SSR (Simple Sequence Repeat) marker combination for identifying Chinese chestnuts

Abstract

The invention discloses an SSR (Simple Sequence Repeat) marker combination for identifying Chinese chestnuts, and belongs to the field of molecular markers and detection thereof. The SSR marker combination covering 12 chromosomes of the Chinese chestnut is developed by utilizing latest Chinese chestnut genome data, and the SSR marker combination marker has the advantages of stability and high inter-variety polymorphism, can be applied to variety identification and genetic relationship analysis of the Chinese chestnut and other Chinese chestnut plants, and has a wide application prospect. Reference is provided for research on genetic diversity research, variety identification, genetic relationship analysis, good product breeding, core germplasm resource construction and the like of Chinese chestnuts and chestnut plants.

Description

Technical Field

[0001] The invention belongs to the field of molecular markers and detection thereof, and particularly relates to an SSR marker combination for chestnut identification. Background Art

[0002] Chestnut has a long history of cultivation and rich germplasm resources. In chestnut production, there are common problems such as mixed varieties, homonyms, and homonyms. Due to the influence of growth cycle and environment, it is difficult to accurately identify chestnut varieties in a short period of time using only morphological methods, which hinders the selection and promotion of excellent varieties. Based on this, a fast, efficient and accurate variety identification method is urgently needed.

[0003] In recent years, with the rapid development of bioinformatics and molecular biology, more and more SSR molecular markers have been widely used in various research areas such as genetic diversity analysis and variety identification of plants. However, there are few reports on the development and application of SSR markers based on the whole genome sequence of chestnut. At present, there is no SSR marker combination covering the 12 chromosomes of chestnut. With the gradual release of high-quality chestnut whole genome sequencing databases by Beijing Agricultural College, Shandong Fruit Research Institute, and Xishuangbanna Institute of the Chinese Academy of Sciences, favorable conditions have been provided for the large-scale development of SSR loci in chestnut. Summary of the invention

[0004] In view of the above-mentioned prior art, the purpose of the present invention is to provide an SSR marker combination for chestnut identification. According to the whole genome sequence information of chestnut, the present invention has developed an SSR marker combination covering 12 chromosomes of chestnut, which provides a reference for more in-depth and comprehensive research on the diversity of chestnut resources and chestnut variety identification.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention provides an SSR molecular marker primer combination for identifying Castanea plant varieties, wherein the SSR molecular marker primer combination covers 12 chromosomes of Castanea plant varieties and includes 48 pairs of primers:

[0007] The nucleotide sequence of the first pair of SSR molecular marker primers is shown in SEQ ID NO.1-2; the nucleotide sequence of the second pair of SSR molecular marker primers is shown in SEQ ID NO.3-4; the nucleotide sequence of the third pair of SSR molecular marker primers is shown in SEQ ID NO.5-6; the nucleotide sequence of the fourth pair of SSR molecular marker primers is shown in SEQ ID NO.7-8; the nucleotide sequence of the fifth pair of SSR molecular marker primers is shown in SEQ ID NO.9-10; the nucleotide sequence of the sixth pair of SSR molecular marker primers is shown in SEQ ID NO.11-12; the nucleotide sequence of the seventh pair of SSR molecular marker primers is shown in SEQ ID NO.13-14; the nucleotide sequence of the eighth pair of SSR molecular marker primers is shown in SEQ ID NO.15-16; the nucleotide sequence of the ninth pair of SSR molecular marker primers is shown in SEQ ID NO.17-18; the nucleotide sequence of the tenth pair of SSR molecular marker primers is shown in SEQ ID NO.19-20; the nucleotide sequence of the eleventh pair of SSR molecular marker primers is shown in SEQ ID NO. NO.21-22; the nucleotide sequence of the 12th pair of SSR molecular marker primers is shown in SEQ ID NO.23-24; the nucleotide sequence of the 13th pair of SSR molecular marker primers is shown in SEQ ID NO.25-26; the nucleotide sequence of the 14th pair of SSR molecular marker primers is shown in SEQ ID NO.27-28; the nucleotide sequence of the 15th pair of SSR molecular marker primers is shown in SEQ ID NO.29-30; the nucleotide sequence of the 16th pair of SSR molecular marker primers is shown in SEQ ID NO.31-32; the nucleotide sequence of the 17th pair of SSR molecular marker primers is shown in SEQ ID NO.33-34; the nucleotide sequence of the 18th pair of SSR molecular marker primers is shown in SEQ ID NO.35-36; the nucleotide sequence of the 19th pair of SSR molecular marker primers is shown in SEQ ID NO.37-38; the nucleotide sequence of the 20th pair of SSR molecular marker primers is shown in SEQ ID NO.39-40; the nucleotide sequence of the 21st pair of SSR molecular marker primers is shown in SEQ ID NO.41-42; the nucleotide sequence of the 22nd pair of SSR molecular marker primers is shown in SEQ ID NO.43-44; the nucleotide sequence of the 23rd pair of SSR molecular marker primers is shown in SEQ ID NO.45-46; the nucleotide sequence of the 24th pair of SSR molecular marker primers is shown in SEQ ID NO.47-48; the nucleotide sequence of the 25th pair of SSR molecular marker primers is shown in SEQ ID NO.49-50; the nucleotide sequence of the 26th pair of SSR molecular marker primers is shown in SEQ ID NO.51-52; the nucleotide sequence of the 27th pair of SSR molecular marker primers is shown in SEQ ID NO.53-54; the nucleotide sequence of the 28th pair of SSR molecular marker primers is shown in SEQ ID NOs.55-56; the nucleotide sequence of the 29th pair of SSR molecular marker primers is shown in SEQ ID NOs.57-58; the nucleotide sequence of the 30th pair of SSR molecular marker primers is shown in SEQ ID NOs.59-60; the nucleotide sequence of the 31st pair of SSR molecular marker primers is shown in SEQ ID NOs.61-62; the nucleotide sequence of the 32nd pair of SSR molecular marker primers is shown in SEQ ID NOs.63-64; the nucleotide sequence of the 33rd pair of SSR molecular marker primers is shown in SEQ ID NOs.65-66; the nucleotide sequence of the 34th pair of SSR molecular marker primers is shown in SEQ ID NOs.67-68; the nucleotide sequence of the 35th pair of SSR molecular marker primers is shown in SEQ ID NOs.69-70; the nucleotide sequence of the 36th pair of SSR molecular marker primers is shown in SEQ ID NOs.71-72; the nucleotide sequence of the 37th pair of SSR molecular marker primers is shown in SEQ ID NO.73-74; the nucleotide sequence of the 38th pair of SSR molecular marker primers is shown in SEQ ID NO.75-76; the nucleotide sequence of the 39th pair of SSR molecular marker primers is shown in SEQ ID NO.77-78; the nucleotide sequence of the 40th pair of SSR molecular marker primers is shown in SEQ ID NO.79-80; the nucleotide sequence of the 41st pair of SSR molecular marker primers is shown in SEQ ID NO.81-82; the nucleotide sequence of the 42nd pair of SSR molecular marker primers is shown in SEQ ID NO.83-284; the nucleotide sequence of the 43rd pair of SSR molecular marker primers is shown in SEQ ID NO.85-86; the nucleotide sequence of the 44th pair of SSR molecular marker primers is shown in SEQ ID NO.87-88; the nucleotide sequence of the 45th pair of SSR molecular marker primers is shown in SEQ ID NO.89-90; the nucleotide sequence of the 46th pair of SSR molecular marker primers is shown in SEQ ID NO.91-92; the nucleotide sequence of the 47th pair of SSR molecular marker primers is shown in SEQ ID NO. NO.93-94; the nucleotide sequence of the 48th pair of SSR molecular marker primers is shown in SEQ ID NO.95-96.

[0008] The Castanea plant is chestnut.

