SSR (Simple Sequence Repeat) marker detection primer for typing identification of potato Chr7 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 identifying homologous chromosome typing of potato Chr7 and application of the SSR marker detection primer, the homologous chromosome of chromosome VII 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 number: 2024110607404, application date: 2024-08-05, patent name: SSR marker detection primers for potato homologous chromosome typing and use Technical Field

[0002] The invention relates to the field of biotechnology, and in particular to an SSR marker detection primer for potato Chr7 homologous chromosome typing and application. Background Art

[0003] Potato is a general term for some species with edible tubers in the genus Solanum of the Solanacea family, native to the Andes Mountains in South America. Its ploidy is highly complex, and there are diploid, triploid, tetraploid, pentaploid, hexaploid and octoploid wild potatoes in nature. Polyploid organisms contain more than two sets of chromosomes. A chromosome set contains a set of chromosomes with different morphologies and functions, but with complementary functions. In polyploid cells, chromosomes with similar morphologies and functions are homologous chromosomes. Current scientific research has found that the gene sequences on homologous chromosomes are not exactly the same. The coding genes that are located in close or identical positions on the chromosome structure have basically similar functions, but their sequences are not exactly the same. This is what is usually called alleles. Generally, the homozygous genotype is the one with exactly the same coding sequence of alleles. To a certain extent, the homozygosity of the allele determines the characteristics of the organism. Therefore, in agriculture, homologous chromosome typing can provide a larger gene selection marker for molecular marker-assisted breeding at the chromosome level, and can better provide a basis for screening the coding region of quantitative trait loci. "Hezuo 88" is one of the potato varieties that is widely produced and planted in southwest my country. This variety can be used for potato chip processing, starch production, fresh eating and other purposes, and has resistance to diseases such as late blight, early blight and viral diseases, making it one of the excellent parents for breeders to crossbreed. Therefore, typing homologous chromosomes of this variety can speed up the progress of potato breeding.

[0004] The existing chromosome analysis and detection technologies mainly include chromosome karyotype analysis, chromosome banding technology and fluorescence in situ hybridization technology. From the perspective of detection purpose, none of the three technologies is dedicated to homologous chromosome typing, so they can only type homologous chromosomes to a limited extent. From the perspective of research objects, all three technologies require cells in the metaphase of mitosis, and to obtain better results, samples need to be taken at a specific time. In terms of time, FISH takes about 4-7 days to get the test results; chromosome banding technology takes more than 7 days; chromosome karyotype analysis takes at least 3-4 days. In terms of technical difficulty, all three technologies require operators to have high experimental skills. The detection throughput of the three is very small, and generally only a single sample can be targeted. From the perspective of equipment, chromosome karyotype analysis and chromosome banding only require ordinary microscopes, while FISH requires fluorescence microscopes, which are expensive. Genome sequencing assembly to the chromosome level is currently the most effective means of homologous chromosome analysis. This technology uses the current second-generation + third-generation sequencing technology to complete DNA nucleotide sequence determination, and uses the powerful computing power of computers, bioinformatics data processing, genetic analysis, etc. to complete the chromosome-level splicing of the entire genome sequence of the organism.

[0005] Bao et al. conducted a study on homologous chromosome typing of potato "Hezuo 88" by sequencing and assembly. The study conducted second-generation sequencing of "Hezuo 88" and 1034 self-pollinated materials, third-generation sequencing of Hifi and ONT of "Hezuo 88", and Hi-C sequencing, which was very expensive. Not only does the preparation of experimental materials require a lot of time and manpower, but the large amount of data obtained by sequencing requires the help of supercomputers to process, and the price of the required equipment is also very high. Data analysis requires the participation of skilled and experienced personnel. Therefore, it is necessary to find a method for homologous chromosome typing of "Hezuo 88", which is very important to speed up the progress of potato breeding. Summary of the invention

[0006] In order to overcome the shortcomings of the above technical defects, the present invention provides an SSR marker detection primer for typing and identification of potato Chr7 homologous chromosomes and its use, which is used for rapid typing and identification of homologous chromosomes of the tetraploid potato variety "Hezuo 88" and its derivative materials.

