SSR marker detection primers for potato Chr4 homologous chromosome typing and identification and their use

Abstract

The present invention relates to the field of biotechnology, and specifically to SSR marker detection primers and uses for potato Chr4 homologous chromosome typing identification. The primers can identify the homologous chromosomes of chromosome IV in "Cooperation 88" and can determine the ploidy of the test sample by combining the results of homologous chromosome typing with other ploidy detection methods. Compared with chromosome karyotype analysis, chromosome banding technology and fluorescence in situ hybridization technology, the detection throughput is high, and the detection results are more objective, more accurate and reproducible. Compared with genome sequencing technology, no special dedicated instruments and equipment are required, the detection cost is lower, the time consumption is shorter, and the operation is simpler. The homologous chromosome typing detection using PCR amplification results has basically no special requirements for the detection materials, and the skill requirements are relatively much lower than sequencing and data processing.

Description

[0001] Parent patent number: 2024110607404, application date: 2024-08-05, patent name: SSR marker detection primers for potato homologous chromosome typing and their uses Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to an SSR marker detection primer for potato Chr4 homologous chromosome typing and application. Background Art

[0003] Potato is a collective name for several species of edible tubers in the genus Solanum, in the Solanaceae family. It is native to the Andes of South America. Its ploidy is highly complex, with wild potatoes occurring in diploid, triploid, tetraploid, pentaploid, hexaploid, and octoploid forms in nature. Polyploid organisms contain more than two sets of chromosomes. Each set contains a set of chromosomes with distinct morphologies and functions, but with complementary functions. Within polyploid cells, chromosomes with similar morphology and function are called homologous chromosomes. Current scientific research has revealed that gene sequences on homologous chromosomes are not completely identical. Genes encoding genes that are located similarly or identically on the chromosome structure and have similar functions may not be identical in sequence, commonly known as alleles. Generally, alleles with identical coding sequences are considered homozygous. To a certain extent, the homozygosity of alleles determines the characteristics of an organism. Therefore, in agriculture, homologous chromosome typing can provide more robust genetic selection markers for molecular marker-assisted breeding at the chromosomal level, providing a better basis for screening coding regions for quantitative trait loci. Hezuo 88 is one of the potato varieties widely cultivated in southwest my country. It can be used for a variety of applications, including potato chip processing, starch production, and fresh consumption. Its resistance to diseases such as late blight, early blight, and viral diseases makes it a promising parent for breeders. Therefore, performing homologous chromosome typing on this variety can accelerate potato breeding.

[0004] Existing chromosome analysis techniques primarily include karyotyping, chromosome banding, and fluorescence in situ hybridization (FISH). None of these techniques is specifically designed for homologous chromosome typing, and therefore, they can only perform limited homologous chromosome typing. Regarding the research subjects, all three require cells in metaphase, and optimal results require sampling at specific times. FISH typically takes 4-7 days to produce results; chromosome banding requires more than 7 days; and karyotyping requires at least 3-4 days. Technically, all three techniques require advanced laboratory skills. Their throughput is low, typically limited to single samples. Regarding equipment, karyotyping and chromosome banding require only a standard microscope, while FISH requires a fluorescence microscope, which is expensive. Currently, genome sequencing down to the chromosome level is the most effective approach for homologous chromosome analysis. This technique utilizes current second- and third-generation sequencing technologies to determine DNA nucleotide sequences, leveraging the powerful computing power of computers, bioinformatics data processing, and genetic analysis to assemble the complete genome sequence at the chromosome level.

[0005] Bao et al. used sequencing and assembly to perform homologous chromosome typing on the potato variety Hezuo 88. This study involved second-generation sequencing of Hezuo 88 and 1,034 inbred materials, third-generation sequencing of Hezuo 88 using HiFi and ONT, and Hi-C sequencing, all at considerable expense. Not only did the preparation of experimental materials require significant time and labor, but the massive amount of data generated by sequencing required the use of expensive supercomputers, and data analysis required highly skilled and experienced personnel. Therefore, developing a method for homologous chromosome typing in Hezuo 88 is crucial to accelerating potato breeding progress. 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 Chr4 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 doubling and self-pollinated populations, obtaining 14 pairs of primers 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 Chr4 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 having nucleotide sequences shown as SEQ ID NO: 27 and SEQ ID NO: 28, respectively.

[0024] The present invention utilizes these 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 represents chromosome IV, Chr5 represents chromosome V, Chr7 represents chromosome VII, and Chr12 represents chromosome XII.

[0028] Furthermore, 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 present invention provides an SSR marker detection primer pair group whose nucleotide sequence has 80% identity with the sequence shown in SEQ ID 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] Illustratively, "stringent conditions" as used herein refer to conditions under which a 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 for 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 potato is "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] (1) 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 (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 maternal 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" self-pollinated population, and it was confirmed that these primers can also be used to identify homologous chromosomes in self-pollinated 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 an SSR marker detection primer pair in identifying the ploidy of potato distant hybridization progeny.

[0065] Furthermore, the potato is "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] (1) 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 maternal plant. 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 potato is "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". The ploidy of the test sample can be determined by combining the typing results of the homologous chromosomes with other ploidy detection methods (such as flow cytometry).