[0009] The 48 pairs of SSR molecular marker primers cover a total of 48 SSR loci on 12 chestnut chromosomes, including 7 SSR loci on chromosome Chr01, 4 SSR loci on chromosome Chr02, 3 SSR loci on chromosome Chr03, 5 SSR loci on chromosome Chr04, 2 SSR loci on chromosome Chr05, 5 SSR loci on chromosome Chr06, 3 SSR loci on chromosome Chr07, 2 SSR loci on chromosome Chr08, 5 SSR loci on chromosome Chr09, 2 SSR loci on chromosome Chr10, 5 SSR loci on chromosome Chr11, and 5 SSR loci on chromosome Chr12.

[0010] In a second aspect, the present invention provides the use of the above-mentioned SSR molecular marker primer combination in the following (A) or (B):

[0011] (A) Genetic analysis of chestnut kinship;

[0012] (B) Identification of chestnut varieties.

[0013] In a third aspect, the present invention provides a method for chestnut variety identification using the above-mentioned SSR molecular marker primer combination, comprising the following steps:

[0014] (1) extracting genomic DNA of chestnut samples to be tested, fluorescently modifying the forward primer of each primer pair in the above-mentioned SSR molecular marker primer combination, using the extracted DNA as a template, and performing PCR amplification using the fluorescently modified forward primer and the corresponding non-fluorescently modified reverse primer;

[0015] (2) The obtained PCR products are grouped and mixed, and then subjected to capillary electrophoresis mixed test, and the capillary electrophoresis results are analyzed to achieve the identification of chestnut varieties.

[0016] The chestnut variety identification may specifically be to identify whether the chestnut variety to be tested belongs to any of the 152 tested chestnut varieties;

[0017] Or, identify whether two unknown chestnut varieties are the same.

[0018] The fluorescent modification uses ROX, TAMRA, FAM or HEX fluorescent molecules.

[0019] The judgment standard for analyzing the capillary electrophoresis results is: if the number of different sites between the genotype of the chestnut to be tested based on the 48 SSR loci and the genotype of a certain variety of chestnut tested based on the 48 SSR loci is 0-2, then the chestnut to be tested and the variety are judged to be similar varieties;

[0020] If the number of difference sites between the genotype of the chestnut to be tested based on the 48 SSR loci and the genotype of a certain chestnut variety among the tested chestnut varieties based on the 48 SSR loci is greater than 2, the chestnut to be tested and the variety are determined to be different varieties.

[0021] The term "difference site number" is used in genetics and molecular biology research to describe the number of differences between two DNA sequences at a specific position. This difference is usually caused by genetic variations such as gene mutations, insertions or deletions. In plant variety identification, the difference site number is used to determine the similarity or difference between two varieties.

[0022] The method for determining the SSR locus genotype of the chestnut to be tested is:

[0023] The genomic DNA of the chestnut to be tested and the genomic DNA of the chestnut test variety are used as templates, and PCR amplification is performed using the primer groups in the SSR marker combination to obtain PCR amplification products; the PCR amplification products are detected to obtain the genotypes of the chestnut to be tested and the chestnut test variety based on the 48 SSR sites.

[0024] The genotype of the SSR locus can be obtained based on the fluorescent signal of the PCR amplification product to obtain the genotype of the chestnut to be tested and the chestnut test variety based on the 48 SSR loci;

[0025] Alternatively, the genotype of the chestnut to be tested and the chestnut test variety based on the 48 SSR loci is obtained according to the fragment size of the detected PCR amplification product.

