CN113493851B

CN113493851B - Application of 32 soybean InDel markers in detection of soybean genetic diversity

Info

Publication number: CN113493851B
Application number: CN202010195219.7A
Authority: CN
Inventors: 闫龙; 李曼; 张洁; 杨春燕; 邸锐; 史晓蕾; 刘兵强; 娜仁; 赵青松; 秦君; 王冬梅; 张孟臣
Original assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Current assignee: Institute of Grain and Oil Crops of Hebei Academy of Agriculture and Forestry Sciences
Priority date: 2020-03-19
Filing date: 2020-03-19
Publication date: 2022-07-12
Anticipated expiration: 2040-03-19
Also published as: CN113493851A

Abstract

The invention discloses application of 32 soybean InDel markers in detection of soybean genetic diversity. The 32 soybean InDel disclosed by the invention are marked as 32 DNA molecules obtained by carrying out PCR amplification on soybeans by using 32 pairs of primers shown in sequences 1-64 in a sequence table. The 32 soybean InDel markers can be used for detecting the genetic diversity of soybeans, identifying soybean varieties or strains or developing molecular markers for identifying the soybean varieties or strains, preparing a soybean genetic map, analyzing the genetic structure of the soybeans and further breeding the soybeans. The 32 soybean InDel markers and the complete set of primers for detecting the 32 soybean InDel markers have wide research prospects.

Description

Application of 32 soybean InDel markers in detection of soybean genetic diversity

Technical Field

The invention relates to the application of 32 soybean InDel markers in detecting soybean genetic diversity in the field of biotechnology.

Background

Soybean (Glycine max (L.) Merrill) originated in china, is a dicotyledonous herbaceous plant evolved by wild variety cultivation, and belongs to leguminosae (Leguminsae), subfamily oroxyli (Papiliontae) and Glycine (Glycine). The soybean provides 1/3 vegetable protein and vegetable oil sources for human beings, and is one of five crops in China. Soybean is a crop related to the basic, economic and strategic nature of the livelihood, and the industry of soybean has great development potential and plays a very important role in agricultural product trade. In recent years, with the adjustment of the reform of the supply side of the national ministry of agriculture, the soybean breeding system has been diversified. The market of the soybean industry in China is very active, a relatively perfect soybean breeding system is established, and a foundation is laid for the development of the soybean industry; the initial scale of soybean seed enterprises accelerates the cultivation and popularization of new varieties; the high-price transfer of the variety rights is also realized when breeding varieties (such as black river 43, zihuang 34 and the like), and the application of the soybean varieties is further accelerated. However, the development of the soybean industry in our country has certain problems: the seed inspection system is imperfect, and the seed purity is low; the market management of the seed industry is not standard, the market of partial regions is disordered, and the phenomena of 'same name and different species' and 'same name and different name' of soybean seeds occur occasionally. In the process of storing the crop germplasm resources, the problem of the same name of materials is a difficult problem in each country. The problem is solved, so that convenience is provided for the research of germplasm resources, and the loss of germplasm resources during storage is reduced.

Disclosure of Invention

The invention aims to solve the technical problem of how to detect the genetic diversity of soybeans.

In order to solve the technical problems, the invention firstly provides application of 32 soybean InDel marks or substances for detecting the 32 soybean InDel marks in detection or auxiliary detection of soybean genetic diversity;

the names of the 32 soybean InDel marks are InDel-10-5, InDel-10-6, InDel-12-4, InDel-12-7, InDel-13-1, InDel-13-4, InDel-14-1, InDel-15-4, InDel-16-3, InDel-1-7, InDel-17-1, InDel-1-8, InDel-18-3, InDel-19-4, InDel-19-8, InDel-20-1, InDel-2-1, InDel-3-4, InDel-3-6, InDel-4-4, InDel-4-6, InDel-5-2, InDel-5-5, InDel-6-1, InDel-7-3, InDel-7-8, InDel-8-1, InDel-9-4, InDel-9-8, InDel-11-6, InDel-2-8 and InDel-6-4;

the InDel-10-5 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-10-5-P; the InDel-10-5-P consists of two single-stranded DNAs shown as

sequences

1 and 2 in a sequence table;

the InDel-10-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-10-6-P; the InDel-10-6-P consists of two single-stranded DNAs shown as

sequences

3 and 4 in a sequence table;

the InDel-12-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-12-4-P; the InDel-12-4-P consists of two single-stranded DNAs shown as

sequences

5 and 6 in a sequence table;

the InDel-12-7 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-12-7-P; the InDel-12-7-P consists of two single-stranded DNAs shown as

sequences

7 and 8 in a sequence table;

the InDel-13-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-13-1-P; the InDel-13-1-P consists of two single-stranded DNAs shown as

sequences

9 and 10 in a sequence table;

the InDel-13-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-13-4-P; the InDel-13-4-P consists of two single-stranded DNAs shown as

sequences

11 and 12 in a sequence table;

the InDel-14-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-14-1-P; the InDel-14-1-P consists of two single-stranded DNAs shown as

sequences

13 and 14 in a sequence table;

the InDel-15-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-15-4-P; the InDel-15-4-P consists of two single-stranded DNAs shown as

sequences

15 and 16 in a sequence table;

the InDel-16-3 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-16-3-P; the InDel-16-3-P consists of two single-stranded DNAs shown as

sequences

17 and 18 in a sequence table;

the InDel-1-7 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-1-7-P; the InDel-1-7-P consists of two single-stranded DNAs shown as

sequences

19 and 20 in a sequence table;

the InDel-17-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-17-1-P; the InDel-17-1-P consists of two single-stranded DNAs shown as

sequences

21 and 22 in a sequence table;

the InDel-1-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-1-8-P; the InDel-1-8-P consists of two single-stranded DNAs shown as

sequences

23 and 24 in a sequence table;

the InDel-18-3 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-18-3-P; the InDel-18-3-P consists of two single-stranded DNAs shown as

sequences

25 and 26 in a sequence table;

the InDel-19-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-19-4-P; the InDel-19-4-P consists of two single-stranded DNAs shown as

sequences

27 and 28 in a sequence table;

the InDel-19-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-19-8-P; the InDel-19-8-P consists of two single-stranded DNAs shown as

sequences

29 and 30 in a sequence table;

the InDel-20-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-20-1-P; the InDel-20-1-P consists of two single-stranded DNAs shown as

sequences

31 and 32 in a sequence table;

the InDel-2-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-2-1-P; the InDel-2-1-P consists of two single-stranded DNAs shown as

sequences

33 and 34 in a sequence table;

the InDel-3-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-3-4-P; the InDel-3-4-P consists of two single-stranded DNAs shown as

sequences

35 and 36 in a sequence table;

the InDel-3-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-3-6-P; the InDel-3-6-P consists of two single-stranded DNAs shown as

sequences

37 and 38 in a sequence table;

the InDel-4-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-4-4-P; the InDel-4-4-P consists of two single-stranded DNAs shown as

sequences

39 and 40 in a sequence table;

the InDel-4-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-4-6-P; the InDel-4-6-P consists of two single-stranded DNAs shown as

sequences

41 and 42 in a sequence table;

the InDel-5-2 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-5-2-P; the InDel-5-2-P consists of two single-stranded DNAs shown as

sequences

43 and 44 in a sequence table;

the InDel-5-5 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-5-5-P; the InDel-5-5-P consists of two single-stranded DNAs shown as sequences 45 and 46 in a sequence table;

the InDel-6-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-6-1-P; the InDel-6-1-P consists of two single-stranded DNAs shown as sequences 47 and 48 in a sequence table;

the InDel-7-3 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-7-3-P; the InDel-7-3-P consists of two single-stranded DNAs shown as sequences 49 and 50 in a sequence table;

the InDel-7-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-7-8-P; the InDel-7-8-P consists of two single-stranded DNAs shown as sequences 51 and 52 in a sequence table;

the InDel-8-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-8-1-P; the InDel-8-1-P consists of two single-stranded DNAs shown as

sequences

53 and 54 in a sequence table;

the InDel-9-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-9-4-P; the InDel-9-4-P consists of two single-stranded DNAs shown as sequences 55 and 56 in a sequence table;

the InDel-9-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-9-8-P; the InDel-9-8-P consists of two single-stranded DNAs shown as sequences 57 and 58 in a sequence table;

the InDel-11-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-11-6-P; the InDel-11-6-P consists of two single-stranded DNAs shown as

sequences

59 and 60 in a sequence table;

the InDel-2-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-2-8-P; the InDel-2-8-P consists of two single-stranded DNAs shown as

sequences

61 and 62 in a sequence table;

the InDel-6-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-6-4-P; the InDel-6-4-P consists of two single-stranded DNAs shown as sequences 63 and 64 in a sequence table.

