SSR Marker Detection Primers for Identifying Homologous Chromosomes of Potato Chr5 and Their Uses

Abstract

The present invention relates to the field of biotechnology, and particularly to SSR marker detection primers for identifying homologous chromosomes of potato Chr5 and their uses. It can identify the homologous chromosomes of chromosome V in "Cooperation 88", and with the help of the homologous chromosome typing results, combined with other ploidy detection methods, the ploidy of the test sample can be determined. Compared with chromosome karyotype analysis, chromosome banding technology and fluorescence in situ hybridization technology, it has a high detection throughput, and the detection results are more objective, more accurate and have good repeatability. Compared with genome sequencing technology, it does not require special dedicated instrument equipment, has a lower detection cost, shorter time consumption and simpler operation; using PCR amplification results for homologous chromosome typing detection has basically no special requirements for the test materials, and compared with sequencing and data processing, the skill requirements are much lower.

Description

[0001] Parent case patent number: 2024110607404, filing date: 2024-08-05, patent title: SSR marker detection primers for potato homologous chromosome typing identification and uses thereof Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to an SSR marker detection primer for potato Chr5 homologous chromosome typing identification and uses thereof. Background Art

[0003] Potato is the general name of some species in the genus Solanum of the Solanaceae family, native to the Andes mountain range in South America. Its ploidy is highly complex, and diploid, triploid, tetraploid, pentaploid, hexaploid and octaploid wild potatoes exist in nature. Polyploid organisms contain more than two chromosome sets. A chromosome set contains a set of chromosomes with different morphological functions but complementary functions in cooperation. In polyploid cells, chromosomes with similar morphology and function are homologous chromosomes to each other. Currently, scientific research has found that the gene sequences on homologous chromosomes are not completely the same. Coding genes that are close or identical in chromosomal structure have basically similar functions, but their sequences are not completely the same, which are the so-called alleles. Generally, alleles with completely identical coding sequences are homozygous genotypes. To a certain extent, the homozygosity of alleles determines the traits of organisms. Therefore, in agriculture, homologous chromosome typing can provide larger gene selection markers for molecular marker-assisted breeding at the chromosomal level, and can better provide a basis for screening the coding regions of quantitative trait loci. "Cooperation 88" is one of the potato varieties widely planted in large areas in southwestern China. This variety can be used for various purposes such as potato chip processing, starch production, and fresh food, and has disease resistance to late blight, early blight, virus disease, etc., thus becoming one of the excellent parents for breeders to carry out hybridization and selection. Therefore, carrying out homologous chromosome typing on this variety can accelerate the potato breeding progress.

[0004] The existing chromosome analysis and detection techniques mainly include chromosome karyotype analysis, chromosome banding techniques, and fluorescence in situ hybridization (FISH). From the perspective of the detection purpose, none of these three techniques is specifically designed for homologous chromosome typing. Therefore, they can only perform limited homologous chromosome typing. In terms of the research object, all three techniques require cells in the metaphase of mitosis. To obtain better results, samples need to be taken at specific times. In terms of time, FISH generally takes about 4 - 7 days to obtain the detection results; chromosome banding techniques take more than 7 days; chromosome karyotype analysis takes at least 3 - 4 days. In terms of technical difficulty, all three techniques require operators to have high experimental skills. The detection throughput of all three is very small, generally only targeting single samples. In terms of equipment, chromosome karyotype analysis and chromosome banding only require ordinary microscopes, while FISH requires a fluorescence microscope, which is expensive. Genome sequencing and assembly to the chromosome level is currently the most effective means of homologous chromosome analysis. This technique uses the current second-generation + third-generation sequencing technology to complete the determination of DNA nucleotide sequences, and relies on the powerful computing power of computers, data processing in bioinformatics, genetic analysis, etc. to complete the chromosome-level assembly of the whole-genome sequence of organisms.

