A combination of ssr markers for identifying chinese chestnut

Abstract

The application discloses a SSR marker combination for identifying Chinese chestnut, and belongs to the field of molecular markers and detection. The latest genome data of Chinese chestnut is used to develop a SSR marker combination covering 12 chromosomes of Chinese chestnut. The SSR marker combination has the advantages of stability and high polymorphism between varieties, and can be applied to variety identification and genetic relationship analysis of Chinese chestnut and other plants of the genus Castanea. The application provides a reference for the researches of genetic diversity, variety identification, genetic relationship analysis, good variety breeding and core germplasm resource construction of Chinese chestnut and plants of the genus Castanea.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular markers and their detection, and particularly relates to a SSR marker combination for identifying Castanea mollissima. BACKGROUND

[0002] Castanea mollissima has a long cultivation history and rich germplasm resources, but there are problems such as mixed varieties, different names for the same thing and different names for the same thing in Castanea mollissima production. Due to the influence of growth cycle and environment, it is difficult to accurately identify Castanea mollissima varieties in a short time by using morphological methods only, which hinders the breeding and promotion of excellent varieties. Therefore, 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 plant genetic diversity analysis and variety identification and other researches. However, there are few reports on the development of SSR markers based on the whole genome sequence of Castanea mollissima and its application. At present, there is no SSR marker combination covering 12 chromosomes of Castanea mollissima. With the gradual disclosure of high-quality whole genome sequencing databases of Castanea mollissima by Beijing Agricultural University, Shandong Fruit Tree Research Institute and Chinese Academy of Sciences Xishuangbanna Institute, it provides favorable conditions for large-scale development of SSR sites of Castanea mollissima. SUMMARY

[0004] In view of the above prior art, the purpose of the present application is to provide a SSR marker combination for identifying Castanea mollissima. According to the whole genome sequence information of Castanea mollissima, the present application develops a SSR marker combination covering 12 chromosomes of Castanea mollissima, which provides a reference for more in-depth and comprehensive study of the diversity of Castanea mollissima resources and the identification of Castanea mollissima varieties.

[0005] To achieve the above purpose, the present application adopts the following technical solutions:

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

[0007] The nucleotide sequences of the first pair of SSR molecular marker primers are shown as SEQ ID NO. 1-2; the nucleotide sequences of the second pair of SSR molecular marker primers are shown as SEQ ID NO. 3-4; the nucleotide sequences of the third pair of SSR molecular marker primers are shown as SEQ ID NO. 5-6; the nucleotide sequences of the fourth pair of SSR molecular marker primers are shown as SEQ ID NO. 7-8; the nucleotide sequences of the fifth pair of SSR molecular marker primers are shown as SEQ ID NO. 9-10; the nucleotide sequences of the sixth pair of SSR molecular marker primers are shown as SEQ ID NO. 11-12; the nucleotide sequences of the seventh pair of SSR molecular marker primers are shown as SEQ ID NO. 13-14; the nucleotide sequences of the eighth pair of SSR molecular marker primers are shown as SEQ ID NO. 15-16; the nucleotide sequences of the ninth pair of SSR molecular marker primers are shown as SEQ ID NO. 17-18; the nucleotide sequences of the tenth pair of SSR molecular marker primers are shown as SEQ ID NO. 19-20; the nucleotide sequences of the eleventh pair of SSR molecular marker primers are shown as SEQ ID NO. 21-22; the nucleotide sequences of the twelfth pair of SSR molecular marker primers are shown as SEQ ID NO. 23-24; the nucleotide sequences of the thirteenth pair of SSR molecular marker primers are shown as SEQ ID NO. 25-26; the nucleotide sequences of the fourteenth pair of SSR molecular marker primers are shown as SEQ ID NO. 27-28; the nucleotide sequences of the fifteenth pair of SSR molecular marker primers are shown as SEQ ID NO. 29-30; the nucleotide sequences of the sixteenth pair of SSR molecular marker primers are shown as SEQ ID NO. 31-32; the nucleotide sequences of the seventeenth pair of SSR molecular marker primers are shown as SEQ ID NO. 33-34; the nucleotide sequences of the eighteenth pair of SSR molecular marker primers are shown as SEQ ID NO. 35-36; the nucleotide sequences of the nineteenth pair of SSR molecular marker primers are shown as SEQ ID NO. 37-38; the nucleotide sequences of the twentieth pair of SSR molecular marker primers are shown as SEQ ID NO. 39-40; the nucleotide sequences of the twenty-first pair of SSR molecular marker primers are shown as SEQ ID NO. 41-42; the nucleotide sequences of the twenty-second pair of SSR molecular marker primers are shown as SEQ ID NO. 43-44; the nucleotide sequences of the twenty-third pair of SSR molecular marker primers are shown as SEQ ID NO. 45-46; the nucleotide sequences of the twenty-fourth pair of SSR molecular marker primers are shown as SEQ ID NO. 47-48; the nucleotide sequences of the twenty-fifth pair of SSR molecular marker primers are shown as SEQ ID NO. 49-50; the nucleotide sequences of the twenty-sixth pair of SSR molecular marker primers are shown as SEQ ID NO. 51-52; the nucleotide sequences of the twenty-seventh pair of SSR molecular marker primers are shown as SEQ ID NO.53-54; the nucleotide sequence of the 28th pair of SSR molecular marker primers is shown as SEQ ID NO. 55-56; the nucleotide sequence of the 29th pair of SSR molecular marker primers is shown as SEQ ID NO. 57-58; the nucleotide sequence of the 30th pair of SSR molecular marker primers is shown as SEQ ID NO. 59-60; the nucleotide sequence of the 31st pair of SSR molecular marker primers is shown as SEQ ID NO. 61-62; the nucleotide sequence of the 32nd pair of SSR molecular marker primers is shown as SEQ ID NO. 63-64; the nucleotide sequence of the 33rd pair of SSR molecular marker primers is shown as SEQ ID NO. 65-66; the nucleotide sequence of the 34th pair of SSR molecular marker primers is shown as SEQ ID NO. 67-68; the nucleotide sequence of the 35th pair of SSR molecular marker primers is shown as SEQ ID NO. 69-70; the nucleotide sequence of the 36th pair of SSR molecular marker primers is shown as SEQ ID NO. 71-72; the nucleotide sequence of the 37th pair of SSR molecular marker primers is shown as SEQ ID NO. 73-74; the nucleotide sequence of the 38th pair of SSR molecular marker primers is shown as SEQ ID NO. 75-76; the nucleotide sequence of the 39th pair of SSR molecular marker primers is shown as SEQ ID NO. 77-78; the nucleotide sequence of the 40th pair of SSR molecular marker primers is shown as SEQ ID NO. 79-80; the nucleotide sequence of the 41st pair of SSR molecular marker primers is shown as SEQ ID NO. 81-82; the nucleotide sequence of the 42nd pair of SSR molecular marker primers is shown as SEQ ID NO. 83-84; the nucleotide sequence of the 43rd pair of SSR molecular marker primers is shown as SEQ ID NO. 85-86; the nucleotide sequence of the 44th pair of SSR molecular marker primers is shown as SEQ ID NO. 87-88; the nucleotide sequence of the 45th pair of SSR molecular marker primers is shown as SEQ ID NO. 89-90; the nucleotide sequence of the 46th pair of SSR molecular marker primers is shown as SEQ ID NO. 91-92; the nucleotide sequence of the 47th pair of SSR molecular marker primers is shown as SEQ ID NO. 93-94; the nucleotide sequence of the 48th pair of SSR molecular marker primers is shown as SEQ ID NO. 95-96.

[0008] The plant of the genus Castanea is Castanea mollissima.

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

[0010] In the second aspect of the present application, the above-mentioned combination of SSR molecular marker primers is applied in (A) or (B) as follows:

[0011] (A) genetic analysis of the relationship of Castanea mollissima;

[0012] (B) identification of Castanea mollissima varieties.