The invention also provides application of the 32 soybean InDel markers or substances for detecting the 32 soybean InDel markers in identification or auxiliary identification of soybean varieties or strains.

The invention also provides application of the 32 soybean InDel markers or substances for detecting the 32 soybean InDel markers in developing molecular markers for identifying or assisting in identifying soybean varieties or strains.

The invention also provides application of the 32 soybean InDel markers or substances for detecting the 32 soybean InDel markers in preparation of a soybean genetic map.

The method for preparing the soybean genetic map may comprise: and detecting the 32 soybean InDel markers in the soybean to be detected, and preparing a genetic map of the soybean to be detected according to the 32 soybean InDel markers.

The invention also provides application of the 32 soybean InDel markers or substances for detecting the 32 soybean InDel markers in soybean breeding.

As above, the substance for detecting the 32 soybean InDel markers comprises a primer set, and the primer set consists of 64 single-stranded DNAs shown in sequences 1-64 in a sequence table.

The substance for detecting the 32 soybean InDel markers can also comprise other reagents except primers required for PCR amplification.

The substance for detecting the 32 soybean InDel markers can be composed of the set of primers and also can be composed of the set of primers and other reagents except the primers required for PCR amplification.

The primer set can be any one of the following primers:

x1, detecting or assisting in detecting soybean genetic diversity;

x2, identification or assisted identification of soybean varieties or lines;

x3, developing a molecular marker for identifying or assisting in identifying the soybean variety or strain;

x4, preparing a soybean genetic map;

x5, soybean breeding.

The substance for detecting the 32 soybean InDel markers also belongs to the protection scope of the invention.

The 32 soybean InDel marks also belong to the protection scope of the invention.

In the invention, the soybean variety or strain to be detected can be Ji bean 12, Zhonghuang 42, Xu bean 16, Qihuang 34, Jiyu 101, Hobbit, HJ117, Zheng 196, ZYD2738, Ji bean 17, Zhonghuang 13 and/or Suiyong 14 and/or any soybean variety or strain from Table 5.

The concentration of the primers in the system for PCR amplification by using the primer pairs in the primer set can be 0.5 mu mol/L. The specific systems for PCR amplification by using the primer pairs in the primer set can be as follows:

genomic DNA (20 ng/. mu.L)^-1)	1μL
		2×Es Taq MasterMix	10μL
Upstream primer (10. mu. mol/L)	1μL
		Downstream primer (10. mu. mol/L)	1μL
ddH₂O	7μL

Wherein, the single-stranded DNA (upstream primer) shown in the sequence 2n-1 and the single-stranded DNA (downstream primer) shown in the sequence 2n form a primer pair, and n is a natural number between 1 and 32. 2 × Es Taq MasterMix is Beijing kang, a product of century Biotechnology Ltd.

The annealing temperature for PCR amplification using the primer pair in the primer set may be 55 ℃. Specifically, the reaction conditions for performing PCR amplification using the primer pair in the set of primers may be: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 34 cycles; final extension at 72 ℃ for 10 min.

The invention obtains 32 soybean InDel marks, can detect the genetic diversity of soybeans by utilizing the 32 soybean InDel marks, can identify soybean varieties or strains or develop molecular marks for identifying the soybean varieties or strains, can also prepare a soybean genetic map, can also be used for analyzing the genetic structure of the soybeans, and further can be used for soybean breeding. The 32 soybean InDel markers and the complete set of primers for detecting the 32 soybean InDel markers have wide research prospects.

Drawings

FIG. 1 shows the result of agarose gel electrophoresis of a part of the PCR product. M is Marker, 1-12 are Ji bean 17, Ji bean 12, Suizong 14, Zhonghuang 13, HJ117, Hobbit, Jiyun 101, Qihuang 34, Xudou 16, ZYD2738, Zheng 196 and Zhonghuang 42; A-J are PCR products of primers InDel-4-6, InDel-5-5, InDel-6-1, InDel-8-1, InDel-8-2, InDel-10-5, InDel-13-4, InDel-16-3, InDel-19-8 and InDel-20-8, respectively.

FIG. 2 shows the result of agarose gel electrophoresis of a part of the PCR product. Lane M, DNA Marker, lanes 1-12 Ji Dou 17, Ji Dou 12, Suiyong 14, Zhonghuang 13, HJ117, Hobbit, Jiyu 101, Qihuang 34, Xu Dou 16, ZYD2738, Zheng 196, Zhonghuang 42; fig. (1) to (16) are respectively: InDel-1-7, InDel-1-8, InDel-4-6, InDel-2-1, InDel-5-2, InDel-2-8, InDel-6-4, InDel-3-4, InDel-6-7, InDel-3-6, InDel-7-3, InDel-4-4, InDel-8-1, InDel-5-5, InDel-6-1, InDel-7-4.

FIG. 3 shows the result of agarose gel electrophoresis of a part of the PCR product. Lane M is DNA Marker, lanes 1-12 are Ji beans 17, Ji beans 12, Suizong 14, Zhonghuang 13, HJ117, Hobbit, Jiyu 101, Qihuang 34, Xudou 16, ZYD2738, Zheng 196, Zhonghuang 42, respectively; fig. (17) to (32) are respectively: InDel-10-5, InDel-7-8, InDel-11-7, InDel-8-2, InDel-12-7, InDel-9-4, InDel-13-1, InDel-9-8, InDel-13-2, InDel-10-6, InDel-14-1, InDel-11-4, InDel-14-3, InDel-11-6, InDel-16-2, and InDel-12-4.

FIG. 4 shows the result of agarose gel electrophoresis of a part of the PCR product. Lane M, DNA Marker, lanes 1-12 Ji Dou 17, Ji Dou 12, Suiyong 14, Zhonghuang 13, HJ117, Hobbit, Jiyu 101, Qihuang 34, Xu Dou 16, ZYD2738, Zheng 196, Zhonghuang 42; fig. (33) to (43) are respectively: InDel-16-3, InDel-13-4, InDel-16-5, InDel-15-4, InDel-16-7, InDel-19-4, InDel-17-1, InDel-19-8, InDel-18-3, InDel-18-5 and InDel-20-1.

FIG. 5 is a graph of genetic distance cluster analysis of soybean material. Note: pop1 is north group material, pop2 is middle group material, and pop3 is south group material.

Fig. 6 is an optimal cluster partitioning for inferring 96 soybean germplasms using Δ K.

FIG. 7 is the genetic structure of 96 soybean germplasm. The abscissa indicates the soybean number, and the soybean names indicated by the numbers are shown in Table 5.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The soybeans in the following examples, and the references described below, are publicly available to the applicant as such biomaterials which are only used for repeating the experiments relevant to the present invention, and are not useful for other purposes:

ji bean 12: zhang Yue Ying, Liu Bing Qiang, Liu Liwei, Yan Long Ji Dou 12 genetic map initially constructs [ J ]. North China agronomy report, 2009,24(02): 60-63.

Medium yellow 42: zhanjian Jun Qing, Zhongguo Zhang, Kongguo, high protein soybean 42 output expression and cultivation technology [ J ] soybean science and technology, 2011(01): 67-68.

Xu Dou 16: wangxiang, Wangzong Biao, Xuzhenju, a breeding and cultivation technique of a new soybean variety Xudou 16 [ J ] soybean science and technology, 2011(06): 64-65.

And (3) full yellow 34: influence of Zhang Mei, Liu Yu celery, Slow sowing time and Density on the yield of Zebra 34 in Soybean variety [ J ]. J.crops, 2016(02): 100-.