[0005] Bao et al. conducted a study on homologous chromosome typing of Solanum tuberosum 'Hexie 88' by sequencing and assembly. This study involved second-generation sequencing of 'Hexie 88' and 1034 self-cross materials, Hifi and ONT third-generation sequencing of 'Hexie 88', and Hi-C sequencing, with huge costs. Not only does the preparation of experimental materials require a large amount of time and manpower, but the large amount of data obtained from sequencing needs to be processed with the help of supercomputers, and the required equipment is also very expensive. Data analysis requires the participation of skilled and experienced personnel. Therefore, it is necessary to find a method for homologous chromosome typing of 'Hexie 88', and it is very important to accelerate the potato breeding process. Summary of the Invention

[0006] In order to overcome the deficiencies of the above technical defects, the present invention provides SSR marker detection primers and uses for homologous chromosome typing and identification of potato Chr5, which are used for rapid typing and identification of homologous chromosomes of the tetraploid potato variety 'Hexie 88' and its derivative materials.

[0007] To achieve the above object, the present invention is realized through the following solutions:

[0008] Perform SSR scanning analysis on the homologous chromosomes of Chr4, Chr5, Chr7, and Chr12 of the known 'Hexie 88' genome, and verify the selected primers in the doubled haploid and self-cross populations of 'Hexie 88' to obtain 14 pairs of primers that can distinguish the homologous chromosomes of Chr4, Chr5, Chr7, and Chr12 of the derivative materials of this variety.

[0009] Therefore, in a first aspect, the present invention provides SSR marker detection primers for potato Chr5 homologous chromosome typing and identification, selected from the primer pairs shown below:

[0010] Primer pair 4-1.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively;

[0011] Primer pair 4-2.2: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively;

[0012] Primer pair 4-3.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 respectively;

[0013] Primer pair 4-4.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8 respectively;

[0014] Primer pair 5-1.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10 respectively;

[0015] Primer pair 5-2.5: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12 respectively;

[0016] Primer pair 5-3.5: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14 respectively;

[0017] Primer pair 5-4.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16 respectively;

[0018] Primer pair 7-1.3: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18 respectively;

[0019] Primer pair 7-2.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 19 and SEQ ID NO: 20 respectively;

[0020] Primer pair 7-3.1: including a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 21 and SEQ ID NO: 22 respectively;

[0021] Primer pair 7-2.10: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 23 and SEQ ID NO: 24 respectively;

[0022] Primer pair 12-1.27: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 25 and SEQ ID NO: 26 respectively;

[0023] Primer pair 12-2.6: It includes a forward primer and a reverse primer with nucleotide sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28 respectively.

[0024] The present invention uses these 14 primer pairs to rapidly genotype and identify homologous chromosomes of chromosomes IV, V, VII, and XII of the potato tetraploid cultivar 'Cooperation 88', which is helpful for molecular marker-assisted breeding at the chromosome scale.

[0025] Further, the primers are divided into four groups, namely Chr4 primer group: primer pair 4-1.1, primer pair 4-2.2, primer pair 4-3.1, primer pair 4-4.1; Chr5 primer group: primer pair 5-1.1, primer pair 5-2.5, primer pair 5-3.5, primer pair 5-4.1; Chr7 primer group: primer pair 7-1.3, primer pair 7-2.1, primer pair 7-3.1, primer pair 7-2.10; Chr12 primer group: primer pair 12-1.27, primer pair 12-2.6.

[0026] In some specific embodiments, primer pair 4-1.1, primer pair 4-2.2, primer pair 4-3.1, and primer pair 4-4.1 are used to identify 4 homologous chromosomes of Chr4 chromosome; primer pair 5-1.1, primer pair 5-2.5, primer pair 5-3.5, and primer pair 5-4.1 are used to identify 4 homologous chromosomes of Chr5 chromosome; primer pair 7-1.3, primer pair 7-2.1, primer pair 7-3.1, and primer pair 7-2.10 are used to identify 4 homologous chromosomes of Chr7 chromosome; primer pair 12-1.27 and primer pair 12-2.6 are used to identify 4 homologous chromosomes of Chr12 chromosome.