[0026] The tested chestnut varieties include: weeping branch, hybrid red chestnut, Dongyue early abundance, N11-1, red chestnut, 227 Dushanxiu, 222 Wangqian big chestnut, osmanthus chestnut, Shangguang, Xixianggou flowerless, Dongmiwu flowerless, Rushan short branch, pointed top oil chestnut, Laixi big chestnut, oak 11, preferred Chushuhong, Jiujiazhong, Hunan Qianyang, Nanganlu, Qianxi No. 23, red chestnut 4, Dagongshu No. 4, Shimen Zaoshu, Xinzhuang No. 2, Dahongpao, Shuhe No. 11, Tancheng 023, red hair early, oak red chestnut, oak 18, Zhongchi chestnut, Beigaozhuang high yield, Chushuhong, Liyuan No. 8, Huaijiu, Chongyin Sticky bottom plate, Shangfeng, Mukouyu early, osmanthus fragrance, bee ball, Baofeng, Tasi 54, Pu 88-big oil chestnut, Huaihuang, Gaodian No. 1, Jiaoza, purple oil chestnut, Yimeng short branch, Shuanghe Dahongpao, Dagongshu No. 2, Jiaoci replacement code, Xigou No. 7, Hunan Baiyun Zao, August Red, June Storm, Zhejiang Zao No. 1, Yufeng, Kuili, Jinzhen chestnut, Guangxi 14-5, Guangxi 14-6, Rongshui, Linxiang Excellent Plant No. 2, Junan No. 5, Yanlong, Shengshan Chestnut, Pingyi Black and Bright, Jiujia Seed, Tancheng 207, Xiangli No. 2, Zhuji Hairy Chestnut, Qingmao Soft Thorn, Dushan Xiushi Shengdaogen, Dali Qing, Huawan No. 1, Huawan No. 2, Zunda, Hongli No. 6, Fu 3, Kaihua Shuangji Li, Hubei Agricultural University No. 1, Yangla Li, Tieli Tou, Dongshan Zao Li, Jingshu Hong, Xiangli Zi, Mingli, July Red, August Fried, Wushan No. 3, Shao Li No. 18, Zhuanqiao Dahongpao, Jinhua No. 1, Pu 279-Dayou Li, Jurong Short Thorn, Lu 35, Stable Yield Jiaozha, Xigou No. 2, Short Thorn Branch Red, Yankui, Baimao Zao, Zaoshui Dahongpao, Machiqing, Queshan No. 10, Yuanling No. 2, Xinhang Chi Li, Deep Thorn Ershui Zao, Jingxian Big Chestnut, Lu 18, Baimao Li, Shallow Thorn Big Chestnut, Hefei Big Hongpao, Shuyang oil chestnut, Tancheng No. 3, Wushan No. 2, orange red peel, Queshan early chestnut, Shuhe No. 7, Jinhua No. 2, short tie, Guangde big oil chestnut, Wushan No. 1, Zaolin No. 13, cocoon head chestnut, Shuyang Dahongpao, Laohongguang, Maozhihong, Zhuanqiao Chushuhong, Youliguang, Nanjing Zaozhuang, Xiaoguihuaxiang, Zaoxiang No. 1, Yuanling No. 1, Dongfeng, Heilin No. 1, De'an double season chestnut, Paoche No. 7, shallow thorn red hair early, Jiujia early, closed mouth red, green hair early, bee ball, King Kong chestnut, flower cover, September cold, green hair soft tie, big bottom green, late thin shell, Chongyangpu, tea chestnut, Gaojiangan and Shenyinou.

[0027] Beneficial effects of the present invention:

[0028] The present invention uses the latest chestnut genome data to develop an SSR marker combination covering 12 chestnut chromosomes. The SSR marker combination markers are stable and have high polymorphism among varieties. They can be applied to variety identification and kinship analysis of chestnut and other Castanea plants, providing a reference for genetic diversity research, variety identification, kinship analysis, breeding of improved varieties and construction of core germplasm resources of chestnut and Castanea plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is a cluster analysis diagram of the SSR marker combination provided by the present invention for 152 tested chestnut varieties.

[0030] Figure 2 This is a diagram showing the single-peak SSR typing effects of the selected 48 primer sets in some tested chestnut varieties.

[0031] Figure 3 This is a diagram showing the bimodal SSR typing effects of the selected 48 primer sets in some tested chestnut varieties. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0033] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present application, rather than to limit the scope of the present invention.

[0034] Example 1: Acquisition of SSR marker combinations

[0035] 1. Collection of Castanea species

[0036] A total of 152 Castanea germplasm resources were collected, and the genomic DNA of the 152 Castanea varieties were extracted using the improved CTAB method. The specific operation steps are as follows:

[0037] (1) Take an appropriate amount (0.2-0.4 g) of young chestnut leaves and place them in a 2 ml centrifuge tube. Add 5 steel balls and place them in liquid nitrogen for 3 min. Grind them into powder at 65 Hz for 60 s. Add 800 ul of CTAB lysis buffer and 16 ul (2%) of mercaptoethanol to each tube and mix them manually.