Jiyu 101: yangguanyu, Wangyang, Maxianpan, Jifeng, Yangchuning, Hujinhai, xuming, Gashuqin, Wang Yingman, high protein soybean new variety Jiyu No. 101 breeding and cultivating technology [ J ] soybean reporting, 2008(02): 45-46.

Hobbit: huxiping, xue Yong, Yuanping, Zhao hai hong, utilization of the U.S. excellent soybean germplasm Hobbit [ J ]. Black Longjiang agricultural science, 2008(03): 25-27.

HJ 117: guo Jingwen, Shixiao bud, Liu xi, Zhao Qing Song, manxue, Liu Bing Qiang, Yan Long, Wang Fengmen, Zhang Meng, Zhao Bao Hua, Yanchun Swallow.

Zheng 196: liu Rui Min, Wang Lu, Li Jimin, Liu Qing Rui, Xia seeding soybean Zheng 196 best seeding period research [ J ] modern agricultural science, 2018(07): 5-7.

ZYD2638: Yan long soybean interspecific cross (Glycine max × g. soja) progeny grain trait QTL location [ D ]. chinese academy of agricultural sciences, 2012.

Jidou 17: genetic basis analysis and important protein mining of Ji beans 17 and Jinf 58 of Huang Huai Hai soybean Wang Jing Huai Hai soybean [ D ]. university of northriver Shi 2015.

Medium yellow 13: wanglian, Zhao Rongjuan, a new high-yield soybean variety-Zhonghuang 13[ J ] agricultural science and technology communication, 2005(06) 40.

The seinhong 14: payashi, the breeding and experience analysis of soybean variety Suiyong 14 (J), Heilongjiang agricultural science, 2002(03), 47-48.

Andou 6023, Handan 15-685, Handan 13-109, Ji 1701, Andou 109, jin family 3, Handan bean 5: the cause of Muxian tree, Lijiaming, Lide mouiao, Lifuqin, Zhangjun, Liu Meng, Xiaojin Ping, Zhang Pu, Huang Huai Hai Xia Soy Tian's disease Qing' is detected and prevented [ J ]. Soy science, 2019,38(05): 770-.

Roc 1408, quotient bean 161, cang bean 11, ru0126: yanwei, Liu national peak, Liwei, Zhang Feng, Daihei Ying, Lin Yan Hui, Wang Cao Jie, Xuran.

Medium crop 11-11, fenbean 103, fenbean 104: SSR marker diversity analysis of newly-bred soybean strains in Huang-Huai-Hai region [ J ] soybean science, 2018,37(02):173 + 178).

Shan Dou No. 1, shan Dou No. 2: the cause of Muxian tree, Lijiaming, Lide mouiao, Lifuqin, Zhangjun, Liu Meng, Xiaojin Ping, Zhang Pu, Huang Huai Hai Xia Soy Tian's disease Qing' is detected and prevented [ J ]. Soy science, 2019,38(05): 770-.

Or 1330: soybean line identification test in Huang-Huai-Hai region of Queen-Huang-Huai-Hai region, soybean science and technology, 2017(01), 3-10.

Andou 6223, Neudou 44, Loudou 4904, Wenk 12102-4, Sheng Dou 103, Zhongshou 11-58, Zhongshou 12-1251, Zhongshou 11-518, Lu Lh284, Ji 1702, Ji 1707, Ji 1708, Handan 16-139, Zhongshou 16067, Zhongshou 1184, Zhongshou J161087, Kedou 26, Candou 1123, Shi 12503, Luzhou 7901, Shengyu 6, Huayun No. 2, Daqiu No. 6, Pan bean 13, Zhonghuang 1312 104, Qinqu 2017, Qinqu 220, Zhongdou 11-719, HN1030, Wan bean 5451, Andou 331, Kedou 31, Zhongshou 08-08, Weiju bean 1897, Shi 11893, Huadou 30, Huaju 21, Huadou 16S379, Kedou 18, Zheng Dou 1677, 0540-4, Handan 617, Lulu bean 617, Zhongju 38105, Zhonghua bean 617, Zhonghua Handan 15, Zhonghua bean 3815, Zhonghua Ju 3815: and in 2018, the identification of important traits of the improved variety attacking soybean of the national soybean, namely the identification of soybean root rot resistance.

Commercial bean 1310: he Xin, Yan onwards, Sun Huajun, New Soybean variety, commercial Bean 1310, was analyzed for productivity, stability and suitability [ J ] Soybean science, 2017(05): 41-49.

Wei bean 12: the distribution of excellent allelic variation of the resistance sites Rhg1 and Rhg4 of the Gliocladium crassifolium Linne in Huanghuai bred varieties [ J ] is the distribution of the excellent allelic variation of the Gliocladium crassifolium Linne, the Lihai dynasty, the Lijinying, the Kingjin society, the Weihe, the Lecheng Fang, the Wuyongkang and the Luo.

Cang bean 09Y 1: cangzhou city academy of agriculture and forestry (Shi 7631X Shi Dou 4).

(1) Expected heterozygogous number: heterozygosity, i.e., gene diversity, abbreviated He, is desired.

He-1- Σ Pi2, where Pi is the frequency of the ith allele in the soybean population, the expected heterozygosity for each site was calculated and averaged to represent the expected heterozygosity for that population.

(2) Nei diversity index: index of Nei diversity.

The Nei diversity index is nHe/(n-1), n is the number of samples, and He is the desired heterozygosity for the population.

(3) Shannon Wiener index: shannon index, abbreviated as SHI.

The SHI of each locus is calculated and the average value represents the SHI of the group.

(4) Polymorphism information content: polymorphism information content, abbreviated as PIC.

Pi and Pj are the frequency of the ith and jth alleles, respectively, in the soybean population, and n is the allele number.

Example 1 use of Soybean marker combinations to detect genetic diversity in Soybean

This example provides a set of soybean InDel marker combinations consisting of 32 insertion/deletion markers (InDel markers) that reflect their genetic diversity in 96 parts of soybean material.

Discovery of soybean InDel marker

The inventors of the present invention performed whole genome re-sequencing of 12 parts of soybean material using Illumia HiSeqTM 2500: extracting plant material DNA, fragmenting the plant material DNA qualified for quality inspection by using ultrasonic waves, then carrying out fragment purification, end repairing, adding A at the 3' end and linking a sequencing joint on the fragmented DNA, selecting the length of a fragment by using agarose gel electrophoresis, carrying out PCR amplification to establish a sequencing library, and sequencing the library qualified for quality inspection by using Illumina HiSeqTM 2500. And (4) performing quality evaluation on the original reads obtained by sequencing, and filtering to obtain Clean reads for subsequent genome sequence assembly.

In sequencing data, the percentage of bases with correct recognition rate higher than Q20 (99.9%) was 97.50%, the percentage of bases with correct recognition rate higher than Q30 (99.9%) was 93.48%, the average of GC content in genome was 36.23%, the average re-sequencing coverage of the sample was 98.70%, and the average sequencing depth was 18.73X. In the sequencing result, the GC content distribution is normal, the sample sequencing coverage rate is high, the sequencing quality is qualified, and the method can be used for subsequent analysis.

The 12 parts of soybean material are: ji bean 12, Zhonghuang 42, Xu bean 16, Qihuang 34, Jiyu 101, Hobbit, HJ117, Zheng 196, ZYD2738, Ji bean 17, Zhonghuang 13 and Suiyong 14.

Performing mutation detection according to the positioning result of Clean reads of 12 parts of soybean materials in a soybean reference genome Glyma.Wm82.a2v1, and detecting two main mutation types of SNP and InDel. The average number of SNPs detected in 12 materials was 1488416, and the average number of InDel detected was 294560.

By alignment with soybean reference genome Glyma.Wm82.a2v1, the number of InDel markers with insertion/deletion fragment lengths of more than 20bp (including 20bp) was detected as 66561, and the number of InDel markers per chromosome was 3262 on average, wherein the number of InDel markers distributed on chromosome 18 was the largest, the number thereof was 4521, and the number of InDel markers distributed on chromosome 12 was the smallest, and the number thereof was 2019.