[0027] Here, Chrx represents the chromosome number, Chr4 is chromosome IV, Chr5 is chromosome V, Chr7 is chromosome VII, and Chr12 is chromosome XII.

[0028] Further, the nucleotide sequences of the primers are (a), (b), or (c) to achieve the same detection purpose;

[0029] (a) Nucleotide sequences shown in SEQ ID NO.1 - SEQ ID NO.28;

[0030] (b) Nucleotide sequences that hybridize under stringent conditions to the nucleotide sequences shown in SEQ ID NOs. 1 - 28 and encode them;

[0031] (c) Nucleotide sequences that have more than 80% homology with the nucleotide sequences shown in SEQ ID NOs. 1 - 28 and encode them.

[0032] In some specific embodiments, the nucleotide sequences of the SSR marker detection primer pair group provided by the present invention have 80% identity with the sequences shown in SEQ ID NOs. 1 - 12; preferably 85% identity, more preferably 90% identity, more preferably 95% identity, and most preferably 99% identity.

[0033] Exemplarily, as used herein, "stringent conditions" refer to conditions under which a probe will hybridize to its target sequence to a detectable degree more than to other sequences (e.g., at least 2 - fold over background). Stringent conditions are sequence - dependent and vary with the environment. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified. Optionally, the stringent conditions can be adjusted to allow some sequence mismatches, such that lower degrees of similarity can be detected. These nucleotide sequences that hybridize under stringent conditions can be used, for example, to express variant proteins of SEQ ID NO. 1 or as primers, probes, exogenous donor sequences, guide RNAs, antisense RNAs, shRNAs, and siRNAs.

[0034] In a second aspect, the present invention provides the use of the SSR marker detection primer pair group in the identification of homologous chromosomes in potatoes.

[0035] Furthermore, it is used for the identification of homologous chromosomes of potato chromosomes Chr4, Chr5, Chr7, and Chr12.

[0036] Furthermore, the potato is "Cooperation 88".

[0037] 8. Furthermore, determine whether the DNA fragments shown in (a) - (p) are included in the sample to be tested;

[0038] (a) The nucleotide sequence as shown in SEQ ID NO: 29;

[0039] (b) The nucleotide sequence as shown in SEQ ID NO: 30;

[0040] (c) The nucleotide sequence as shown in SEQ ID NO: 31;

[0041] (d) The nucleotide sequence shown in SEQ ID NO: 32;

[0042] (e) The nucleotide sequence shown in SEQ ID NO: 33;

[0043] (f) The nucleotide sequence shown in SEQ ID NO: 34;

[0044] (g) The nucleotide sequence shown in SEQ ID NO: 35;

[0045] (h) The nucleotide sequence shown in SEQ ID NO: 36;

[0046] (i) The nucleotide sequence shown in SEQ ID NO: 37;

[0047] (j) The nucleotide sequence shown in SEQ ID NO: 38;

[0048] (k) The nucleotide sequence shown in SEQ ID NO: 39;

[0049] (l) The nucleotide sequence shown in SEQ ID NO: 40;

[0050] (m) The nucleotide sequence shown in SEQ ID NO: 41;

[0051] (n) The nucleotide sequence shown in SEQ ID NO: 42;

[0052] (o) The nucleotide sequence shown in SEQ ID NO: 43;

[0053] (p) The nucleotide sequence shown in SEQ ID NO: 44;

[0054] (q) The nucleotide sequence shown in SEQ ID NO: 45;

[0055] (r) The nucleotide sequence shown in SEQ ID NO: 46;

[0056] (s) The nucleotide sequence shown in SEQ ID NO: 47;

[0057] (t) The nucleotide sequence shown in SEQ ID NO: 48;

[0058] (u) The nucleotide sequence shown in SEQ ID NO: 49;

[0059] (v) The nucleotide sequence shown in SEQ ID NO: 50;

[0060] (w) The nucleotide sequence shown in SEQ ID NO: 51.