[0013] In the third aspect of the present application, a method for identifying the varieties of Castanea mollissima by using the above-mentioned combination of SSR molecular marker primers is provided, which comprises the following steps:

[0014] (1) extracting the genomic DNA of the sample Castanea mollissima to be tested, and performing fluorescence modification on the forward primers of each pair of primers in the above-mentioned combination of SSR molecular marker primers, and using the extracted DNA as a template to perform PCR amplification by using the forward primers with fluorescence modification and the corresponding reverse primers without fluorescence modification;

[0015] (2) mixing the obtained PCR products by groups and then performing capillary electrophoresis detection, analyzing the capillary electrophoresis results, and realizing the identification of the varieties of Castanea mollissima.

[0016] The identification of the varieties of Castanea mollissima can specifically be the identification of whether the Castanea mollissima to be tested belongs to any of the 152 tested Castanea mollissima varieties;

[0017] Or, the identification of whether two unknown Castanea mollissima varieties are the same variety.

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

[0019] The determination standard for analyzing the capillary electrophoresis results is that if the difference in the number of loci between the genotype of the Castanea mollissima to be tested based on the 48 SSR loci and the genotype of a certain variety of the tested Castanea mollissima based on the 48 SSR loci is 0-2, then the Castanea mollissima to be tested is determined as a similar variety to the variety.

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

[0021] The term "difference in the number of loci" refers to the number of differences in specific positions between two DNA sequences in the research of genetics and molecular biology. Such differences are usually caused by genetic mutations, insertions or deletions, etc. In plant variety identification, the difference in the number of loci is used to judge the similarity or difference between two varieties.

[0022] The SSR locus genotype determination method of the to-be-tested chestnut is as follows:

[0023] The genomic DNA of the to-be-tested chestnut and the genomic DNA of the chestnut test variety are used as templates, respectively, and the primers in the primer set in the SSR marker combination are used for PCR amplification, respectively, to obtain PCR amplification products; the PCR amplification products are detected to obtain the genotype of the to-be-tested chestnut and the chestnut test variety based on 48 SSR loci.

[0024] The genotype of the SSR locus can be obtained based on the fluorescence signal of the detected PCR amplification products to obtain the genotype of the to-be-tested chestnut and the chestnut test variety based on the 48 SSR loci.

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

[0026] The test chestnut varieties include: Chuanzhi, hybrid red chestnut, Dongyuezao, N11-1, red chestnut, 227 Dushanxiu, 222 Wangqian Dali, Guihuachestnut, Shangguang, Xixianggou Huahua, Dongmiwu Huahua, Rushan short branch, sharp top oil chestnut, Lexi Dabanchestnut, Zao 11, Youxuanchushu red, nine family species, Hunan Qianyang, Nannanlu, Qianxi 23, red chestnut No. 4, Daguoshu No. 4, Shimenzaosuo, Xinzhuang No. 2, Dahongpao, Shuhe No. 11, Tancheng 023, Hongmao early, Zao red chestnut, Zao 18, Zhongdichestnut, Beigaogongzhaofeng, Chushu red, Chestnut Garden No. 8, Huaijiu, Zhongyin sticky bottom plate, Shangfeng, Mukouyu early, Guihuaxiang, Honey ball, Baofeng, Tasi 54, Garden 88 - large oil chestnut, Huaihuang, Gaodian No. 1, Jiazu, Ziyouchestnut, Yimeng short branch, double combination Dahongpao, Daguoshu No. 2, Jia Cai Dicai, Xigou No. 7, Hunan Baiyun early, August red, June violent, Zhe early No. 1, Yufeng, Quili, Jinzhenli, Guangxi 14-5, Guangxi 14-6, Rongshui, Lixiang excellent strain No. 2, Junan No. 5, Yanlong, Shengshan chestnut, Pingyi black and bright, nine family species, Tancheng 207, Xiangli No. 2, Zhoushi Mao chestnut, Qingmao soft thorn, Dushanxiu seedling, Daliqing, Huawan No. 1, Huawan No. 2, Zundan, Hongli No. 6, E3, Kaihua double season chestnut, Hubei Agricultural University No. 1, Yanglali, Tie chestnut head, Dongshan early chestnut, Jingshu red, Xiangli, Mingli, July red, August explosion, Wushan No. 3, Shaoli No. 18, Zhuanqiao Dahongpao, Jinhua No. 1, Garden 279 - large oil chestnut, Jencang short thorn, Lu 35, stable yield Jiazha, Xigou No. 2, short thorn branch red, Yan Kui, baimao early, Zhaoshui Dahongpao, Malachite, Queshan No. 10, Yuanling No. 2, New Hangdichestnut, deep thorn early, Jingxian Dabanchestnut, Lu 18, baimao chestnut, shallow thorn Dabanchestnut, Hefei Dahongpao, Shuyang oil chestnut, Tancheng No. 3, Wushan No. 2, orange red skin, Queshan early chestnut, Shuhe No. 7, Jinhua No. 2, short Za, Guangde Dali, Wushan No. 1, Zaolin No. 13, Canhead chestnut, Shuyang Dahongpao, old red light, Mao branch red, Zhuanqiao Chushu red, Youli light, Nanjing Zaizhuang, small Guihuaxiang, early Xiang No. 1, Yuanling No. 1, Dongfeng, Heilin No. 1, De'an double season chestnut, Cannon car No. 7, shallow thorn Hongmao early, nine family early, closed mouth red, Qingmao early, honey ball, jingang chestnut, Huagai, September cold, Qingmao soft Za, large bottom Qing, late thin shell, Zhongyangpu, tea chestnut, Gaokan, Shenyinou.

