map of the 8-parental MAGIC population of tobacco, collinearity diagrams of the genetic linkage map can be drawn using software such as ggplot2, plyr, and reshape2. Figure 5 ),Depend on Figure 5 It can be seen that the SNP markers on the 24 linkage groups in the constructed genetic linkage map are completely consistent with the base sequence on the 24 chromosomes in the K326 reference genome, that is, the quality of the SNP marker genetic linkage map of the MAGIC population of tobacco 8 parents is very high.

[0067] In summary, this invention designs a method for constructing a genetic linkage map of an 8-parental MAGIC population of tobacco. First, the frequency distribution of progeny genotypes and parental genotypes in the 8-parental MAGIC population of tobacco is quality controlled. Second, a strategy for constructing a genetic linkage map of the 8-parental MAGIC population of tobacco is proposed, which significantly improves the accuracy of constructing the genetic linkage map of the 8-parental MAGIC population.

[0068] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for constructing a genetic linkage map of an 8-parental MAGIC population in tobacco, characterized in that, The method includes the following steps: (1) Obtain genotype data of the MAGIC population of tobacco parental lines 8, wherein the genotype data includes SNPs and Indels; (2) Screening polymorphic loci between parents that meet the requirements of the 8-parent MAGIC population type and typing offspring individuals. The 8-parent MAGIC population has four MAF distributions of 0.125, 0.25, 0.375 and 0.5, and each MAF corresponds to one or more offspring genotype distributions. Based on the percentage of each genotype in the offspring before self-fertilization, the frequency of each genotype in the nth generation is calculated using the following formula: ， ， ； in, p aa , p Aa , p AA This represents the percentage of each genotype in the offspring before self-fertilization. (3) Perform linkage disequilibrium pruning on chromosomes, and group genotype data and construct genetic linkage maps.

2. The method for constructing a genetic linkage map of an 8-parental MAGIC population of tobacco according to claim 1, characterized in that, The method for obtaining genotype data in step (1) includes performing genome resequencing and detecting SNP and Indel variant information based on the genome resequencing data; Optionally, the depth of the genome resequencing is 5× or 10×.

3. The method for constructing a genetic linkage map of an 8-parental MAGIC population of tobacco according to claim 2, characterized in that, The genome resequencing data underwent preprocessing, which included filtering quality control and alignment with a reference genome.

4. The method for constructing a genetic linkage map of an 8-parental MAGIC population of tobacco according to any one of claims 1-3, characterized in that, Step (3) of linkage disequilibrium pruning includes filtering genotype data within 100 kb on chromosomes; Optionally, the filtering method includes retaining only one genotype with the lowest deletion rate and the highest minimum allele frequency per 100 kb.

5. The method for constructing a genetic linkage map of an 8-parental MAGIC population according to any one of claims 1-4, characterized in that, The method for grouping genotypes in step (3) includes dividing the genotype data into 24 chromosomes.

6. The method for constructing a genetic linkage map of an 8-parental MAGIC population of tobacco according to any one of claims 1-5, characterized in that, The method for constructing a genetic linkage map in step (3) includes using genetic analysis mapping software.

7. The method for constructing a genetic linkage map of a MAGIC population of 8 tobacco parents according to claim 6, characterized in that, The method for constructing a genetic linkage map in step (3) specifically includes the following steps: (3-1) Generate a pedigree corresponding to the 8-parent MAGIC population. Before self-pollination, there are n offspring, each offspring has m F1 plants. Then, self-pollinate continuously for x generations, x>3. (3-2) The recombination rate was calculated using the estimateRFSingleDesign function; (3-3) Use the clusterOrderCross and orderCross functions to perform repeated sorting of the graph and set the parameter randomStart = T so that the function sorts the graph starting from a random starting point. (3-4) Use the impute function to fill in the missing parts of the recombination rate, and use estimateMap to construct a linkage map based on the recombination rate, and set maxOffset=200; (3-5) The linkage map was constructed using the GAPL tool. The filtering criteria for each chromosome were set as follows: deletion rate less than 0.05 and deviation P value less than 0.

05. At the same time, the optimal marker within the same Bin was selected according to the deletion rate to remove redundancy.

8. The method for constructing a genetic linkage map of a MAGIC population of 8 parents of tobacco according to any one of claims 1-7, characterized in that, The method also includes a step of quality assessment of the constructed genetic linkage map, including drawing a collinearity map between the constructed genetic linkage map and the reference genome.

9. A genetic linkage map of a MAGIC population of eight parents in tobacco, characterized in that, The genetic linkage map is obtained by the method for constructing a genetic linkage map of the MAGIC population of tobacco 8 parents as described in any one of claims 1-8.

10. The application of the genetic linkage map of the MAGIC population of tobacco parent 8 in claim 9 in the mapping of quantitative trait loci (QTLs) in tobacco.

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