[0061] In some specific embodiments, 14 pairs of primers (the nucleotide sequences of which are shown in SEQ ID NO.1-SEQ ID NO.28) can amplify 16 DNA fragments (the nucleotide sequences of which are shown in SEQ ID NO.29-SEQ ID NO.44) in the female parent, and can identify the homologous chromosomes of chromosomes Chr4, Chr5, Chr7 and Chr12 in "Cooperation 88".

[0062] Furthermore, using these 14 pairs of primers to identify homologous chromosomes in the self-cross population of "Cooperation 88" confirmed that these primers can also be used for the identification of homologous chromosomes in the self-cross population ( Figure 2 ), and can estimate the flow of parental genes.

[0063] Furthermore, the uses include nucleic acid hybridization detection, molecular markers, preparation of gene chips, preparation of molecular probes, and preparation of detection kits.

[0064] In a third aspect, the present invention provides the use of the SSR marker detection primer pair group in the ploidy identification of the progeny of distant hybridization of potatoes.

[0065] Furthermore, the potato is "Cooperation 88".

[0066] Furthermore, determine whether the DNA fragments shown in (a)-(p) are included in the sample to be tested; (a) the nucleotide sequence shown in SEQ ID NO: 29;

[0067] (b) the nucleotide sequence shown in SEQ ID NO: 30;

[0068] (c) the nucleotide sequence shown in SEQ ID NO: 31;

[0069] (d) the nucleotide sequence shown in SEQ ID NO: 32;

[0070] (e) the nucleotide sequence shown in SEQ ID NO: 33;

[0071] (f) the nucleotide sequence shown in SEQ ID NO: 34;

[0072] (g) the nucleotide sequence shown in SEQ ID NO: 35;

[0073] (h) the nucleotide sequence shown in SEQ ID NO: 36;

[0074] (i) the nucleotide sequence shown in SEQ ID NO: 37;

[0075] (j) The nucleotide sequence as shown in SEQ ID NO: 38;

[0076] (k) The nucleotide sequence as shown in SEQ ID NO: 39;

[0077] (l) The nucleotide sequence as shown in SEQ ID NO: 40;

[0078] (m) The nucleotide sequence as shown in SEQ ID NO: 41;

[0079] (n) The nucleotide sequence as shown in SEQ ID NO: 42;

[0080] (o) The nucleotide sequence as shown in SEQ ID NO: 43;

[0081] (p) The nucleotide sequence as shown in SEQ ID NO: 44;

[0082] (q) The nucleotide sequence as shown in SEQ ID NO: 45;

[0083] (r) The nucleotide sequence as shown in SEQ ID NO: 46;

[0084] (s) The nucleotide sequence as shown in SEQ ID NO: 47;

[0085] (t) The nucleotide sequence as shown in SEQ ID NO: 48;

[0086] (u) The nucleotide sequence as shown in SEQ ID NO: 49;

[0087] (v) The nucleotide sequence as shown in SEQ ID NO: 50;

[0088] (w) The nucleotide sequence as shown in SEQ ID NO: 51.

[0089] In some specific embodiments, 14 pairs of primers (the nucleotide sequences of which are as shown in SEQ ID NO.1 - SEQ ID NO.28) can amplify 16 DNA fragments (the nucleotide sequences of which are as shown in SEQ ID NO.29 - SEQ ID NO.44) in the female parent in total. According to the presence or absence of these DNA fragments, the homologous chromosome typing results of chromosomes Chr4, Chr5, Chr7, and Chr12 in "Cooperation 88" can be obtained, and combined with other ploidy detection methods (such as flow cytometry, etc.), the ploidy of the detection sample can be determined.

[0090] Further, the potato is "Cooperation 88".

[0091] Further, the uses include nucleic acid hybridization detection, molecular markers, preparation of gene chips, preparation of molecular probes, and preparation of detection kits.