[0027] The beneficial effects of the present application are:

[0028] The present application utilizes the latest chestnut genome data to develop a combination of SSR markers covering 12 chromosomes of chestnut, which has the advantages of stability and high polymorphism between varieties, and can be applied to variety identification and genetic relationship analysis of chestnut and other Castanea plants, thereby providing a reference for genetic diversity research, variety identification, genetic relationship analysis, good variety breeding and core germplasm resource construction of chestnut and Castanea plants. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1The SSR marker combination provided by the application is used for clustering analysis of 152 test Chinese chestnut varieties.

[0030] Figure 2 The single-peak SSR typing effect diagram of the selected 48 primer groups in part of the test Chinese chestnut varieties.

[0031] Figure 3 The double-peak SSR typing effect diagram of the selected 48 primer groups in part of the test Chinese chestnut varieties. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0033] The specific embodiments of the application will be further described in conjunction with the examples. The following detailed description is exemplary and is intended to provide further explanation of the application, rather than limit the scope of the application.

[0034] Example 1: Acquisition of SSR marker combination

[0035] 1. Collection of Chinese chestnut varieties

[0036] A total of 152 Chinese chestnut germplasm resources were collected, and the genomic DNA of the 152 test Chinese chestnut varieties was extracted by using the improved CTAB method, and the specific operation steps are as follows:

[0037] (1) Take a proper amount (0.2-0.4g) of young leaves of Chinese chestnut in a 2ml centrifuge tube, add 5 steel balls, grind into powder in liquid nitrogen for 3min at 65Hz, 60s, add 800ul CTAB lysis solution, add 16ul (2%) of mercaptoethanol to each tube, and mix evenly by hand.

[0038] (2) 65℃ water bath for 30min, mix well every 10min.

[0039] (3) Add an equal volume of 800ul chloroform:isopropyl alcohol (24:1), mix well, stand for 2min, centrifuge at 12000rpm at room temperature for 10min.

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

[0041] (5) The DNA precipitate was left to dry at room temperature for about 20 minutes. 100 μL of ddH2O was added to fully dissolve the precipitate, and the extracted chestnut genomic DNA was detected for mass and concentration using a Nano-100 instrument. The qualified DNA (A260 / A280 ratio of about 1.8, A260 / A230 ratio greater than 1.8, and concentration of the chestnut variety genomic DNA for testing of 30-50 ng / μL) was stored in a -20°C refrigerator for standby use.