[0007] To achieve the above object, the present invention is implemented through the following scheme:

[0008] SSR scanning analysis was performed on the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 of the known "Hezuo 88" genome, and the screened primers were verified in the "Hezuo 88" reduced-polyploidy and self-pollinated populations, and 14 pairs of primers were obtained that can distinguish the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 of the derived materials of this variety.

[0009] Therefore, in a first aspect, the present invention provides SSR marker detection primers for potato Chr7 homologous chromosome typing and identification, selected from the primer pairs shown below:

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

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

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

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

[0014] 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;

[0015] 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;

[0016] 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;

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

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

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

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

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

[0022] Primer pair 12-1.27: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively;

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

[0024] The present invention utilizes the 14 pairs of primers to rapidly type and identify homologous chromosomes of chromosomes IV, V, VII and XII of the potato tetraploid variety "Hezuo 88", which is helpful for molecular marker-assisted breeding on a chromosome scale.

[0025] Furthermore, 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 the four 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 the four 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 the four homologous chromosomes of Chr7 chromosome; primer pair 12-1.27 and primer pair 12-2.6 are used to identify the four 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 sequence of the primer is (a), (b) or (c), achieving the same detection purpose;

[0029] (a) the nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.28;

[0030] (b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO.1 to SEQ ID NO.28 under stringent conditions;

[0031] (c) A nucleotide sequence encoding a nucleotide sequence having 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 1 to SEQ ID NO. 28.

[0032] In some specific embodiments, the nucleotide sequence of the SSR marker detection primer pair provided by the present invention has 80% identity with the sequence shown in SEQID NO.1-SEQ ID NO.12; preferably, it has 85% identity, more preferably, it has 90% identity, more preferably, it has 95% identity, and most preferably, it has 99% identity.

[0033] Exemplarily, "stringent conditions" as described herein refer to conditions under which the probe will hybridize to its target sequence to a detectable extent exceeding hybridization with other sequences (e.g., at least 2 times the background). Stringent conditions are sequence-dependent and vary depending on the environment. By controlling the stringency of hybridization and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified. Alternatively, stringent conditions can be adjusted to allow some sequence mismatches so that a lower degree of similarity is detected. These nucleotide sequences that hybridize under stringent conditions can be used, for example, to express variant proteins of SEQ ID NO.1 or can be used as primers, probes, exogenous donor sequences, guide RNA, antisense RNA, shRNA and siRNA.

[0034] In a second aspect, the present invention provides use of an SSR marker detection primer pair in potato homologous chromosome typing and identification.

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

[0036] Furthermore, the potatoes are "Cooperation 88".

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

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

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

[0040] (c) the nucleotide sequence 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 (whose nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.28) can amplify a total of 16 DNA fragments (whose nucleotide sequences are shown in SEQ ID NO.29-SEQ ID NO.44) in the parent plant, which can identify the homologous chromosomes of chromosomes Chr4, Chr5, Chr7 and Chr12 in "Hezuo 88".

[0062] Furthermore, these 14 primer pairs were used to identify homologous chromosomes in the "Hezuo 88" selfing population, confirming that these primers can also be used to identify homologous chromosomes in selfing populations ( Figure 2 ) and can estimate the direction of parental gene flow.

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

[0064] In a third aspect, the present invention provides use of a primer pair for detecting SSR markers in identifying the ploidy of potato distant hybridization progeny.

[0065] Furthermore, the potatoes are "Cooperation 88".

[0066] Further, determining 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 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 shown in SEQ ID NO: 38;

[0076] (k) the nucleotide sequence 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) the nucleotide sequence 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 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 shown in SEQ ID NO:51.

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

[0090] Furthermore, the potatoes are "Cooperation 88".

[0091] Furthermore, the uses include nucleic acid hybridization detection, molecular labeling, 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 be used to identify the homologous chromosomes of chromosome IV, chromosome V, chromosome VII, and chromosome XII in "Hezuo 88", and the ploidy of the test sample can be determined by combining the typing results of homologous chromosomes with other ploidy detection methods (such as flow cytometry, etc.);

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

[0095] (3) Compared with genome sequencing technology, it does not require special instruments and equipment, and the detection cost is lower, the time consumption is shorter, and the operation is simpler. The PCR amplification results are used to detect homologous chromosome typing, and there are basically no special requirements for the detection materials. Compared with sequencing and data processing, the skill requirements are relatively much lower. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0097] Figure 2 This is the chromosome IV homologous chromosome typing test diagram;