[0092] Compared with the prior art, the present invention has the following beneficial effects:

[0093] (1) Fourteen pairs of SSR marker primers can identify the homologous chromosomes of chromosome IV, chromosome V, chromosome VII, and chromosome XII in "Cooperation 88", and with the help of the homologous chromosome typing results, combined with other ploidy detection methods (such as flow cytometry, etc.), the ploidy of the test sample can be determined;

[0094] (2) Compared with chromosome karyotype analysis, chromosome banding technology, and fluorescence in situ hybridization technology, the detection throughput is high, the detection results are more objective, more accurate, and have good repeatability.

[0095] (3) Compared with genome sequencing technology, it does not require special dedicated instrument equipment, the detection cost is lower, the time consumption is shorter, and the operation is simpler; using the PCR amplification results for homologous chromosome typing detection, there are basically no special requirements for the test materials, and compared with sequencing and data processing, the skill requirements are much lower. Description of the Drawings

[0096] Figure 1 It is a flow chart for screening SSR marker detection primers;

[0097] Figure 2 It is a detection map for homologous chromosome typing of chromosome IV;

[0098] Figure 3 It is a detection map for homologous chromosome typing of chromosome V;

[0099] Figure 4 It is a detection map for homologous chromosome typing of chromosome VII;

[0100] Figure 5 It is a detection map for homologous chromosome typing of chromosome XII;

[0101] Figure 6 It is a peak map of the clone sequencing result of the amplification product of primer pair 4-1.1;

[0102] Figure 7 It is a peak map of the clone sequencing result of the amplification product of primer pair 7-1.1. Detailed Embodiments

[0103] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below in conjunction with specific embodiments. For the experimental methods without specific conditions noted in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. Unless otherwise stated, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are for illustrative purposes only.

[0104] Based on the present invention, ordinary cultivated potatoes are tetraploids, with 4 chromosome sets in the cells, and each chromosome has 4 homologous chromosomes. However, since the gene sequences on the homologous chromosomes are not completely the same, the different homologous chromosomes contained in the derived offspring determine the genotype of the offspring and have a great influence on the phenotype. Therefore, the inventor believes that homologous chromosome typing is a good means of molecular marker-assisted breeding at the chromosome scale.

[0105] The technical solution of the present invention will be further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0106] Based on the present invention, after analyzing the "Cooperation 88" genome, the inventor developed SSR marker detection primers suitable for rapid typing and identification of the homologous chromosomes of "Cooperation 88", which can be used for rapid typing and identification of the homologous chromosomes of the tetraploid potato variety "Cooperation 88" and its derived materials, and combined with other ploidy detection means (such as flow cytometry, etc.) to determine the ploidy of the test sample.

[0107] Example 1

[0108] The genome sequence of "Cooperation 88" downloaded from the potato genome resource website Spud DB ( http: / / spuddb.uga.edu / c88_potato_ download.shtml ) was used to analyze the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 by using bioinformatics software. A total of 39,106 SSR loci were found. Primers were screened for these loci according to the following criteria: (1) The expected amplification fragment is not greater than 400 bp and not less than 100 bp; (2) The primer binding site is unique on all 48 chromosomes; (3) There is a binding site only on the homologous chromosomes of one chromosome, but the amplified fragment sizes on the homologous chromosomes are different; When meeting (2) or (3), the screening conditions are satisfied.

[0109] Primers were designed by extending 100 - 200 bp on both wings of the obtained SSR fragments. First, fragments with low GC% content at both ends that could not be used for designing amplification primers were removed; then, the designed primers were subjected to ePCR in this genome, and fragments and primers that could distinguish homologous chromosomes were retained; finally, the selected primers were synthesized, and PCR detection verification and product electrophoresis detection were carried out in the self - crossing population of "Cooperation 88". According to the detection and analysis results, 14 pairs of molecular marker primers that could be used for homologous chromosome typing were determined. The selected primers were verified in the reduced - ploidy and self - crossing populations of "Cooperation 88", and the amplification products were subjected to electrophoresis detection. Screening was carried out according to the electrophoresis results, and 14 pairs of SSR marker detection primers that could be used for the identification of homologous chromosome typing of Chr4, Chr5, Chr7, and Chr12 were determined (the screening process is shown in Figure 1 ).