[0038] (2) Incubate in a 65°C water bath for 30 min, inverting and mixing every 10 min.

[0039] (3) Add 800 μl of chloroform:isoamyl alcohol (24:1) of equal volume, mix by inversion, let stand for 2 min, and centrifuge at 12,000 rpm at room temperature for 10 min.

[0040] (4) Transfer 600 μL of supernatant to a new 2 ml centrifuge tube, add 600 μL of chloroform and repeat the extraction. Transfer the supernatant to a new 1.5 ml centrifuge tube, add an equal volume of -20°C pre-cooled isopropanol to the supernatant, centrifuge at 15000 rpm, 4°C for 15 min, discard the supernatant, and add 500 μL of 70% ethanol to wash the precipitate twice.

[0041] (5) Allow the DNA pellet to dry at room temperature for about 20 minutes. Add 100 μL ddHO 2 O, fully dissolve the precipitate, and use Nano-100 instrument to detect the quality and concentration of the extracted chestnut genomic DNA. Qualified DNA (A260 / A280 ratio is about 1.8, A260 / A230 ratio is greater than 1.8, and the concentration of genomic DNA of the tested chestnut variety is 30-50ng / μL) is stored in a -20°C refrigerator for future use.

[0042] Table 1: 152 varieties of materials:

[0043]

[0044]

[0045]

[0046] 2. Design SSR marker combinations

[0047] The applicant designed SSR loci based on the published chestnut genome sequence information and combined the whole genome resequencing data of 152 germplasm resources. These 152 chestnut resources are rich in types, covering the main ecological types and agronomic traits of chestnuts available on the market, and can reflect the representativeness of germplasm as much as possible, with high genetic diversity. Subsequently, 48 pairs of SSR primer molecular markers with good polymorphism were obtained through polyacrylamide gel electrophoresis screening to form an SSR marker combination, which covers 12 chromosomes of Castanea species.

[0048] Table 2: Basic information of 48 pairs of SSR primer molecules

[0049]

[0050]

[0051]

[0052] The 5′ end of the forward primer of each primer pair in the SSR marker combination was fluorescently modified, and 48 primer pairs were used to perform PCR amplification using the genomic DNA of 152 tested chestnut varieties as templates to obtain PCR amplification products. The PCR amplification reaction system used 15 μL: 7.5 μL 2x TSINGKE Master Mix (Qingke Biotechnology Co., Ltd.), 1 μL 50 ng / μL DNA template, 0.3 μL 10 μM / μL fluorescently modified forward primer, 0.3 μL 10 μM / μL reverse primer, 5.9 μL ddH 2The PCR amplification reaction program was as follows: 94°C pre-denaturation for 3 min; 94°C denaturation for 30 s, 57°C annealing for 30 s, 72°C extension for 45 s, for a total of 35 cycles; 72°C full extension for 10 min, and storage at 4°C.

[0053] The PCR amplification products were diluted to a concentration of 1 ng / μL, and the PCR amplification products were analyzed by fluorescent capillary detection using a fully automatic capillary electrophoresis instrument. The capillary electrophoresis detection results of the 152 tested chestnut varieties were analyzed by MEGA7 software, that is, cluster analysis was performed based on the genotypes of the tested chestnuts based on 48 SSR loci. The cluster analysis of the 152 tested chestnut varieties based on 48 primer pairs is shown in Figure 2. Figure 1 As shown. The cluster analysis results showed that the 48 primer pairs can completely distinguish the 152 tested chestnut varieties and objectively reflect the relationship between varieties. The analysis of genetic diversity based on 48 primer pairs (Table 3) and genetic diversity based on 152 genetic groups (Table 4) showed that the SSR marker combination provided by the present invention has good polymorphism and stability, and can be used for genetic analysis and variety identification of Castanea resources including chestnut.

