SSR (Simple Sequence Repeat) marker detection primer for typing identification of potato Chr5 homologous chromosome and application of SSR marker detection primer

Abstract

The invention relates to the technical field of biology, in particular to an SSR (Simple Sequence Repeat) marker detection primer for potato Chr5 homologous chromosome typing identification and application, the homologous chromosome of a chromosome V in 'Cooperation 88' can be identified, and the ploidy of a detection sample can be determined by combining a homologous chromosome typing result with other ploidy detection means. Compared with a chromosome karyotype analysis technology, a chromosome banding technology and a fluorescence in-situ hybridization technology, the detection flux is high, the detection result is more objective and more accurate, and the repeatability is good. Compared with a genome sequencing technology, special instruments and equipment are not needed, the detection cost is lower, the consumed time is shorter, and the operation is simpler; the homologous chromosome typing detection is carried out by adopting a PCR amplification result, the detection material is basically not specially required, and compared with sequencing and data processing, the skill requirement is relatively low.

Description

[0001] Parent Patent No.: 2024110607404, Application Date: 2024-08-05, Patent Title: SSR Marker Detection Primers for Homologous Chromosome Typing Identification in Potatoes and Their Uses Technical Field

[0002] This invention relates to the field of biotechnology, specifically to an SSR marker detection primer for identifying potato Chr5 homologous chromosomes and its uses. Background Technology

[0003] Potatoes are a general term for some species of the genus *Solanum* in the family Solanaceae, native to the Andes Mountains of South America. Their ploidy is highly complex, with diploid, triploid, tetraploid, pentaploid, hexaploid, and octoploid wild potatoes existing in nature. Polyploid organisms contain more than two sets of chromosomes. A single chromosome set contains a set of chromosomes with different morphologies and functions, but whose functions complement each other. Within polyploid cells, chromosomes with similar morphology and function are called homologous chromosomes. Current scientific research has found that the gene sequences on homologous chromosomes are not completely identical. Genes that are structurally close or identical on chromosomes have similar functions, but their sequences are not completely identical; these are what are commonly referred to as alleles. Generally, alleles with completely identical coding sequences are homozygous genotypes. To a certain extent, the homozygosity of alleles determines the traits of an organism. Therefore, in agriculture, homologous chromosome typing can provide greater gene selection markers at the chromosome level for marker-assisted selection, and can better provide a basis for screening coding regions of quantitative trait loci. "Hezuo 88" is one of the potato varieties widely cultivated in southwestern my country. This variety can be used for various purposes, including potato chip processing, starch production, and fresh consumption. It also exhibits resistance to diseases such as late blight, early blight, and viral diseases, making it an excellent parent variety for crossbreeding. Therefore, homologous chromosome typing of this variety can accelerate the progress of potato breeding.

[0004] Existing chromosome analysis and detection technologies mainly include chromosome karyotype analysis, chromosome banding, and fluorescence in situ hybridization (FISH). From the perspective of detection purpose, none of these three technologies are specifically designed for homologous chromosome typing; therefore, they can only perform limited typing of homologous chromosomes. In terms of research subjects, all three technologies require cells in metaphase of mitosis, and sampling at specific times is necessary for good results. In terms of time, FISH generally takes 4-7 days to obtain results; chromosome banding takes more than 7 days; and chromosome karyotype analysis takes at least 3-4 days. In terms of technical difficulty, all three technologies require operators with high experimental skills. The throughput of all three is very low, generally only applicable to 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 at the chromosome level is currently the most effective method for homologous chromosome analysis. This technology uses current second- and third-generation sequencing technologies to determine the DNA nucleotide sequence, and leverages the powerful computing capabilities of computers, bioinformatics data processing, and genetic analysis to complete the chromosome-level assembly of the entire genome sequence.

[0005] Bao et al. conducted homologous chromosome typing of the potato variety "Hezuo 88" using a sequencing assembly method. This study involved second-generation sequencing of "Hezuo 88" and 1034 self-pollinated materials, Hi-Fi and ONT third-generation sequencing of "Hezuo 88," and Hi-C sequencing, incurring significant costs. Not only did the preparation of experimental materials require substantial time and manpower, but the large volume of sequencing data necessitated supercomputer processing, requiring expensive equipment, and data analysis required highly skilled and experienced personnel. Therefore, finding a method for homologous chromosome typing of "Hezuo 88" is crucial to accelerating potato breeding progress. Summary of the Invention

[0006] To overcome the shortcomings of the above-mentioned technical defects, the present invention provides an SSR marker detection primer for potato Chr5 homologous chromosome typing and identification and its application, which is used for rapid typing and identification of homologous chromosomes of tetraploid potato variety "Cooperation 88" and its derivative materials.