[0110] They are primer pair 4 - 1.1 (the nucleotide sequences are shown as SEQ ID NO: 1 and SEQ ID NO: 2 respectively), primer pair 4 - 2.2 (the nucleotide sequences are shown as SEQ ID NO: 3 and SEQ ID NO: 4 respectively), primer pair 4 - 3.1 (the nucleotide sequences are shown as SEQ ID NO: 5 and SEQ ID NO: 6 respectively), primer pair 4 - 4.1 (the nucleotide sequences are shown as SEQ ID NO: 7 and SEQ ID NO: 8 respectively), primer pair 5 - 1.1 (the nucleotide sequences are shown as SEQ ID NO: 9 and SEQ ID NO: 10 respectively), primer pair 5 - 2.5 (the nucleotide sequences are shown as SEQ ID NO: 11 and SEQ ID NO: 12 respectively), primer pair 5 - 3.5 (the nucleotide sequences are shown as SEQ ID NO: 13 and SEQ ID NO: 14 respectively), primer pair 5 - 4.1 (the nucleotide sequences are shown as SEQ ID NO: 15 and SEQ ID NO: 16 respectively), primer pair 7 - 1.3 (the nucleotide sequences are shown as SEQ ID NO: 17 and SEQ ID NO: 18 respectively), primer pair 7 - 2.1 (the nucleotide sequences are shown as SEQ ID NO: 19 and SEQ ID NO: 20 respectively), primer pair 7 - 3.1 (the nucleotide sequences are shown as SEQ ID NO: 21 and SEQ ID NO: 22 respectively), primer pair 7 - 2.10 (the nucleotide sequences are shown as SEQ ID NO: 23 and SEQ ID NO: 24 respectively), primer pair 12 - 1.27 (the nucleotide sequences are shown as SEQ ID NO: 25 and SEQ ID NO: 26 respectively), primer pair 12 - 2.6 (the nucleotide sequences are shown as SEQ ID NO: 27 and SEQ ID NO: 28 respectively).

[0111] Example 2

[0112] The total leaf DNA of "Cooperation 88" and its chromosome-doubled and self-crossed populations was extracted by the improved CTAB method. Fourteen pairs of SSR marker detection primers were screened and used for PCR amplification and detection verification in "Cooperation 88". The amplification system and amplification program are shown in Table 1 and Table 2:

[0113] Table 1 Amplification system

[0114]

[0115] Table 2 Amplification program

[0116]

[0117]

[0118] The amplification products were detected by electrophoresis with 2% agarose (see Figures 2 - 5 ), and it was found that 14 pairs of primers could identify the homologous chromosomes of Chr4, Chr5, Chr7, and Chr12 in "Cooperation 88". Figures 2 - 5 Among them, M is Maker, lane 1 is C88; lane 2 is H2O; lanes 3 - 22 are diploids in the chromosome-doubled population of C88; from Figure 2 it can be seen that primer pair 4-1.1, primer pair 4-2.2, primer pair 4-3.1, and primer pair 4-4.1 have binding sites on the 4 homologous chromosomes (Chr4-1 - 4) of chromosome IV (Chr4), and 4 amplification products were obtained respectively, namely Chr4-1.1 (its nucleotide sequence is shown in SEQ ID NO: 29), Chr4-2.2 (its nucleotide sequence is shown in SEQ ID NO: 30), Chr4-3.1 (its nucleotide sequence is shown in SEQ ID NO: 31), and Chr4-4.1 (its nucleotide sequence is shown in SEQ ID NO: 32), with lengths of 328bp, 392bp, 259bp, and 131bp respectively;

[0119] After separately cutting the electrophoresis band of primer pair 4-1.1, the band was recovered, ligated to the vector, and sequenced. Part of the sequencing peaks are as shown in Figure 6 , which is consistent with the expected fragment in the reference genome of "Cooperation 88".