Of 66561 InDel markers detected, the number of InDel markers located at the intron was 8222 (12.35%), the number of InDel markers located at the upstream and downstream sequences of the gene were 17190 (25.83%) and 13602 (20.44%), respectively, the number of InDel markers located at the intergenic region was 22893 (34.39%), the number of InDel markers located between the genes was 125 (0.19%), and the number of InDel markers located at the 3'-UTR and 5' -UTR were 1004 (1.51%) and 617 (0.93%), respectively.

The average length of 66561 detected InDel marker insertion/deletion fragments is 41bp, the number of InDel markers with the fragment length size between 20 and 40bp is 42453, the number of InDel markers with the fragment length size between 41 and 60bp is 13044, the number of InDel markers with the fragment length size between 61 and 80bp is 5034, the number of InDel markers with the fragment length size between 81 and 100bp is 2285, and the number of InDel markers with the fragment length size more than 100bp is 2413.

66561 InDel-labeled PIC, Nei diversity index, Shannon index in these 12 soybean materials were analyzed using the POPGENE software, with PIC ranging from 0.067-0.375, Nei diversity index ranging from 0.710-0.667, and Shannon index ranging from 0.154-0.693. The PIC concentrated distribution range is 0.100-0.200 and 0.300-0.400, and the proportion is 35.58% and 33.01% respectively; the centralized distribution range of the Nei diversity index is 0.100-0.200, and the proportion is 33.56%; the concentration distribution range of the shannon index is 0.200-0.300 and 0.600-0.700, and the proportion is 29.30% and 24.09% respectively.

Clean reads were compared to the reference genomic sequence (glyma. wm82.a2v1) and the InDel markers were detected using GATK software with the following criteria:

(1) selecting a site with an insertion/deletion fragment larger than 20 bp;

(2) there were a maximum of 2 allelic variations per site.

160 long fragment insertion/deletion sites were selected, 160 of which satisfied the following condition:

(1) 8 pairs of evenly distributed loci are selected on each chromosome of the soybean;

(2) the length of the insertion/deletion fragment is more than 40bp (including 40 bp);

(3) each site has and only has two allelic variations;

(4) and selecting the sites with the polymorphism information content of more than 0.25.

Primers are designed on the upstream and downstream of the 160 InDel marked sites, and 160 pairs of primers are obtained.

Of these 160 primers, 51 primers failed to produce a band from each of the 12 soybean material parts at an annealing temperature of 55 ℃ and 109 primers stably produced a band from each of the 12 soybean material parts. Among 109 pairs of primers which stably amplified bands, 43 pairs of primers have clear amplification bands, the insertion/deletion type of the fragment can be distinguished by naked eyes, 66 pairs of primer amplification bands can not distinguish the insertion/deletion type of the fragment easily, the detailed information of 43 InDel markers amplified by 43 pairs of primers is shown in Table 1, and the primer information is shown in Table 2. The results of agarose gel electrophoresis detection of a portion of the InDel labeled primers are shown in FIG. 1.

TABLE 1 soybean InDel marker combination information

InDel name	Chromosome	Position of	InDel name	Chromosome	Position of
						InDel-1-7	1	38601796	InDel-11-4	11	24374193
InDel-1-8	1	50513481	InDel-11-6	11	15581830
						InDel-2-1	2	22242181	InDel-12-4	12	17649495
InDel-2-8	2	2237743	InDel-12-7	12	11949731
						InDel-3-4	3	30874756	InDel-13-1	13	31358622
InDel-3-6	3	21939196	InDel-13-2	13	16930542
						InDel-4-4	4	48358162	InDel-13-4	13	37748838
InDel-4-6	4	45230392	InDel-14-1	14	4424007
						InDel-5-2	5	34356071	InDel-14-3	14	43544210
InDel-5-5	5	39857792	InDel-15-4	15	15360421
						InDel-6-1	6	20039692	InDel-16-2	16	25311303
InDel-6-4	6	10896943	InDel-16-3	16	156095
						InDel-6-7	6	48773671	InDel-16-5	16	31016663
InDel-7-3	7	17704210	InDel-16-7	16	29753588
						InDel-7-4	7	7458176	InDel-17-1	17	39593964
InDel-7-8	7	37866069	InDel-18-3	18	51025175
						InDel-8-1	8	47711883	InDel-18-5	18	8476744
InDel-8-2	8	9409779	InDel-19-4	19	41727589
						InDel-9-4	9	49917537	InDel-19-8	19	8157745
InDel-9-8	9	29913558	InDel-20-1	20	36027621
						InDel-10-5	10	49646363	InDel-20-8	20	37390649
InDel-10-6	10	11287611

TABLE 2 primer information for amplification of 43 soybean InDel markers

The system for performing PCR amplification of genomic DNAs of 12 parts of soybean material using the 43 pairs of primers was as follows:

wherein 2 × Es Taq MasterMix is Beijing kang, a product of century Biotechnology Ltd.

The PCR amplification conditions were: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 34 cycles; final extension at 72 ℃ for 10 min.

The PCR products of the 43 pairs of primers were detected by 1.0% agarose gel electrophoresis, 50bp ladder was used as the quality standard for PCR amplification products, and the products were subjected to 120V electrophoresis for 35min and observed under a gel imager. The results show that each primer can obtain PCR products with clear bands, and the electrophoresis detection images of 43 pairs of stably amplified primers are shown in FIGS. 2-4.

The amplification results of the 43 InDel markers in 12 test materials were analyzed by Popgene32 software, and the genetic diversity index reflected by each pair of primers was calculated using different sizes of PCR products of each InDel marker as alleles, and the PIC of each InDel marker is shown in Table 3.

TABLE 3 PIC of 43 InDel flags

InDel name	PIC	InDel name	PIC	InDel name	PIC	InDel name	PIC
								InDel-1-7	0.3538	InDel-6-4	0.3411	InDel-11-4	0.3538	InDel-16-3	0.3538
InDel-1-8	0.3047	InDel-6-7	0.3249	InDel-11-6	0.2149	InDel-16-5	0.3538
								InDel-2-1	0.3249	InDel-7-3	0.3633	InDel-12-4	0.3750	InDel-16-7	0.3249
InDel-2-8	0.3698	InDel-7-4	0.3750	InDel-12-7	0.3249	InDel-17-1	0.2149
								InDel-3-4	0.2800	InDel-7-8	0.3633	InDel-13-1	0.3249	InDel-18-3	0.3538
InDel-3-6	0.3698	InDel-8-1	0.2149	InDel-13-2	0.2800	InDel-18-5	0.3047
								InDel-4-4	0.2800	InDel-8-2	0.3750	InDel-13-4	0.3538	InDel-19-4	0.2149
InDel-4-6	0.3411	InDel-9-4	0.2800	InDel-14-1	0.3750	InDel-19-8	0.2503
								InDel-5-2	0.3698	InDel-9-8	0.2800	InDel-14-3	0.3249	InDel-20-1	0.3047
InDel-5-5	0.3698	InDel-10-5	0.3698	InDel-15-4	0.3698	InDel-20-8	0.2149
								InDel-6-1	0.3538	InDel-10-6	0.3538	InDel-16-2	0.3538

Two, 32 InDel markers analysis of genetic diversity

32 InDel-1-7, InDel-1-8, InDel-2-1, InDel-2-8, InDel-3-4, InDel-3-6, InDel-4-4, InDel-4-6, InDel-5-2, InDel-5-5, InDel-6-1, InDel-6-4, InDel-7-3, InDel-7-8, InDel-8-1, InDel-9-4, InDel-9-8, InDel-10-5, InDel-10-6, InDel-11-6, InDel-12-4, InDel-12-7, InDel-13-1, InDel-13-4, and InDel-14-1 are selected from 43 InDel markers in the first step, InDel-15-4, InDel-17-1, InDel-16-3, InDel-18-3, InDel-19-4, InDel-19-8 and InDel-20-1) to form a soybean InDel marker combination consisting of 32 InDel markers, and respectively carrying out PCR amplification on the genomic DNA of 96 other soybean materials by using the 32 pairs of primers, wherein the PCR amplification system is as follows:

TABLE 4,

Composition (I)	Dosage of
		Genomic DNA (20 ng/. mu.L)^-1)	1μL
2×Es Taq MasterMix	10μL
		Upstream primer (10. mu. mol/L)	1μL
Downstream primer (10. mu. mol/L)	1μL
		ddH₂O	7μL

The 96 parts of soybean material used were divided into three groups, north, middle and south by geographical origin, see table 5 for details.