[0054] Table 3: Genetic diversity based on 48 primer pairs

[0055] N Na Ne I Ho He BL-01 147 25 8.238 2.566 0.245 0.879 BL-02 151 45 21.280 3.377 0.550 0.953 BL-04 150 20 6.996 2.330 0.560 0.857 BL-06 151 13 3.380 1.574 0.099 0.704 BL-10 151 10 4.099 1.641 0.821 0.756 BL-12 150 42 19.231 3.247 0.887 0.948 BL-13 152 25 6.324 2.371 0.678 0.842 BL-21 148 34 16.130 3.129 0.419 0.938 BL-24 152 30 14.449 2.874 0.395 0.931 BL-26 148 32 11.937 2.930 0.574 0.916 BL-29 149 31 11.391 2.825 0.228 0.912 BL-30 151 15 7.306 2.185 0.093 0.863 BL-31 152 17 3.172 1.576 0.158 0.685 BL-32 143 20 6.5 2.203 0.727 0.846 BL-34 152 37 9.168 2.749 0.171 0.891 BL-38 143 27 7.800 2.499 0.287 0.872 BL-39 151 16 4.019 1.856 0.576 0.751 BL-42 151 16 4.551 1.905 0.503 0.780 BL-43 150 42 19.036 3.248 0.953 0.947 BL-46 152 21 6.521 2.332 0.112 0.847 BL-47 152 37 10.435 2.876 0.033 0.904 BL-50 152 30 7.177 2.505 0.658 0.861 BL-51 150 27 8.349 2.495 0.840 0.880 BL-52 148 15 2.585 1.477 0.122 0.613 BL-53 151 11 2.875 1.274 0.874 0.652 BL-54 152 27 2.483 1.696 0.276 0.597 BL-55 145 27 3.944 2.118 0.076 0.746 BL-57 152 52 22.607 3.474 0.553 0.956 BL-59 149 24 3.599 1.812 0.356 0.722 BL-65 151 11 3.084 1.447 0.358 0.676 BL-68 150 39 6.778 2.717 0.487 0.852 BL-70 152 3 1.061 0.144 0.007 0.058 BL-72 152 16 3.187 1.595 0.586 0.686 BL-75 142 17 3.149 1.698 0.183 0.682 BL-77 148 22 3.224 1.749 0.182 0.690 BL-79 150 24 12.363 2.720 0.813 0.919 BL-85 152 17 3.753 1.683 0.079 0.734 BL-87 150 25 7.859 2.444 0.547 0.873 BL-93 152 11 4.625 1.788 0.375 0.784 BL-95 151 18 5.548 2.079 0.987 0.820 BL-97 140 51 19.272 3.405 0.821 0.948 BL-98 151 10 3.085 1.413 0.060 0.676 BL-99 142 31 5.799 2.448 0.282 0.828 BL-101 152 50 25.843 3.519 0.757 0.961 BL-108 146 33 8.191 2.676 0.390 0.878 BL-115 152 19 5.522 2.072 0.645 0.819 BL-117 122 22 7.366 2.427 0.090 0.864 BL-120 152 20 9.020 2.510 0.132 0.889

[0056] Note: In Table 3, N represents population size, Na represents the observed number of alleles, Ne represents the effective number of alleles, I represents Shannon's information index, Ho represents the observed heterozygosity, and He represents the expected heterozygosity.

[0057] Table 4: Genetic diversity based on 152 genetic populations

[0058] Na Ne I Ho He uHe F Genetic population 25.041 8.180 2.283 0.435 0.807 0.810 0.475

[0059] Note: In Table 4, Na represents the observed number of alleles, Ne represents the effective number of alleles, I represents Shannon's information index, Ho represents the observed heterozygosity, He represents the expected heterozygosity, uHe represents the unbiased expected heterozygosity, and F represents the genetic differentiation index.