[0042] Table 1: 152 varieties of materials:

[0043]

[0044]

[0045]

[0046] 2. Design of SSR marker combination

[0047] Applicants designed SSR sites based on published chestnut whole genome sequence information, combined with whole genome resequencing data of 152 germplasm resources. The 152 chestnut resources are rich in types, covering the main ecological types and agronomic traits of chestnuts currently available on the market, and can represent the germplasm as much as possible, with high genetic diversity. Subsequently, 48 pairs of SSR primer molecular markers with good polymorphism were obtained by polyacrylamide gel electrophoresis screening, forming a SSR marker combination. The SSR marker combination covers 12 chromosomes of Castanea plants.

[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 pair of primers in the SSR marker combination is respectively modified with fluorescence, and the genomic DNA of 152 test chestnut varieties is respectively used as a template to perform PCR amplification using 48 primer groups, to obtain PCR amplification products. The PCR amplification reaction system uses 15 μL: 7.5 μL of 2x TSINGKE Master Mix (Tsingke Biological Company), 1 μL of 50 ng / μL DNA template, 0.3 μL of 10 μM / μL fluorescence-modified forward primer, 0.3 μL of 10 μM / μL reverse primer, and 5.9 μL of ddH2O to make up 15 μL of the system. The PCR amplification reaction program is: 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, a total of 35 cycles; 72°C for 10 min for sufficient extension, and 4°C for storage.

[0053] The PCR amplification products are diluted to a concentration of 1 ng / μL, and the PCR amplification products are subjected to fluorescence capillary detection analysis using a full-automatic capillary electrophoresis instrument. The capillary electrophoresis detection results of the 152 test chestnut varieties are analyzed by clustering based on the genotypes of the test chestnut varieties at 48 SSR loci using MEGA7 software. The clustering analysis of the 152 test chestnut varieties based on the 48 primer groups is shown in Figure 1 The clustering analysis results show that the 48 primer groups can completely distinguish the 152 test chestnut varieties and objectively reflect the genetic relationship between the varieties. The genetic diversity based on the 48 primer groups (Table 3) and the genetic diversity based on the 152 genetic groups (Table 4) show that the SSR marker combination provided by the present application 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 groups

[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 the population size, Na represents the observed number of alleles, Ne represents the effective number of alleles, I represents the Shannon's information index, Ho represents the observed heterozygosity, and He represents the expected heterozygosity.

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

[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 the 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 identifying Chinese chestnut varieties by SSR molecular marker primer combination

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

[0062] According to the description in Example 1, the genomic DNA of the to-be-tested Chinese chestnut variety is extracted by the improved CTAB method, and the PCR product of the to-be-tested Chinese chestnut variety is 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 to-be-tested Chinese chestnut variety are recorded. If there is only one allelic variation at a certain site, and the size is 100 bp, then the genotype of the main allelic variation at this site is recorded as 100 / 100. If there are two allelic variations at a certain site, and the sizes are 150 bp and 100 bp, respectively, then the genotype of the main allelic variation at this site is recorded as 150 / 100.

[0063] The 48 SSR site data of the to-be-tested Chinese chestnut variety are obtained respectively. If the difference site number between the to-be-tested Chinese chestnut variety and a certain tested Chinese chestnut variety is 0-2, then the to-be-tested Chinese chestnut variety and the tested Chinese chestnut variety are determined as similar Chinese chestnut varieties. If the difference site number between the to-be-tested Chinese chestnut variety and the tested Chinese chestnut variety is more than 2, then the to-be-tested Chinese chestnut variety does not belong to any of the 152 tested Chinese chestnut varieties.

[0064] (2) Detecting whether two unknown to-be-tested Chinese chestnut varieties are the same variety

[0065] According to the description in Example 1, the genomic DNA of the two to-be-tested Chinese chestnut varieties is extracted by the improved CTAB method, and the PCR product of the two to-be-tested Chinese chestnut varieties is 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 to-be-tested Chinese chestnut varieties are recorded. If there is only one allelic variation at a certain site, and the size is 100 bp, then the genotype of the main allelic variation at this site is recorded as 100 / 100. If there are two allelic variations at a certain site, and the sizes are 150 bp and 100 bp, respectively, then the genotype of the main allelic variation at this site is recorded as 150 / 100.