TABLE 5, 96 parts Soybean Material information

Serial number

Name of Material

Group of

Serial number

Name of Material

Group of

Serial number

Name of Material

Group of

1

Row 1408

North group

33

Stone 11893

North group

65

Week 12102-4

Middle group

2

Row 1407

North group

34

Holy bean 102

North group

66

Commercial bean 1310

Middle group

3

Shan Dou No. 1

North group

35

Sheng Dou 103

North group

67

Commercial bean 161

Middle group

4

Chinese medicine 16067

North group

36

Hua Dou 30

North group

68

Qin bean 2017

Middle group

5

Intermediate product 1184

North group

37

Autumn 37

North group

69

Qin bean 220

Middle group

6

Selection of corridor profile 4

North group

38

Luodao 4904

Middle group

70

Zhonghuang 320 (Chinese rhubarb)

Middle group

7

Selection of corridor profile 2

North group

39

Luodan 7901

Middle group

71

Lotus bean 44

Middle group

8

HN2018

North group

40

Hua Dou 21

Middle group

72

Pudou 5136

Middle group

9

Zhongdong J161087

North group

41

Shengyu No. 5

Middle group

73

Kedou 18

Middle group

10

Kedou 26

North group

42

Shengyu No. 6

Middle group

74

Zhongdong 11-518

Middle group

11

Zhongmao 12-1251

North group

43

Huayu No. 2

Middle group

75

Zhongdong 11-719

Middle group

12

Cang beans 1123

North group

44

No. 6 Tao autumn

Middle group

76

Zheng 1677

Middle group

13

Cang Dou 1312

North group

45

Pan bean 13

Middle group

77

Zheng 16175

Middle group

14

Wing 1708

North group

46

Pan bean 19

Middle group

78

JiJ 14187

Middle group

15

Jidou 12

North group

47

Lu Lh284

Middle group

79

Fendou 104

Middle group

16

Wing 1702

North group

48

Shan Dou No. 2

Middle group

80

JiG 15073

Middle group

17

Wing 1701

North group

49

Row 1337

Middle group

81

Fendou 103

Middle group

18

Handan 16-139

North group

50

Ro 1330

Middle group

82

Yongyu No. 1

Middle group

19

Handan 15-324

North group

51

Chinese rhubarb 104

Middle group

83

Lu 0540-4

Middle group

20

River which rises in the northeastern part of Anhui Province Ke56

North group

52

Zhonghuang 105 (Chinese rhubarb)

Middle group

84

HN1030

Middle group

21

River which rises in the northeastern part of Anhui Province Ke 55

North group

53

Zhongmao 11-11

Middle group

85

Jin Ke No. 3

Middle group

22

Andou 6223

North group

54

Zhongmao 11-54

Middle group

86

Jin Huang No. 11

Middle group

23

Andou 331

North group

55

Weiqi bean 1897

Middle group

87

Jin Huang No. 12

Middle group

24

Jin Yi 59

North group

56

Stone 16S379

Middle group

88

Andou 109

Middle group

25

Jin Yi 57

North group

57

Andou 6023

Middle group

89

Wandou 920

South group

26

Kedou 31

North group

58

River which rises in the northeastern part of Anhui Province Ke 57

Middle group

90

River which rises in the northeastern part of Anhui Province Ke 58

South group

27

Zhonghuang 103

North group

59

Handan 15-685

Middle group

91

Commercial bean 18

South group

28

Zhongmao 08-08

North group

60

Handan Bean No. 5

Middle group

92

Shan Dou No. 3

South group

29

Zhongmao 11-58

North group

61

Handan 13-109

Middle group

93

Lu 0126

South group

30

Weiqi bean 1897

North group

62

Wing 1707

Middle group

94

Handan 617

South group

31

Wei bean 12

North group

63

Cang Dou 11

Middle group

95

Andou 115

South group

32

Stone 12503

North group

64

Cang Do 09Y1

Middle group

96

Andou 5451

South group

Detecting the obtained PCR product by using 1.0% agarose gel electrophoresis, taking 50bp ladder as the quality standard of the PCR amplification product, carrying out 120V electrophoresis for 35min, and observing under a gel imager. The results show that each primer can obtain a PCR product with clear bands.

The different size PCR products for each InDel marker were used as alleles and analyzed for the Nei diversity index, shannon index and PIC for 32 markers in 96 soybean material populations using Popgene32 software, with the results shown in table 6. The results show that the Nei diversity index of each InDel marker is distributed between 0.1486 and 0.4999, and the mean value is 0.3935; SHI distribution is 0.2807-0.6931, mean value is 0.5779; PIC distribution was 0.1528-0.5953 with an average value of 0.3963. The Nei diversity index, the SHI and the PIC have larger amplitude, which indicates that the 32 InDel markers can better reflect the rich genetic diversity information in 96 parts of soybean materials.

TABLE 6 Nei diversity index, SHI, PIC for 32 InDel markers

InDel tag name	Number of allelic variation	Index of Nei diversity	SHI	PIC
					InDel-1-7	3.0000	0.3457	0.5297	0.2963
InDel-1-8	3.0000	0.3592	0.5449	0.3904
					InDel-2-1	2.0000	0.3225	0.5033	0.3277
InDel-2-8	2.0000	0.2710	0.4423	0.2778
					InDel-3-4	3.0000	0.3047	0.4826	0.2863
InDel-3-6	2.0000	0.4531	0.6913	0.5099
					InDel-4-4	2.0000	0.4831	0.6455	0.4707
InDel-4-6	2.0000	0.2571	0.4253	0.2188
					InDel-5-2	3.0000	0.4831	0.6581	0.4946
InDel-5-5	2.0000	0.4808	0.6762	0.4674
					InDel-6-1	2.0000	0.3150	0.4946	0.2963
InDel-6-4	2.0000	0.3775	0.5652	0.3852
					InDel-7-3	2.0000	0.4885	0.6716	0.4824
InDel-7-8	2.0000	0.4510	0.6433	0.4373
					InDel-8-1	2.0000	0.2342	0.3966	0.1919
InDel-9-4	2.0000	0.4512	0.6435	0.4370
					InDel-9-8	2.0000	0.4349	0.6266	0.4387
InDel-10-5	2.0000	0.4999	0.6931	0.5000
					InDel-10-6	2.0000	0.4167	0.6074	0.3670
InDel-11-6	3.0000	0.2615	0.4307	0.2389
					InDel-12-4	2.0000	0.4346	0.6262	0.4420
InDel-12-7	3.0000	0.4824	0.6755	0.4900
					InDel-13-1	2.0000	0.3852	0.5735	0.3931
InDel-13-4	2.0000	0.4987	0.6918	0.5000
					InDel-14-1	2.0000	0.4964	0.6896	0.5953
InDel-15-4	2.0000	0.2891	0.4641	0.2963
					InDel-17-1	2.0000	0.4375	0.6293	0.4373
InDel-16-3	2.0000	0.4938	0.6870	0.4965
					InDel-18-3	2.0000	0.4167	0.6074	0.5623
InDel-19-4	2.0000	0.1486	0.2807	0.1528
					InDel-19-8	2.0000	0.3567	0.5421	0.3299
InDel-20-1	3.0000	0.4613	0.6539	0.4714
					Mean value of	2.2188	0.3935	0.5779	0.3963

The 32 InDel markers were analyzed for Nei diversity index, Shannon index and PIC in 37 North cohort materials using Popgene32 software, and the results are shown in Table 7. The Nei diversity index of each InDel marker is distributed between 0.1184 and 0.5000, and the mean value is 0.3990; SHI distribution is 0.3365-0.6931, mean value is 0.5680; PIC distribution 0.1928-0.4938, mean 0.3859.