[0120] From Figure 3As can be seen, primer pairs 5-1.1, 5-2.5, 5-3.5, and 5-4.1 have binding sites on four homologous chromosomes (Chr5-1 to 4) of chromosome V (Chr5), respectively, and four amplification products are obtained, namely Chr5-1 (whose nucleotide sequence is shown in SEQ ID NO: 33), Chr5-2 (whose nucleotide sequence is shown in SEQ ID NO: 34), Chr5-3 (whose nucleotide sequence is shown in SEQ ID NO: 35), and Chr5-4 (whose nucleotide sequence is shown in SEQ ID NO: 36), with lengths of 155 bp, 208 bp, 278 bp, and 144 bp bands, respectively.

[0121] From Figure 4 As can be seen, primer pairs 7-1.3, 7-2.1, 7-3.1, and 7-2.10 have binding sites on four homologous chromosomes (Chr7-1 to 4) of chromosome VII (Chr7), respectively, and four amplification products are obtained, namely Chr7-1 (whose nucleotide sequence is shown in SEQ ID NO: 37), Chr7-2 (whose nucleotide sequence is shown in SEQ ID NO: 38), Chr7-3 (whose nucleotide sequence is shown in SEQ ID NO: 39), and Chr7-4 (whose nucleotide sequence is shown in SEQ ID NO: 40), with lengths of 365 bp, 183 bp, 337 bp, and 214 bp, respectively.

[0122] After cutting the electrophoresis band of primer pair 7-1.1 separately, the band was recovered, ligated to the vector, and sequenced. Some of the sequencing peaks are as Figure 7 shown, which is consistent with the expected fragment in the reference genome of "Cooperation 88".

[0123] From Figure 5 As can be seen, primer pairs 12-1.27 and 12-2.6 have binding sites on four homologous chromosomes (Chr12-1 to 4) of the homologous chromosomes (Chr12-1 to 4) of chromosome XII (Chr12). Among them, the amplification product of primer pair 12-1.27 is Chr12-3 (whose nucleotide sequence is shown in SEQ ID NO: 41), with a length of 371 bp. Primer pair 12-2.6 obtained three amplification products, namely Chr12-1 (whose nucleotide sequence is shown in SEQ ID NO: 42), Chr12-2 (whose nucleotide sequence is shown in SEQ ID NO: 43), and Chr12-4 (whose nucleotide sequence is shown in SEQ ID NO: 43), with lengths of 274 bp, 334 bp, and 259 bp, respectively.

[0124] Through these DNA fragments, the homologous chromosomes of Chr4, Chr5, Chr7, and Chr12 in "Cooperation 88" can be identified. Specifically, the presence or absence of Chr4-1 is determined by primer pair 4-1.1, the presence or absence of Chr4-2 is determined by primer pair 4-2.2, the result of primer pair 4-3.1 determines Chr4-3, and the result of primer pair 4-4.1 determines Chr4-4; similarly, primer pairs 5-2.5, 5-3.5, and 5-4.1 distinguish the four homologous chromosomes Chr5-1, Chr5-2, Chr5-3, and Chr5-4 of Chr5; similarly, primer pairs 7-1.3, 7-2.1, and 7-3.1 respectively determine the presence or absence of Chr7-1, Chr7-2, and Chr7-3, and primer pair 7-2.10 determines the presence or absence of Chr7-4; primer pair 12-1.27 determines the presence or absence of Chr12-3, and primer pair 12-2.6 can determine the presence or absence of Chr12-1, Chr12-2, and Chr12-4.

[0125] In addition, the inventors found that 14 pairs of primers can also amplify some variant DNA fragments with a small number of insertions and deletions, which are also within the detection range. The variant DNA fragments are Chr5-2.2 (whose nucleotide sequence is as shown in SEQ ID NO: 45), Chr5-2.3 (whose nucleotide sequence is as shown in SEQ ID NO: 46), Chr5-2.4 (whose nucleotide sequence is as shown in SEQ ID NO: 47), Chr7-1.2 (whose nucleotide sequence is as shown in SEQ ID NO: 48), Chr7-3.2 (whose nucleotide sequence is as shown in SEQ ID NO: 49), Chr7-3.3 (whose nucleotide sequence is as shown in SEQ ID NO: 50), and Chr7-4.2 (whose nucleotide sequence is as shown in SEQ ID NO: 51).