[0007] To achieve the above objectives, the present invention is implemented through the following solution:

[0008] SSR scanning analysis was performed on the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 of the known "Cooperation 88" genome. The selected primers were validated in the "Cooperation 88" down-multiplication and self-pollinated populations, and 14 pairs of primers that can distinguish the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 of the derived materials of this variety were obtained.

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

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

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

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

[0013] Primer pairs 4-4.1: include forward and reverse primers with nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively;

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

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

[0016] Primer pair 5-3.5: includes the forward and reverse primers with nucleotide sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively;

[0017] Primer pairs 5-4.1: include forward and reverse primers with nucleotide sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively;

[0018] Primer pair 7-1.3: includes the forward and reverse primers with nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively;

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

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

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

[0022] Primer pairs 12-1.27: include forward and reverse primers with nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

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

[0024] This invention utilizes these 14 pairs of primers to rapidly identify homologous chromosomes of chromosomes IV, V, VII, and XII of the tetraploid potato variety "Cooperation 88," which is helpful for molecular marker-assisted breeding at the chromosome scale.

[0025] Furthermore, the primers are divided into four groups: 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 pairs 4-1.1, 4-2.2, 4-3.1, and 4-4.1 are used to identify the four homologous chromosomes of chromosome Chr4; primer pairs 5-1.1, 5-2.5, 5-3.5, and 5-4.1 are used to identify the four homologous chromosomes of chromosome Chr5; primer pairs 7-1.3, 7-2.1, 7-3.1, and 7-2.10 are used to identify the four homologous chromosomes of chromosome Chr7; and primer pairs 12-1.27 and 12-2.6 are used to identify the four homologous chromosomes of chromosome Chr12.

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

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

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

[0030] (b) Nucleotide sequences that hybridize with and encode the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.28 under stringent conditions;

[0031] (c) A nucleotide sequence that has more than 80% homology with the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.28 and encodes a nucleotide sequence.

[0032] In some specific embodiments, the present invention provides an SSR marker detection primer pair set whose nucleotide sequence has 80% identity with the sequences shown in SEQ ID NO.1-SEQ ID NO.12; preferably 85% identity, more preferably 90% identity, even more preferably 95% identity, and most preferably 99% identity.

[0033] For example, "stringent conditions" as described herein refer to conditions under which a probe hybridizes to its target sequence to a detectable degree exceeding that of hybridization with other sequences (e.g., at least twice the background). Stringent conditions are sequence-dependent and vary depending on the environment. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified. Alternatively, stringent conditions can be modulated to allow some sequence mismatches, resulting in the detection of a lower degree of similarity. These nucleotide sequences hybridized under stringent conditions can be used, for example, to express variant proteins of SEQ ID NO. 1 or can serve as primers, probes, exogenous donor sequences, guide RNAs, antisense RNAs, shRNAs, and siRNAs.

[0034] Secondly, this invention provides the use of SSR marker detection primer pairs in the identification of homologous chromosomes in potatoes.

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

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

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

[0038] (a) A 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 shown in SEQ ID NO: 31;

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

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

[0043] (f) The nucleotide sequence as 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) Nucleotide sequence as 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) A nucleotide sequence as 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 as 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 as shown in SEQ ID NO: 51.

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

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

[0063] Furthermore, the applications include nucleic acid hybridization detection, molecular markers, gene chip preparation, molecular probe preparation, and detection kit preparation.

[0064] Thirdly, the present invention provides the use of SSR marker detection primer pairs in the identification of ploidy in distant hybrid offspring of potatoes.

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

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

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

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

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

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

[0071] (f) The nucleotide sequence as 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 shown in SEQ ID NO: 38;

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

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

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

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

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

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

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

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

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

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

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

[0087] (v) The nucleotide sequence 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 (with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.28) can amplify a total of 16 DNA fragments (with nucleotide sequences as shown in SEQ ID NO.29-SEQ ID NO.44) in the maternal parent. Based on 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), the ploidy of the test sample can be determined.

[0090] Furthermore, the potato in question is "Cooperation 88".

[0091] Furthermore, the applications include nucleic acid hybridization detection, molecular markers, gene chip preparation, molecular probe preparation, and detection kit preparation.

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

[0093] (1) Using 14 pairs of SSR marker primers, homologous chromosomes of chromosome IV, chromosome V, chromosome VII and chromosome XII in “Cooperation 88” can be identified. Furthermore, the homologous chromosome typing results can be combined with other ploidy detection methods (such as flow cytometry) to determine the ploidy of the test sample.