[0060] Example 2: Method for chestnut variety identification using SSR molecular marker primer combination

[0061] (1) Detect whether the chestnut variety to be tested belongs to one of the 152 chestnut varieties tested

[0062] According to the description in Example 1, the genomic DNA of the chestnut variety to be tested was extracted by the modified CTAB method, and the PCR product of the chestnut variety to be tested was obtained by using the configuration of the SSR marker combination and the PCR reaction system. The fragment sizes of the 48 SSR amplification products of the chestnut variety to be tested were recorded. If the sample to be tested has only one allele variation at a certain site, the size of which is 100bp, the genotype of the main allele variation at the site is recorded as 100 / 100; if a sample has two allele variations at a certain site, the sizes of which are 150bp and 100bp respectively, the genotype of the main allele variation at the site is recorded as 150 / 100.

[0063] The 48 SSR loci data of the chestnut varieties to be tested were obtained respectively. If the number of different loci between the chestnut variety to be tested and a certain chestnut variety to be tested is 0-2, the chestnut variety to be tested and the chestnut variety to be tested are determined to be similar chestnut varieties; if the number of different loci between the chestnut variety to be tested and the chestnut variety to be tested is more than 2, the chestnut variety to be tested does not belong to any of the 152 chestnut varieties to be tested.

[0064] (2) Detect whether two unknown chestnut varieties are the same variety

[0065] According to the description in Example 1, the genomic DNA of the two chestnut varieties to be tested was extracted by the modified CTAB method, and the PCR products of the two chestnut varieties to be tested were obtained by using the SSR marker combination and the configuration of the PCR reaction system. The fragment sizes of the 48 SSR amplification products of the two chestnut varieties to be tested were recorded. If the sample to be tested has only one allele variation at a certain site, the size of which is 100bp, the genotype of the main allele variation at the site is recorded as 100 / 100; if a sample has two allele variations at a certain site, the sizes of which are 150bp and 100bp respectively, the genotype of the main allele variation at the site is recorded as 150 / 100.

[0066] The 48 SSR site data of the two chestnut varieties to be tested were obtained respectively. If the number of difference sites in the above 48 SSR sites of the two chestnut varieties to be tested is 2 or more, the two chestnut varieties to be tested are judged to be different chestnut varieties; the more the number of difference sites, the more distant the genetic relationship; if the number of different sites in the above 48 SSR sites of the two chestnut varieties to be tested is 0-2, the two chestnut varieties to be tested are judged to be similar chestnut varieties.

[0067] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A SSR molecular marker primer combination for identifying Castanea species, characterized in that: The SSR molecular marker primer combination covers 48 SSR sites on 12 chromosomes of Castanea species, and includes 48 pairs of primers, and the nucleotide sequences of the 48 pairs of primers are shown in SEQ ID NO.1-SEQ ID NO.

96.

2. The SSR molecular marker primer combination according to claim 1, characterized in that: The Castanea plant is chestnut.

3. Use of the SSR molecular marker primer combination according to claim 1 in the following (A) or (B): (A) Genetic analysis of chestnut kinship; (B) Identification of chestnut varieties.

4. A method for chestnut variety identification using the SSR molecular marker primer combination of claim 1, characterized in that: The steps include: (1) extracting genomic DNA of chestnut samples to be tested, fluorescently modifying the forward primer of each primer pair in the SSR molecular marker primer combination of claim 1, and using the extracted DNA as a template to perform PCR amplification using the fluorescently modified forward primer and the corresponding non-fluorescently modified reverse primer; (2) The obtained PCR products are grouped and mixed, and then subjected to capillary electrophoresis mixed test, and the capillary electrophoresis results are analyzed to achieve the identification of chestnut varieties.

5. The method according to claim 4, characterized in that The fluorescent modification uses ROX, TAMRA, FAM or HEX fluorescent molecules.

6. The method according to claim 4, characterized in that The judgment standard for analyzing the capillary electrophoresis results is: if the number of different sites between the genotype of the chestnut to be tested based on the 48 SSR loci and the genotype of a certain variety of chestnut tested based on the 48 SSR loci is 0-2, then the chestnut to be tested and the variety are judged to be similar varieties; If the number of difference sites between the genotype of the chestnut to be tested based on the 48 SSR loci and the genotype of a certain chestnut variety among the tested chestnut varieties based on the 48 SSR loci is greater than 2, the chestnut to be tested and the variety are determined to be different varieties.