TABLE 7 Nei diversity index, SHI, PIC for North group materials InDel markers

InDel tag name	Index of Nei diversity	SHI	PIC
				InDel-1-7	0.3878	0.5763	0.3682
InDel-1-8	0.4321	0.6237	0.4383
				InDel-2-1	0.3133	0.4926	0.3200
InDel-2-8	0.3878	0.5763	0.3944
				InDel-3-4	0.3324	0.5147	0.3067
InDel-3-6	0.4512	0.4307	0.4918
				InDel-4-4	0.4178	0.6086	0.4244
InDel-4-6	0.2659	0.4362	0.2337
				InDel-5-2	0.4654	0.6581	0.4704
InDel-5-5	0.3944	0.5835	0.375
				InDel-6-1	0.3068	0.4850	0.2778
InDel-6-4	0.3006	0.4777	0.3068
				InDel-7-3	0.4778	0.6708	0.4821
InDel-7-8	0.4321	0.6237	0.4178
				InDel-8-1	0.2778	0.4506	0.2841
InDel-9-4	0.4861	0.6792	0.4800
				InDel-9-8	0.3615	0.5474	0.3389
InDel-10-5	0.5000	0.6931	0.4753
				InDel-10-6	0.4821	0.6751	0.3225
InDel-11-6	0.3133	0.4926	0.4753
				InDel-12-4	0.4996	0.6928	0.4762
InDel-12-7	0.4704	0.6633	0.2801
				InDel-13-1	0.2778	0.4506	0.4938
InDel-13-4	0.4909	0.6840	0.4938
				InDel-14-1	0.4967	0.6899	0.4938
InDel-15-4	0.1928	0.3425	0.1975
				InDel-16-3	0.4963	0.6895	0.4795
InDel-17-1	0.4501	0.6424	0.4558
				InDel-18-3	0.3068	0.485	0.3133
InDel-19-4	0.1884	0.3365	0.1928
				InDel-19-8	0.4114	0.6017	0.3944
InDel-20-1	0.4114	0.6017	0.3944
				Mean value of	0.3990	0.5680	0.3859

The Nei diversity index, shannon index and PIC of the 32 InDel markers in 51 sets of material were analyzed using Popgene32 software and the results are shown in table 8. The Nei diversity index of each InDel marker is distributed in 0.1420-0.4983, and the mean value is 0.3798; SHI distribution is 0.2712-0.6914, mean value is 0.5548; the PIC distribution was 0.1446-0.4992 with an average value of 0.3711.

TABLE 8 Nei diversity index, SHI, PIC for InDel markers for middle group materials

The data show that there is a large difference in genetic diversity of materials between different groups, indicating that the 32 InDel markers can be used to identify genetic diversity between different materials. The genetic diversity index of soybean material was sorted by size into north > middle group.

As can be further seen from Table 6, the Nei diversity index and SHI of InDel10-5 are highest, and PIC is also higher than that of most markers, indicating that the markers have strong ability to distinguish test materials. Respectively calculating the genetic diversity index of each InDel marker in different groups, wherein the different groups show larger difference, for example, the Nei diversity index and the SHI of InDel10-5 in north group materials are the highest, and the material distinguishing capability is strong; the Nei diversity index and the SHI of InDel 13-4 in the medium group of materials are the highest, and the material distinguishing capability is strong.

UPGMA-based cluster analysis

And (3) performing clustering analysis on 96 parts of soybean materials by using UPGMA of UTSYS-pc 2.0 software according to the sizes of PCR products of 32 pairs of primers of 96 parts of soybean materials in the second step, wherein the steps are as follows:

(1) compared with the soybean reference genome Glyma.Wm82.a2v1, the site with the mutation type of insertion is recorded as 1, the site with the mutation type of deletion is recorded as 0, the site with the mutation type not detected is recorded as 999, and the data are converted into a format supported by UPGMA.

(2) And (4) importing the data into UPGMA, setting parameters, operating a program and carrying out genetic distance analysis.

(3) And obtaining a clustering analysis result.

The genetic distances for the three groups of soybean material are shown in fig. 5, north group material and middle group material are grouped into the first group, the genetic distance for the two groups is 0.95, south group material is one group alone, and north group and middle group material are further in genetic distance, and soybean material can be classified into 3 categories at a genetic similarity coefficient of 0.57 (table 9).

TABLE 9 clustering results of 96 parts of soybean materials

Fourth, Structure group genetic Structure analysis based on Bayesian method

And (4) according to the size of the PCR product of 32 pairs of primers of 96 parts of soybean materials in the second step, carrying out genetic structure analysis on 96 parts of materials by utilizing structure2.3.4 software based on a Bayesian method. The method comprises the following steps:

(1) compared with soybean reference genome Glyma.Wm82.a2v1, the site with the mutation type of insertion is marked as 1, the site with the mutation type of deletion is marked as 0, the site without the mutation type is marked as-9, and the data is converted into a format supported by the Structure.

(2) And determining an optimal population group K value. The software K value range is set to be 2-7, and the repetition number is set to be 2. Calculate Δ K using the Admixure model as a measure of the best K worth.

The results are shown in FIG. 6. When K equals 5, Δ K reaches a maximum value. The bar plot in the Simulation Result plots the proportion of each soybean variety when K is 5 (fig. 7). As can be seen in FIG. 7, the Structure software groups 96 portions of soybean material into five major groups, each indicated by 5 colors, and the histogram illustrates the proportion of the different varieties divided into five groups.

According to the Q value (probability that the genome variation of the ith material is derived from k groups) distribution of all soybean varieties, when the Q value of a certain variety is more than or equal to 0.6, the college of the variety is relatively single; if the number is less than 0.6, the variety is considered to have a mixed source. To more clearly illustrate the genetic background of each material, 96 soybean material were analyzed for Q-value, of which 69 varieties (71.88%) were single in consanguinity and divided into 5 cluster groups. The distribution of soybean germplasm from different sources in the field (north, middle, south) in 5 clusters is shown in table 10. In addition to the south group, the north and middle group materials were distributed across 5 clustered populations, indicating that the north and middle group materials have abundant types of genetic variation.

TABLE 10 distribution of soybean germplasm of different sources in 5 clustering groups

Fifthly, construction of fingerprint

According to the size of the PCR product of 32 pairs of primers of 96 parts of soybean materials in the second step, compared with the soybean reference genome Glyma.Wm82.a2v1, under the same migration rate, a band with fragment insertion variation as the electrophoresis detection result is marked as 1, a band with fragment deletion as the detection result is marked as 0, and a band with no detection or more than two allelic variations as 999, the genetic map consisting of 96 parts of soybean materials is obtained.

Through verification, the purity of the tested soybean material is 96.84%, and no phenomenon of 'synonym and synonym' occurs, so that the genetic map constructed based on 32 InDel markers can be used for identifying soybean varieties.

<110> institute of grain and oil crops of academy of agriculture and forestry, Hebei province

Application of <120> 32 soybean InDel markers in detection of soybean genetic diversity

<160> 64

<170> PatentIn version 3.5

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<213> Artificial sequence (Artificial sequence)