[0098] Figure 3 This is the chromosome V homologous chromosome typing test map;

[0099] Figure 4 This is the chromosome VII homologous chromosome typing test diagram;

[0100] Figure 5 This is the chromosome XII homologous chromosome typing test map;

[0101] Figure 6 This is the peak diagram of the cloning and sequencing results of the product amplified by primer pair 4-1.1;

[0102] Figure 7 This is the peak diagram of the cloning and sequencing results of the product amplified by primer pair 7-1.1. DETAILED DESCRIPTION

[0103] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0104] Based on the present invention, the commonly cultivated potato is a tetraploid, containing 4 chromosome sets in the cell, each chromosome has 4 homologous chromosomes, but because 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 is 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 genome of "Hezuo 88", the inventors developed SSR marker detection primers suitable for rapid typing and identification of homologous chromosomes of "Hezuo 88", which can be used for rapid typing and identification of homologous chromosomes of the tetraploid potato variety "Hezuo 88" and its derivative materials, and combined with other ploidy detection methods (such as flow cytometry, etc.) to determine the ploidy of the test sample.

[0107] Example 1

[0108] Downloaded from Spud DB (Potato genome resource website http: / / spuddb.uga.edu / c88_potato_ download.shtml ) was used to generate the "Cooperation 88" genome sequence. Bioinformatics software was used to analyze the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12, and a total of 39,106 SSR sites were found. Primers were selected for these sites according to the following criteria: (1) the expected amplified fragment was no larger than 400 bp and no smaller than 100 bp; (2) the primer binding site was unique on all 48 chromosomes; (3) there was a binding site only on the homologous chromosome of one chromosome, but the sizes of the amplified fragments on the homologous chromosomes were different. The screening criteria were met when (2) or (3) was met.

[0109] The obtained SSR fragments were extended by 100-200bp on both wings for primer design. First, the fragments with low GC% content at both ends of the fragments that could not be used to design amplification primers were removed; then, the designed primers were subjected to ePCR in the genome to retain the fragments and primers that could distinguish homologous chromosomes; finally, the screened primers were synthesized and verified by PCR detection and product electrophoresis detection in the "Hezuo 88" self-pollinated population. According to the detection and analysis results, 14 pairs of molecular marker primers that can be used for homologous chromosome typing were determined. The screened primers were verified in the "Hezuo 88" reduced-fold and self-pollinated populations, and the amplified products were detected by electrophoresis. Based on the electrophoresis results, 14 pairs of SSR marker detection primers that can be used for typing and identification of homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 were determined (see the screening process). Figure 1 ).

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

[0111] Example 2

[0112] The total DNA of leaves of "Hezuo 88" and its reduced-polyploidy population and self-pollinated population was extracted by the improved CTAB method. The 14 pairs of SSR marker detection primers screened were used for PCR amplification detection and verification in "Hezuo 88". The amplification system and amplification procedure are shown in Tables 1 and 2:

[0113] Table 1 Amplification system

[0114]

[0115] Table 2 Amplification procedures

[0116]

[0117]

[0118] The amplified products were detected by electrophoresis on 2% agarose gel (see Figure 2-5 ), and found that 14 pairs of primers could identify the homologous chromosomes of Chr4, Chr5, Chr7 and Chr12 in "Cooperation 88". Figure 2-5 In the figure, M is Maker, lane 1 is C88; lane 2 is H2O; lanes 3 to 22 are diploids in the C88 reduced fold population; Figure 2 As can be seen, 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 four homologous chromosomes (Chr4-1 to 4) of chromosome IV (Chr4), and four amplification products are 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 328 bp, 392 bp, 259 bp and 131 bp, respectively;

[0119] The electrophoresis band of primer pair 4-1.1 was cut separately, and the band was recovered and connected to the vector for sequencing. Some sequencing peaks were as follows Figure 6 As shown, it is consistent with the expected fragment in the “Cooperation 88” reference genome.

[0120] from Figure 3It can be seen that primer pair 5-1.1, primer pair 5-2.5, primer pair 5-3.5, and primer pair 5-4.1 have binding sites on the four homologous chromosomes (Chr5-1~4) of chromosome V (Chr5), 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 155bp, 208bp, 278bp and 144bp, respectively.