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

[0095] (3) Compared with genome sequencing technology, it does not require special instruments and equipment, and the detection cost is lower, the time is shorter, and the operation is simpler. The detection of homologous chromosomes by using PCR amplification results has no special requirements for detection materials. Compared with sequencing and data processing, the skill requirements are much lower. Attached Figure Description

[0096] Figure 1 Flowchart for primer screening for SSR marker detection;

[0097] Figure 2 This is a diagram of chromosome IV homologous chromosome typing.

[0098] Figure 3 This is a chromosome V homologous chromosome typing detection diagram;

[0099] Figure 4 This is a chromosomal typing diagram for chromosome VII homologous chromosomes;

[0100] Figure 5 This is a chromosomal XII homologous chromosome typing detection image;

[0101] Figure 6 The peak diagram shows the cloning and sequencing results of the amplification product of primer pair 4-1.1.

[0102] Figure 7 This is a peak diagram of the cloning and sequencing results of the amplification product of primer pair 7-1.1. Detailed Implementation

[0103] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be described in detail below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise specified, the test materials used in the following embodiments were purchased from conventional biochemical reagent stores. Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0104] Based on this invention, conventionally cultivated potatoes are tetraploid, containing four sets of chromosomes within their cells. Each chromosome has four homologous chromosomes. However, because the gene sequences on homologous chromosomes are not entirely identical, the different homologous chromosomes present in the offspring determine the genotype of the offspring and have a significant impact on the phenotype. Therefore, the inventors believe that homologous chromosome typing is an excellent method for marker-assisted breeding at the chromosome scale.

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

[0106] Based on this invention, after analyzing the genome of “Hezuo 88”, the inventors developed SSR marker detection primers suitable for rapid typing and identification of homologous chromosomes of “Hezuo 88”. These primers can be used for rapid typing and identification of homologous chromosomes of tetraploid potato variety “Hezuo 88” and its derivative materials, and can be combined with other ploidy detection methods (such as flow cytometry) to determine the ploidy of the test sample.

[0107] Example 1

[0108] Downloaded from the potato genome resource website Spud DB ( http: / / spuddb.uga.edu / c88_potato_ download.shtml The genome sequence of "Cooperation 88" was analyzed using bioinformatics software on the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12, and 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 no greater than 400 bp and no less than 100 bp; (2) the primer binding site is unique on all 48 chromosomes; (3) the binding site is only on the homologous chromosome of one chromosome, but the amplification fragments on the homologous chromosomes are different in size; when (2) or (3) is met, the screening conditions are met.

[0109] Primers were designed by extending the obtained SSR fragments 100-200 bp to each side. First, fragments with low GC content at both ends that could not be used for primer design were removed. Then, the designed primers were used for ePCR on the genome, retaining fragments and primers that could distinguish homologous chromosomes. Finally, the selected primers were synthesized and validated by PCR and electrophoresis in the "Cooperation 88" self-pollinated population. Based on the analysis results, 14 pairs of molecular marker primers suitable for homologous chromosome typing were identified. The selected primers were validated in the "Cooperation 88" folding and self-pollinated populations, and the amplification products were detected by electrophoresis. Based on the electrophoresis results, 14 pairs of SSR marker detection primers suitable for Chr4, Chr5, Chr7, and Chr12 homologous chromosome typing were identified (screening procedure see [link to screening process]). Figure 1 ).

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

[0111] Example 2

[0112] Total DNA was extracted from the leaves of "Hezuo 88" and its multiplier and self-pollinated populations using a modified CTAB method. Fourteen pairs of screened SSR marker detection primers were then used for PCR amplification and verification in "Hezuo 88". The amplification system and procedure are shown in Tables 1 and 2.

[0113] Table 1 Amplification System

[0114]

[0115] Table 2 Amplification Procedure

[0116]

[0117]

[0118] The amplification products were detected by electrophoresis with 2% agarose (see [link]). Figure 2-5 ), and found that 14 pairs of primers can identify the homologous chromosomes Chr4, Chr5, Chr7 and Chr12 in “Cooperation 88”. Figure 2-5 In the diagram, M stands for Maker, lane 1 represents C88; lane 2 represents H2O; lanes 3-22 represent diploids within the C88 down-multiplication population; from... Figure 2 As can be seen, primer pairs 4-1.1, 4-2.2, 4-3.1, and 4-4.1 have binding sites on the four homologous chromosomes (Chr4-1 to 4) of chromosome IV (Chr4), and four amplification products were obtained, 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 individually cutting the electrophoretic bands of primer pair 4-1.1, the bands were recovered, ligated into the vector, and sequenced. Partial sequencing peaks were observed. Figure 6 As shown, it is consistent with the expected fragment in the “Cooperation 88” reference genome.