<400> 21

gttcttcttt ggttgtgttg 20

<210> 22

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

ggatattttg accttacagc 20

<210> 23

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

aaacactcac tctccacaac 20

<210> 24

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

atttcacttc cccctgcgat 20

<210> 25

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

gaaactaccc aatcgagatc 20

<210> 26

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

ggactttggt tagactgttt 20

<210> 27

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

tgctatttca aggagagaag 20

<210> 28

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

ccggagagga aaagaataaa 20

<210> 29

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

agcacactct aacacattct 20

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

aatggcctga tacaatagct 20

<210> 31

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

aaataagcca tacacagtgc 20

<210> 32

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

agagctatca caaaaaccag 20

<210> 33

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

gcaatgtcca accttttctt 20

<210> 34

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

ctaggttgtg ggtcaggtca 20

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

ggcagttatc caacacactc 20

<210> 36

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

ggtatcgggt cctttctaaa 20

<210> 37

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

ggttactcac aacagaaagg 20

<210> 38

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

catgccaagc tctagggact 20

<210> 39

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

cgtcgcataa ccaatgtagt 20

<210> 40

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

ccttcccttt ctatttgcaa 20

<210> 41

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

actttctttg ttgctgaggg 20

<210> 42

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

gcccaccatc tttcactttg 20

<210> 43

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

ataagaatgc ccaaacgaag 20

<210> 44

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

gcaacaaaag aaaggaagtg 20

<210> 45

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

cgaaccatgt agaggaaaat 20

<210> 46

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

gaaagacaac tttcatccac 20

<210> 47

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

gtttgcacca tctcccgctt 20

<210> 48

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

gccattaggg acaaagtctt 20

<210> 49

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

ataggggcaa atggtcattc 20

<210> 50

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

ggccgctaaa tgacaacgta 20

<210> 51

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

gcttgctcac tcttgccaat 20

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 52

acataagccc ttctcccgtc 20

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

ccaacgaaaa tagggagcag 20

<210> 54

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 54

caggcagtat gctgagtgaa 20

<210> 55

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

tagtgttaaa tgggagagcc 20

<210> 56

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 56

ctttatgctc ggtaacctgt 20

<210> 57

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

tgcttcttat ccttcccatc 20

<210> 58

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 58

gggtgttaga gcttgtatta 20

<210> 59

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

tcagacagcc attttgctag 20

<210> 60

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 60

caattgtgat gtagggttca 20

<210> 61

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

gttgctttct ttcactatgc 20

<210> 62

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 62

ggtcagtagc acaacaatag 20

<210> 63

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 63

agtctcttct ccaatcgttg 20

<210> 64

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 64

gagaacctac acctcaaacg 20

Claims

1.32 soybean InDel marks or the application of detecting the 32 soybean InDel marked substances in auxiliary detection of soybean genetic diversity;

the InDel-10-5 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-10-5-P; the InDel-10-5-P consists of two single-stranded DNAs shown as sequences 1 and 2 in a sequence table;

the InDel-10-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-10-6-P; the InDel-10-6-P consists of two single-stranded DNAs shown as sequences 3 and 4 in a sequence table;

the InDel-12-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-12-4-P; the InDel-12-4-P consists of two single-stranded DNAs shown as sequences 5 and 6 in a sequence table;

the InDel-12-7 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-12-7-P; the InDel-12-7-P consists of two single-stranded DNAs shown as sequences 7 and 8 in a sequence table;

the InDel-13-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-13-1-P; the InDel-13-1-P consists of two single-stranded DNAs shown as sequences 9 and 10 in a sequence table;

the InDel-13-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-13-4-P; the InDel-13-4-P consists of two single-stranded DNAs shown as sequences 11 and 12 in a sequence table;

the InDel-14-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-14-1-P; the InDel-14-1-P consists of two single-stranded DNAs shown as sequences 13 and 14 in a sequence table;

the InDel-15-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-15-4-P; the InDel-15-4-P consists of two single-stranded DNAs shown as sequences 15 and 16 in a sequence table;

the InDel-16-3 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-16-3-P; the InDel-16-3-P consists of two single-stranded DNAs shown as sequences 17 and 18 in a sequence table;

the InDel-1-7 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-1-7-P; the InDel-1-7-P consists of two single-stranded DNAs shown as sequences 19 and 20 in a sequence table;

the InDel-17-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-17-1-P; the InDel-17-1-P consists of two single-stranded DNAs shown as sequences 21 and 22 in a sequence table;

the InDel-1-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-1-8-P; the InDel-1-8-P consists of two single-stranded DNAs shown as sequences 23 and 24 in a sequence table;

the InDel-18-3 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-18-3-P; the InDel-18-3-P consists of two single-stranded DNAs shown as sequences 25 and 26 in a sequence table;

the InDel-19-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-19-4-P; the InDel-19-4-P consists of two single-stranded DNAs shown as sequences 27 and 28 in a sequence table;

the InDel-19-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-19-8-P; the InDel-19-8-P consists of two single-stranded DNAs shown as sequences 29 and 30 in a sequence table;

the InDel-20-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-20-1-P; the InDel-20-1-P consists of two single-stranded DNAs shown as sequences 31 and 32 in a sequence table;

the InDel-2-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-2-1-P; the InDel-2-1-P consists of two single-stranded DNAs shown as sequences 33 and 34 in a sequence table;

the InDel-3-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-3-4-P; the InDel-3-4-P consists of two single-stranded DNAs shown as sequences 35 and 36 in a sequence table;

the InDel-3-6 is a DNA molecule obtained by performing PCR amplification on soybean genome DNA by using a primer pair named InDel-3-6-P; the InDel-3-6-P consists of two single-stranded DNAs shown as sequences 37 and 38 in a sequence table;

the InDel-4-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-4-4-P; the InDel-4-4-P consists of two single-stranded DNAs shown as sequences 39 and 40 in a sequence table;

the InDel-4-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-4-6-P; the InDel-4-6-P consists of two single-stranded DNAs shown as sequences 41 and 42 in a sequence table;

the InDel-5-2 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-5-2-P; the InDel-5-2-P consists of two single-stranded DNAs shown as sequences 43 and 44 in a sequence table;

the InDel-8-1 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-8-1-P; the InDel-8-1-P consists of two single-stranded DNAs shown as sequences 53 and 54 in a sequence table;

the InDel-11-6 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-11-6-P; the InDel-11-6-P consists of two single-stranded DNAs shown as sequences 59 and 60 in a sequence table;

the InDel-2-8 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-2-8-P; the InDel-2-8-P consists of two single-stranded DNAs shown as sequences 61 and 62 in a sequence table;

the InDel-6-4 is a DNA molecule obtained by carrying out PCR amplification on soybean genome DNA by using a primer pair named InDel-6-4-P; the InDel-6-4-P consists of two single-stranded DNAs shown as sequences 63 and 64 in a sequence table;

the soybean is selected from Zhonghuang 42, Xue Dou 16, Qihuang 34, Ji Yu 101, Hobbit, HJ117, Zheng 196, ZYD2738, Ji Dou 17, Zhonghuang 1, Suiyong 14, Luo 1408, Shi 11893, Zhong12102-4, Ji 1702, Shaan Dou No. 2, JiG 15073, Lu 1407, Sheng Dou 102, Shang Dou 1310, Ji 1701, Luo 1337, Fen Dou 103, Shaan Dou No. 1, Sheng Dou 103, Shang 161, Handan-139, Luo Yuan 1, Zhongpin 16067, Hua Dou 30, Qin Dou 2017, Handan-324, Zhonghuang 104, Lu 0540-4, Zhongpin 1184, Daoqiu 37, Qin Dou 220, Jun 52, Zhonghuang 105, HN1030, Luo Lu 4, Luo 4904, Zhonghuang Huang 320, river which rises in the northeastern part of Anhui Province, Zhonghua Zhongbo Miao 11-11, Luo 3, Luo 627, Zhonghua Lu Hua Dou No. 19, Zhonghua No. 8, Zhonghua Luo Huang No. 8, jin Rou No. 897, jin Huang No. 11, jin Rou No. 8, jin Rou No. 11, jin Rou No. 2, jin Rou No. 11, jin Rou No. 8, jin Rou No. 11, jin, The Weiqi beans 18, the Weiqi beans 59, the Weiqi beans 16S379, the Andou 109, the Weiqi beans 26, the Shengyu No. 6, the Zhongchang 11-518, the Jinyi 57, the Andou 6023, the Anhui 920, the Zhongchang 12-1251, the Huayu No. 2, the Zhongchang 11-719, the Weidou 31, the river which rises in the northeastern part of Anhui Province, the Shenzhou 57, the river which rises in the northeastern part of Anhui Province, the Cangdou 58, the Cangdou 1123, the Daqiu Qiu 6, the Zhenghuang 1677, the Zhonghuang 103, the Handan-685, the Shandou 18, the Cangdou 1312, the Xidou 13, the Zheng16175, the Zhongchang 08-Handan, the Weidou No. 5, the Shaandou No. 3, the Ji 1708, the Pandou 19, the Jiju J14187, the Zhongchang 11-58, the Handan-109, the 0126, the Ji beans 12, the Llu 284, the Fenghou 104, the Weidou 897, the Ji 1257, the Jilu beans 17025, the Weijulu beans 09, the Weidou 617, the Yilu 12, the Yilu beans 115, the Anhulu beans 115, and/or the Anhulu beans 11.