7. The method according to claim 6, characterized in that The method for determining the SSR locus genotype of the chestnut to be tested is: The genomic DNA of the chestnut to be tested and the genomic DNA of the chestnut test variety are used as templates, and PCR amplification is performed using the primer groups in the SSR marker combination to obtain PCR amplification products; the PCR amplification products are detected to obtain the genotypes of the chestnut to be tested and the chestnut test variety based on the 48 SSR sites.

8. The method according to claim 4, characterized in that There are 152 varieties of chestnut for testing, including: weeping branch, hybrid red chestnut, Dongyue Zaofeng, N11-1, red chestnut, 227 Dushanxiu, 222 Wangqian big chestnut, osmanthus chestnut, Shangguang, Xixianggou flowerless, Dongmiwu flowerless, Rushan short branch, pointed oil chestnut, Laixi big chestnut, oak 11, preferred Chushuhong, Jiujiazhong, Hunan Qianyang, Nanganlu, Qianxi No. 23, red chestnut 4, Dagongshu No. 4, Shimen Zaoshu, Xinzhuang No. 2, Dahongpao, Shuhe No. 11, Tancheng 023, red hair early, oak red chestnut, oak 18, Zhongchi chestnut, Beigaozhuang high yield, Chushuhong, Liyuan No. 8, Huaijiu, Chongyin sticky bottom plate, Shangfeng, Mukouyu early, osmanthus fragrance, bee ball, Baofeng, Tasi 54, Pu 88-big oil chestnut, Huaihuang, Gaodian No. 1, Jiaoza, purple oil chestnut, Yimeng short branch, Shuanghe Dahongpao, Dagongshu No. 2, Jiaoci replacement code, Xigou No. 7, Hunan Baiyun Zao, August Red, June Storm, Zhejiang Zao No. 1, Yufeng, Kuili, Jinzhen chestnut, Guangxi 14-5, Guangxi 14-6, Rongshui, Linxiang Excellent Plant No. 2, Junan No. 5, Yanlong, Shengshan chestnut, Pingyi Black and Bright, Jiujiazhong, Tancheng 207, Xiangli No. 2, Zhuji Hairy Chestnut, Qingmao Soft Thorn, Dushan Xiushi Sheng Descendants , Daliqing, Huawan No. 1, Huawan No. 2, Zunda, Hongli No. 6, Fu 3, Kaihua Shuangji Li, Hubei Agricultural University No. 1, Yangla Li, Tieli Tou, Dongshan Zao Li, Jingshu Hong, Xiangli Zi, Mingli, July Red, August Fried, Wushan No. 3, Shaoli No. 18, Zhuanqiao Dahongpao, Jinhua No. 1, Pu 279-Dayou Li, Jurong Short Thorn, Lu 35, Stable Yield Jiaozha, Xigou No. 2, Short Thorn Branch Red, Yankui, Baimao Zao, Zaoshui Dahongpao, Machiqing, Queshan No. 10, Yuanling No. 2, Xinhang Chi Li, Deep Thorn Ershui Zao, Jingxian Daban Li, Lu 18, Baimao Li, Shallow Thorn Daban Li, He Feidahongpao, Shuyang oil chestnut, Tancheng No. 3, Wushan No. 2, orange red peel, Queshan early chestnut, Shuhe No. 7, Jinhua No. 2, short tie, Guangde big oil chestnut, Wushan No. 1, Zaolin No. 13, cocoon head chestnut, Shuyang Dahongpao, Laohongguang, Maozhihong, Zhuanqiao Chushuhong, Youliguang, Nanjing Zaozhuang, Xiaoguihuaxiang, Zaoxiang No. 1, Yuanling No. 1, Dongfeng, Heilin No. 1, De'an double season chestnut, Paoche No. 7, shallow thorn red hair early, Jiujia early, closed mouth red, green hair early, bee ball, King Kong chestnut, flower cover, September cold, green hair soft tie, big bottom green, late thin shell, Chongyangpu, tea chestnut, Gaojiangan and Shenyinou.

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