[0066] The 48 SSR site data of the two to-be-tested Chinese chestnut varieties are obtained respectively. If the difference site number between the two to-be-tested Chinese chestnut varieties is more than 2 at the above-mentioned 48 SSR sites, then the two to-be-tested Chinese chestnut varieties are determined as different Chinese chestnut varieties. The more the difference site number is, the farther the genetic kinship is. If the difference site number between the two to-be-tested Chinese chestnut varieties is 0-2 at the above-mentioned 48 SSR sites, then the two to-be-tested Chinese chestnut varieties are determined as similar Chinese chestnut varieties.

[0067] The above descriptions are only the preferred embodiments of the present application, and are not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modifications, equivalent replacements, improvements, 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 chestnut varieties, characterized in that: The SSR molecular marker primer combination covers 48 SSR sites on 12 chromosomes of chestnut varieties and includes 48 pairs of primers. The nucleotide sequences of the 48 pairs of primers are shown in SEQ ID NO.1 to SEQ ID NO.

96.

2. 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.

3. A method for chestnut variety identification using the SSR molecular marker primer combination according to claim 1, characterized in that: The steps include: (1) Extracting genomic DNA of chestnut samples to be tested, fluorescently modifying the forward primers of each primer pair in the SSR molecular marker primer combination of claim 1, and using the extracted DNA as a template, performing PCR amplification using the fluorescently modified forward primers and the corresponding non-fluorescently modified reverse primers; (2) The obtained PCR products were mixed in groups and then subjected to capillary electrophoresis for mixed testing. The capillary electrophoresis results were analyzed to identify the chestnut varieties.

4. The method according to claim 3, characterized in that The fluorescent modification uses ROX, TAMRA, FAM or HEX fluorescent molecules.

5. The method according to claim 3, 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 chestnut variety based on the 48 SSR loci is 0-2, then the chestnut to be tested and the variety are determined to be similar varieties; If the number of different sites between the genotype of the chestnut to be tested based on the 48 SSR sites and the genotype of a certain chestnut variety based on the 48 SSR sites is greater than 2, the chestnut to be tested and the variety are determined to be different varieties.

6. The method according to claim 5, 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 the primer groups in the SSR marker combination are used for PCR amplification 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.

7. The method according to claim 3, characterized in that The chestnut varieties tested were 152, including: 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 early Shuo, 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, Pasi 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 early, August red, June storm, Zhejiang early 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 species, Tancheng 207, Xiangli No. 2, Zhuji hairy chestnut, Qingmao soft thorn, Dushan Xiushi Sheng descendants , Da Li Qing, Huawan No. 1, Huawan No. 2, Zunda, Hongli No. 6, Fu 3, Kaihua double-season chestnut, Hubei Agricultural University No. 1, Yang La Li, Tie Li Tou, Dongshan Early Chestnut, Jingshu Red, Xiang Li Zi, Ming Li, July Red, August Explosion, Wushan No. 3, Shao Li No. 18, Zhuanqiao Dahongpao, Jinhua No. 1, Pu 279-Big Oil Chestnut, Jurong Short Thorn, Lu 35, Stable Yield Jiao Zha, Xigou No. 2, Short Thorn Branch Red, Yan Kui, Bai Mao Zao, Zhashui Dahongpao, Ma Chi Qing, Queshan No. 10, Yuanling No. 2, Xinhang Late Chestnut, Deep Thorn Ershui Early, Jingxian Big Chestnut, Lu 18, White Hair Chestnut, Shallow Thorn Big Chestnut, He Feidahongpao, Shuyang oil chestnut, Tancheng No. 3, Wushan No. 2, orange 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, small osmanthus fragrance, early fragrance 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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