2. Use of the 32 soybean InDel markers or detecting the 32 soybean InDel marked substances in claim 1 to assist in identifying soybean varieties or lines;

the soybean variety or strain is Zhonghuang 42, Xuhua bean 16, Qihuang 34, Jiyu 101, Hobbit, HJ117, Zheng 196, ZYD2738, Zhonghuang 1, Suiyong 14, Luo 1408, Shi 11893, Zhong12102-4, Ji 1702, Shaan bean No. 2, Ji G15073, Luo 1407, Sheng bean 102, Shandong 1310, Ji 1701, Luo 1337, Fengbean 103, Shaan bean No. 1, Sheng bean 103, Shandong 161, Handan 16-139, Luo 1330, Yongyu No. 1, Zhongpin 16067, Hua bean 30, Qin bean 2017, Handan 15-324, Zhonghuang 104, Lu 0540-4, Zhongpin 1184, Daoqiu 37, Qin bean 220, Qin river which rises in the northeastern part of Anhui Province family 56, Zhonghuang yellow 105, HN1030, Luo Hua 4, Luo 4904, Zhonghuang yellow 320, Zhonghua bean river which rises in the northeastern part of Anhui Province family 11-11, Zhongcao family Rou family 3, jin Rou family 622, Zhonghua bean No. 11, Zhonghua bean No. 19, Zhonghua bean No. 8, Zhonghua bean No. 897, Zhonghua bean No. 8, jin bean No. 897, jin bean No. 11, jin bean No. 8, jin bean No. 11, jin Hua No. 2, jin bean No. 11, jin Hua No. 8, jin bean 088, jin bean No. 2, jin Hua, jin bean No. 2, jin Hua No. 11, jin bean No. 2, jin Hua, jin bean No. 2, jin Hua No. 2, jin bean No. 11, jin Hua, jin bean No. 6, jin bean No. 2, jin Hua, jin bean No. 2, jin Hua No. 3, jin Hua, jin bean No. 2, jin bean No. 6, jin Hua No. 2, jin Hua, jin bean No. 2, jin Hua, jin bean No. 6, jin bean No. 2, jin bean No. 6, jin Hua, jin bean No. 2, jin bean No. 3, jin bean No. 2, jin Hua, jin bean No. 2, jin bean No. 3, weiyu No. 5, Weiyu No. 18, Weiyu 59, Weiyu 16S379, Weiyu 109, Weiyu 26, Shengyu No. 6, Zhongzhao 11-518, Jinyi 57, Andou 6023, Anhui bean 920, Zhongzhao 12-1251, Huayu No. 2, Zhongzhao 11-719, Weidou 31, river which rises in the northeastern part of Anhui Province, 57, river which rises in the northeastern part of Anhui Province, 58, Candou 1123, Daqiu No. 6, Zheng 1677, Zhonghuang 103, Handan 15-685, Shandou 18, Candou 1312, Pandou 13, Zheng 16175, Zhongzhao 08-08, Handan, Shaandou No. 3, Ji 1708, Shudou 19, Jiji 14187, Zhongzhao 11-58, Handan 13-109, Lu 0126, Llu 284, Fenghu 104, Weidou 897, Ji 1257, Handan 617, Weiyu 03, Weiya Y09, Weiya 1, Xilu 38, Andou 51 and/or Weidou 115.

3. Use of the 32 soybean InDel markers or substances for detecting the 32 soybean InDel markers of claim 1 for developing molecular markers for assisting in identifying soybean varieties or lines;

4. Use of the 32 soybean InDel markers or the substance for detecting the 32 soybean InDel markers in claim 1 for preparing a soybean genetic map;

the soybean is selected from Zhonghuang 42, xu bean 16, Qihuang 34, Jiyu 101, Hobbit, HJ117, Zheng 196, ZYD2738, Ji bean 17, Zhonghuang 1, Suiyong 14, Luo 1408, Shi 11893, Zhong12102-4, Ji 1702, Shaan bean No. 2, JiG 15073, Lu 1407, Sheng bean 102, Shang bean 1310, Ji 1701, Lu 1337, Feng bean 103, Shaan bean 1, Sheng bean 103, Shang bean 161, Handan-139, Lo 1330, Yongyu No. 1, Zhongpin 16067, Hua bean 30, Qin bean 2017, Handan-324, Zhonghuang 104, Lu 0540-4, Zhongwei 1184, Daqiu 37, Qin bean 220, river which rises in the northeastern part of Anhui Province family 56, Zhonghuang 105, HN1030, Rou select No. 4, Huahe bean 4904, Zhonghuang 320, river which rises in the northeastern part of Anhui Province family 55, Zhongwei family 11-11, Jinhong Hua lake No. 3, Jinyun # 2, Shangjin # 1617, Jinyu # 15223, Zhonghua # 11, Zhonghua bean 16, Zhonghua # 1jin bean 12h, Zhonghua bean 1jin bean 112h, jin bean 11118, Zhonghua # 11, Jishan bean 12, Zhonghua bean 12, Jihong bean 12, Jishan bean 2 # 18, Jizhong Hua bean 2 # 18, Jizhong bean 2, Jizhong Hua bean 2 # 1h, Jizhong Hua bean 2, Jizhong Hua bean 12, Jizhong bean 2, Jizhong, Ji bean 2, Jizhong # 1hua bean 2, Jizhong bean 12, Jizhong # 1h bean 12, Jizhong bean 2, Jizhong # 1h bean 2, Jizhong # 1hua bean 2, Jizhong bean 2, Jihua bean 2, Ji bean 2, Jihua bean 2, Jizhong bean 2, Jihua bean 2, Ji # 1h bean 1, Ji bean 2, Ji # 1h bean 2 et h, Ji bean 2, Jihua # 1h, Jihua bean 2 et h, Jihua # 1h, Ji # 9, Jihua # 9, Ji bean 2, Jihua bean 2, Ji bean 2 et h bean 2, Ji # 9, Ji bean 2, Jihua bean 2, Ji bean 2 et h bean 2, Ji bean 2, Jihua bean 2 et al, Ji bean 2 et h, Jihua bean 2, Ji bean 2 et al, Ji bean 2, Ji # 9, Ji, The Weiqi beans 18, the Weiqi beans 59, the Weiqi beans 16S379, the Andou 109, the Weiqi beans 26, the Shengyu No. 6, the Zhongchang 11-518, the Jinyi 57, the Andou 6023, the Anhui 920, the Zhongchang 12-1251, the Huayu No. 2, the Zhongchang 11-719, the Weidou 31, the river which rises in the northeastern part of Anhui Province, the Shenzhou 57, the river which rises in the northeastern part of Anhui Province, the Cangdou 58, the Cangdou 1123, the Daqiu Qiu 6, the Zhenghuang 1677, the Zhonghuang 103, the Handan-685, the Shandou 18, the Cangdou 1312, the Xidou 13, the Zheng16175, the Zhongchang 08-Handan, the Weidou No. 5, the Shaandou No. 3, the Ji 1708, the Pandou 19, the Jiju J14187, the Zhongchang 11-58, the Handan-109, the 0126, the Ji beans 12, the Llu 284, the Fenghou 104, the Weidou 897, the Ji 1257, the Jilu beans 17025, the Weijulu beans 09, the Weidou 617, the Yilu 12, the Yilu beans 115, the Anhulu beans 115, and/or the Anhulu beans 11.

5. Application of the 32 soybean InDel markers or substances for detecting the 32 soybean InDel markers in claim 1 in soybean breeding

6. Use according to any one of claims 1 to 5, characterized in that: the substance for detecting the 32 soybean InDel markers comprises a primer set, wherein the primer set consists of 64 single-stranded DNAs shown in sequences 1-64 in a sequence table.

7. Use according to claim 6, characterized in that: the substance for detecting the 32 soybean InDel markers also comprises other reagents except primers required for PCR amplification.

8. The method of detecting the 32 soybean InDel-labeled substances recited in any one of claims 1-7.

9. The 32 soybean InDel markers of claim 1.