[0121] from Figure 4 As can be seen, primer pair 7-1.3, primer pair 7-2.1, primer pair 7-3.1, and primer pair 7-2.10 have binding sites on the four homologous chromosomes (Chr7-1 to 4) of chromosome VII (Chr7), 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] The electrophoresis band of primer pair 7-1.1 was cut separately, and the band was recovered and connected to the vector for sequencing. Some sequencing peaks were as follows Figure 7 As shown, it is consistent with the expected fragment in the “Cooperation 88” reference genome.

[0123] from Figure 5 It can be seen that primer pair 12-1.27 and primer pair 12-2.6 have binding sites on the four homologous chromosomes (Chr12-1~4) of the homologous chromosomes (Chr12-1~4) of chromosome XII (Chr12), respectively. 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 371bp, and primer pair 12-2.6 obtains 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 274bp, 334bp and 259bp, respectively.

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

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

[0126] Example 3

[0127] The material pair that has been preliminarily judged to be the diploid "Hezuo 88" by the homologous chromosome pair of Chr4 is verified for ploidy. The primer pair 4-1.1, primer pair 4-2.2, primer pair 4-3.1 and primer pair 4-4.1 are used to perform PCR amplification detection and verification in the material to be tested. If there are only two homologous chromosomes of two Chr4, it means that the material is a diploid material. It can be seen that the ploidy of the test sample can be determined based on the typing results of the homologous chromosomes of 4-1.1, 4-2.2, 4-3.1 and 4-4.1. Similarly, if other chromosomes are used to confirm the ploidy, the corresponding primer sets are used. For example, if the Chr5 chromosome is used to determine the ploidy, primer pair 5-1.1, primer pair 5-2.5, primer pair 5-3.5, and primer pair 5-4 are selected for PCR amplification detection and verification; if the Chr7 chromosome is used to determine the ploidy, primer pair 7-1.3, primer pair 7-2.1, primer pair 7-3.1, and primer pair 7-2.10 are selected for PCR amplification detection and verification; if the Chr12 chromosome is used to determine the ploidy, primer pair 12-1.27 and primer pair 12-2.6 are selected for PCR amplification detection and verification.

[0128] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. An SSR marker detection primer for potato Chr7 homologous chromosome typing identification, characterized in that: Select from the following primer pairs: Primer pair 7-1.3: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; Primer pair 7-2.1: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; Primer pair 7-3.1: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; Primer pair 7-2.10: comprises a forward primer and a reverse primer whose nucleotide sequences are shown as SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

2. Use of the primers described in claim 1 in typing and identification of Chr7 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 (i)-(l), (t)-(w); (i) the nucleotide sequence shown in SEQ ID NO: 37; (j) the nucleotide sequence shown in SEQ ID NO: 38; (k) the nucleotide sequence shown in SEQ ID NO: 39; (l) the nucleotide sequence shown in SEQ ID NO: 40; (t) the nucleotide sequence shown in SEQ ID NO: 48; (u) the nucleotide sequence shown in SEQ ID NO: 49; (v) the nucleotide sequence shown in SEQ ID NO: 50; (w) The nucleotide sequence shown in SEQ ID NO:

51.

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 7-1.3: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively; Primer pair 7-2.1: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; Primer pair 7-3.1: comprising a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; Primer pair 7-2.10: comprises a forward primer and a reverse primer whose nucleotide sequences are shown as SEQ ID NO: 23 and SEQ ID NO: 24, respectively.

5. The use according to claim 4, characterized in that: Determine whether the sample to be tested includes the DNA fragments shown in (i)-(l), (t)-(w); (i) the nucleotide sequence shown in SEQ ID NO: 37; (j) the nucleotide sequence shown in SEQ ID NO: 38; (k) the nucleotide sequence shown in SEQ ID NO: 39; (l) the nucleotide sequence shown in SEQ ID NO: 40; (t) the nucleotide sequence shown in SEQ ID NO: 48; (u) the nucleotide sequence shown in SEQ ID NO: 49; (v) the nucleotide sequence shown in SEQ ID NO: 50; (w) The nucleotide sequence shown in SEQ ID NO:

51.

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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