[0126] Example 3

[0127] The homologous chromosome pairs of Chr4 have been preliminarily used to verify the ploidy of the materials that are initially judged to be the diploid materials of "Cooperation 88". The primer pairs 4-1.1, 4-2.2, 4-3.1 and 4-4.1 are used for PCR amplification detection and verification in the material to be tested. If only two homologous chromosomes of the two Chr4 are present, it indicates that the material is a diploid material. It can be seen that according to the homologous chromosome typing results of 4-1.1, 4-2.2, 4-3.1 and 4-4.1, the ploidy of the test sample can be judged. Similarly, when using other chromosomes to confirm the ploidy, the corresponding primer sets are used. For example, when using Chr5 chromosome to determine the ploidy, the primer pairs 5-1.1, 5-2.5, 5-3.5, 5-4 are selected for PCR amplification detection and verification; when using Chr7 chromosome to determine the ploidy, the primer pairs 7-1.3, 7-2.1, 7-3.1, 7-2.10 are selected for PCR amplification detection and verification; when using Chr12 chromosome to determine the ploidy, the primer pairs 12-1.27, 12-2.6 are selected for PCR amplification detection and verification.

[0128] Finally, it should be noted that the above description is only the preferred embodiment of the present invention. Those skilled in the art can make various similar representations under the inspiration of the present invention without violating the purpose and claims of the present invention. Such transformations all fall within the protection scope of the present invention.

Claims

1. An SSR marker detection primer for Chr5 homologous chromosome typing identification of potato "Hezuo 88", characterized in that: Include the following primer pairs: Primer pair 5-1.1: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; Primer pair 5-2.5: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; Primer pair 5-3.5: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; Primer pair 5-4.1: comprises a forward primer and a reverse primer whose nucleotide sequences are shown as SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

2. Use of the primers described in claim 1 in typing and identification of Chr5 homologous chromosomes of potato "Hezuo 88".

3. The use according to claim 2, characterized in that: Determine whether the sample to be tested includes the DNA fragments shown in (e)-(h), (q)-(s); (e) the nucleotide sequence shown in SEQ ID NO: 33; (f) the nucleotide sequence shown in SEQ ID NO: 34; (g) the nucleotide sequence shown in SEQ ID NO: 35; (h) the nucleotide sequence shown in SEQ ID NO: 36; (q) the nucleotide sequence shown in SEQ ID NO: 45; (r) the nucleotide sequence shown in SEQ ID NO: 46; (s) the nucleotide sequence shown in SEQ ID NO:

47.

4. Use of the primers according to claim 1 in identifying the ploidy of distant hybridization progeny of potato "Hezuo 88", characterized in that: The primers include the following primer pairs: Primer pair 5-1.1: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively; Primer pair 5-2.5: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively; Primer pair 5-3.5: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively; Primer pair 5-4.1: comprises a forward primer and a reverse primer whose nucleotide sequences are shown as SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

5. The use according to claim 4, characterized in that: Determine whether the sample to be tested includes the DNA fragments shown in (e)-(h), (q)-(s); (e) the nucleotide sequence shown in SEQ ID NO: 33; (f) the nucleotide sequence shown in SEQ ID NO: 34; (g) the nucleotide sequence shown in SEQ ID NO: 35; (h) the nucleotide sequence shown in SEQ ID NO: 36; (q) the nucleotide sequence shown in SEQ ID NO: 45; (r) the nucleotide sequence shown in SEQ ID NO: 46; (s) the nucleotide sequence shown in SEQ ID NO:

47.

6. The use according to claim 2, characterized in that: The use is for preparing a detection kit.

7. The use according to claim 4, characterized in that: The use is for preparing a detection kit.

Images