[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 the four homologous chromosomes (Chr5-1 to 4) of chromosome V (Chr5), and four amplification products were obtained respectively: Chr5-1 (nucleotide sequence as shown in SEQ ID NO: 33), Chr5-2 (nucleotide sequence as shown in SEQ ID NO: 34), Chr5-3 (nucleotide sequence as shown in SEQ ID NO: 35), and Chr5-4 (nucleotide sequence as shown in SEQ ID NO: 36), with lengths of 155bp, 208bp, 278bp, and 144bp, 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 the four homologous chromosomes (Chr7-1 to 4) of chromosome VII (Chr7), and four amplification products were obtained, namely Chr7-1 (its nucleotide sequence is shown in SEQ ID NO: 37), Chr7-2 (its nucleotide sequence is shown in SEQ ID NO: 38), Chr7-3 (its nucleotide sequence is shown in SEQ ID NO: 39), and Chr7-4 (its nucleotide sequence is shown in SEQ ID NO: 40), with lengths of 365bp, 183bp, 337bp, and 214bp, respectively.

[0122] After individually cutting the electrophoretic bands of primer pair 7-1.1, the bands were recovered, ligated into the vector, and sequenced. Partial sequencing peaks were observed. Figure 7 As shown, it is consistent with the expected fragment in the “Cooperation 88” reference genome.

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

[0124] These DNA fragments can be used to identify homologous chromosomes of Chr4, Chr5, Chr7, and Chr12 in "Cooperation 88". Specifically, primer pair 4-1.1 is used to determine the presence of Chr4-1, primer pair 4-2.2 to determine the presence of Chr4-2, primer pair 4-3.1 to determine Chr4-3, and primer pair 4-4.1 to determine Chr4-4. Similarly, primer pairs 5-2.5, 5-3.5, and 5-4.1 are used to distinguish the four homologous chromosomes of Chr5. Chromosomes Chr5-1, Chr5-2, Chr5-3, and Chr5-4; similarly, primer pairs 7-1.3, 7-2.1, and 7-3.1 determine the existence of Chr7-1, Chr7-2, and Chr7-3, respectively, while primer pair 7-2.10 determines the existence of Chr7-4; primer pair 12-1.27 determines the existence of Chr12-3, while primer pair 12-2.6 can determine the existence of Chr12-1, Chr12-2, and Chr12-4.

[0125] In addition, the inventors discovered that the 14 primer pairs 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 (nucleotide sequence as shown in SEQ ID NO: 45), Chr5-2.3 (nucleotide sequence as shown in SEQ ID NO: 46), Chr5-2.4 (nucleotide sequence as shown in SEQ ID NO: 47), Chr7-1.2 (nucleotide sequence as shown in SEQ ID NO: 48), Chr7-3.2 (nucleotide sequence as shown in SEQ ID NO: 49), Chr7-3.3 (nucleotide sequence as shown in SEQ ID NO: 50), and Chr7-4.2 (nucleotide sequence as shown in SEQ ID NO: 51).

[0126] Example 3

[0127] Based on the homologous chromosome pairs of Chr4, the material was initially identified as "Cooperation 88" diploid. Pluralism verification was performed using primer pairs 4-1.1, 4-2.2, 4-3.1, and 4-4.1. If only two homologous chromosomes of Chr4 were present, the material was considered diploid. Therefore, the ploidy of the sample can be determined based on the homologous chromosome typing results from primers 4-1.1, 4-2.2, 4-3.1, and 4-4.1. Similarly, when using other chromosomes to confirm ploidy, the corresponding primer sets are used. For example, when using Chr5 chromosome to determine ploidy, primer pairs 5-1.1, 5-2.5, 5-3.5, and 5-4 are selected for PCR amplification and verification. When using Chr7 chromosome to determine ploidy, primer pairs 7-1.3, 7-2.1, 7-3.1, and 7-2.10 are selected for PCR amplification and verification. When using Chr12 chromosome to determine ploidy, primer pairs 12-1.27 and 12-2.6 are selected for PCR amplification and verification.

[0128] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Those skilled in the art, under the guidance of the present invention, can make various similar representations without departing from the spirit and claims of the present invention, and such modifications all fall within the protection scope of the present invention.

Claims

1. An SSR marker detection primer for potato Chr5 homologous chromosome typing identification, characterized in that: Select from 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 offspring 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.

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