[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 detection diagram;

[0099] Figure 4 This is the chromosome VII homologous chromosome typing detection 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 specified in the following examples are usually based on conventional conditions or 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 equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0104] According to the present invention, commonly cultivated potatoes are tetraploid, containing four chromosome sets within a cell, each with four homologous chromosomes. However, because the gene sequences on homologous chromosomes are not identical, the different homologous chromosomes present in derived offspring determine the offspring's genotype and significantly influence their phenotype. Therefore, the inventors believe that homologous chromosome typing is an effective tool for molecular marker-assisted breeding at the chromosomal scale.

[0105] The technical solutions of the present invention are 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 sequence the "Cooperation 88" genome. 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 amplified fragments on the homologous chromosomes were of different sizes; when (2) or (3) was met, the screening conditions were met.

[0109] The primers were designed by extending 100-200bp on both wings of the obtained SSR fragments. First, the fragments with low GC% content at both ends 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. Based on 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 shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively), primer pair 4-2.2 (nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively), primer pair 4-3.1 (nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively), primer pair 4-4.1 (nucleotide sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively), primer pair 5-1.1 (nucleotide sequences shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively), primer pair 5-2.5 (nucleotide sequences shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively), primer pair 5-3.5 (nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively), primer pair 5-4.1 (nucleotide sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16, respectively), primer pair 7-1.3 (nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively). ID NO: 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, its reduced-polyploidy population, and self-pollinated population was extracted using the modified CTAB method. The 14 pairs of SSR marker detection primers screened were used for PCR amplification 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] The amplified products were detected by electrophoresis on 2% agarose gel (see Figure 2-5 ), 14 pairs of primers were found to 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 ploidy 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), respectively, and four amplification products were obtained, namely Chr4-1.1 (the nucleotide sequence of which is shown in SEQ ID NO: 29), Chr4-2.2 (the nucleotide sequence of which is shown in SEQ ID NO: 30), Chr4-3.1 (the nucleotide sequence of which is shown in SEQ ID NO: 31), and Chr4-4.1 (the nucleotide sequence of which is shown in SEQ ID NO: 32), with lengths of 328 bp, 392 bp, 259 bp, and 131 bp, respectively;

[0118] 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.

[0119] 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 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 155bp, 208bp, 278bp and 144bp, respectively.

[0120] 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 were obtained, namely Chr7-1 (the nucleotide sequence of which is shown in SEQ ID NO: 37), Chr7-2 (the nucleotide sequence of which is shown in SEQ ID NO: 38), Chr7-3 (the nucleotide sequence of which is shown in SEQ ID NO: 39), and Chr7-4 (the nucleotide sequence of which is shown in SEQ ID NO: 40), with lengths of 365 bp, 183 bp, 337 bp, and 214 bp, respectively;

[0121] 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.

[0122] 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 the homologous chromosomes (Chr12-1 to 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 371 bp. 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 274 bp, 334 bp and 259 bp, respectively.

[0123] These DNA fragments can be used to identify the homologous chromosomes of chromosomes Chr4, Chr5, Chr7 and Chr12 in "Cooperation 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.

[0124] 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).

[0125] Example 3

[0126] The material, which was initially identified as a diploid "Hezuo 88" by the homologous chromosome pair of Chr4, was subjected to ploidy verification. PCR amplification detection and verification was performed on the test material using primer pair 4-1.1, primer pair 4-2.2, primer pair 4-3.1, and primer pair 4-4.1. If only two homologous chromosomes of Chr4 were present, the material was diploid. Therefore, the ploidy of the test sample can be determined based on the typing results of the homologous chromosomes 4-1.1, 4-2.2, 4-3.1, and 4-4.1. Similarly, if other chromosomes are used to confirm ploidy, the corresponding primer sets are used. For example, if the Chr5 chromosome is used to determine 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 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 ploidy, primer pair 12-1.27 and primer pair 12-2.6 are selected for PCR amplification detection and verification.

[0127] 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 various similar expressions without violating the purpose and claims of the present invention. Such changes fall within the scope of protection of the present invention.

Claims

1. An SSR marker detection primer for potato Chr4 homologous chromosome typing identification, characterized in that: Included primer pairs are as follows: 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; 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; 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; Primer pair 4-4.1: comprises a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

2. Use of the primers according to claim 1 in typing and identifying the Chr4 homologous chromosomes of potato "Hezuo 88".

3. The use according to claim 2, characterized in that: Determining whether the sample to be tested includes the DNA fragments shown in (a)-(d); (a) the nucleotide sequence shown in SEQ ID NO: 29; (b) the nucleotide sequence shown in SEQ ID NO: 30; (c) the nucleotide sequence shown in SEQ ID NO: 31; (d) The nucleotide sequence shown in SEQ ID NO:

32.

4. Use of the primers according to claim 1 in identifying the ploidy of distant hybrid offspring of potato "Hezuo 88", characterized in that: The primers include the following primer pairs: 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; 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; 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; Primer pair 4-4.1: comprises a forward primer and a reverse primer having nucleotide sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

5. The use according to claim 4, characterized in that: Determining whether the sample to be tested includes the DNA fragments shown in (a)-(d); (a) the nucleotide sequence shown in SEQ ID NO: 29; (b) the nucleotide sequence shown in SEQ ID NO: 30; (c) the nucleotide sequence shown in SEQ ID NO: 31; (d) The nucleotide sequence shown in SEQ ID NO:

32.

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

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

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