CN111719013A

CN111719013A - Method for identifying authenticity of watermelon variety and special SSR primer combination thereof

Info

Publication number: CN111719013A
Application number: CN202010624618.0A
Authority: CN
Inventors: 许勇; 温常龙; 张建; 罗江; 杨静静; 刘慧�; 张海英
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: JINGYAN YINONG (BEIJING) SEED SCI-TECH Co.,Ltd.
Priority date: 2019-10-11
Filing date: 2020-07-02
Publication date: 2020-09-29
Anticipated expiration: 2040-07-02
Also published as: CN111719013B

Abstract

The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a method for identifying the authenticity of a watermelon variety and a special SSR primer combination thereof. The SSR primer combination provided by the invention can be used for early identification of watermelon varieties in seed or seedling stages, so that the authenticity of the varieties is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for the protection of watermelon germplasm resources and new varieties. The invention can identify that: whether the watermelon variety to be detected belongs to one of the standard watermelon varieties or not and which watermelon variety is to be detected. Therefore, the method can be used for identifying the unknown watermelon varieties and also identifying the authenticity of the known varieties. The invention has the advantages of high flux, accuracy, low cost, simple operation, manpower and material resource saving and the like, and has very wide application prospect.

Description

Method for identifying authenticity of watermelon variety and special SSR primer combination thereof

Technical Field

The invention belongs to the field of molecular markers and detection thereof, and particularly relates to a method for identifying the authenticity of a watermelon variety and a special SSR primer combination thereof.

Background

China is the first major country for watermelon production and consumption, the annual sowing area reaches 182.8 ten thousand hectares, and the annual sowing area accounts for 7.9 percent of the cultivation area of all vegetable crops. The watermelon breeding in China realizes the updating and updating of at least 5 defective varieties, breeds the primary variety of the watermelon in different periods, and greatly promotes the development of the watermelon industry in China. However, problems also follow up, and due to the fact that variety approval is not coordinated among provinces and cities, variety management and germplasm monitoring of watermelons cannot follow up in time, a large number of phenomena of same species and different names and different species appear on the market, the seed quality cannot be guaranteed, and the seed market is seriously affected. According to statistics, since registration of a non-main crop new variety is implemented in 4 months in 2017, 3126 watermelon varieties are applied for registration in two short years, 1971 watermelon new varieties are authorized, and the number is far more than the sum of the number of all new variety protected watermelon varieties in the past. According to the requirements of the registration guidelines for non-major crop varieties, DNA detection results can be directly submitted for the variety trait specifications and related traits involved in the variety DUS test reports, such as clear associated genes. Therefore, establishing a set of molecular markers for effectively identifying the authenticity and purity of watermelon varieties based on DNA fingerprints is very important. The set of molecular markers can cover the whole genome and represent the variation sites of the whole genome genetic background as much as possible; and the genetic variation sites can be combined as few as possible, so that the genetic diversity of the variety can be reflected to the maximum extent.

In recent years, SSR molecular markers are widely applied to the identification of variety authenticity by virtue of the advantages of large quantity, rich variation, stable heredity and the like. At present, China develops an SSR molecular marker method (NYT 2472-2013) for identifying watermelon varieties for seven years, and the number of the current burst watermelon varieties cannot be met. The traditional SSR locus development lacks the support of big data of watermelon variation groups, the number of varieties to be identified is limited, and unreal, false positive and false negative results are easily caused by a detection mode.

Therefore, in the work of identifying watermelon varieties by using SSR molecular markers, a stable and efficient SSR locus and determination method based on large-scale watermelon re-sequencing and analysis of watermelon variant group information are needed.

The inventor subject group can master the real sequence variation condition of SSR Motif internal and PCR amplified fragments by analyzing the big data of the watermelon genome variation group. Based on the multiplex PCR amplification technology, the method can meet the requirement of large-scale, stable and efficient watermelon variety identification.

Disclosure of Invention

The invention provides a method for identifying the authenticity of watermelon varieties and a special SSR primer combination thereof, which can obtain a stable and high-efficiency identification result: whether the watermelon variety to be detected belongs to one of the standard watermelon varieties or not, and specifically which watermelon variety to be detected belongs to the standard watermelon variety.

The invention is realized by the following technical scheme:

an SSR locus for identifying the authenticity of a watermelon variety, selected from any 1 to 18 of the following first to eighteenth SSR loci: a first SSR locus which is located at the No. 1 chromosome 30509464-30509481 of the watermelon reference genome or an interspecies homologous genome fragment thereof; a second SSR locus which is positioned at the 13593981-13593992 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome; a third SSR locus which is positioned at the 3380588-3380608 site of the 4 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the third SSR locus; the fourth SSR locus is positioned at the No. 4 chromosome 3380588-3380608 of the watermelon reference genome or the homologous genome segment among varieties of the watermelon reference genome; a fifth SSR locus which is located at the 19330086-19330093 th chromosome 5 of the watermelon reference genome or an interspecies homologous genome segment thereof; the sixth SSR locus is positioned at the No. 5 chromosome 27924990-27925007 of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome; the seventh SSR locus is positioned at the 13260487-13260494 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome; the eighth SSR locus is positioned at the 16630830-16630839 th chromosome of the watermelon reference genome or an interspecies homologous genome segment thereof; a ninth SSR locus located at position 29257614-29257639 of chromosome 5 of the reference genome of watermelon, or an interspecies homologous genome fragment thereof; the tenth SSR locus is positioned at the No. 8 chromosome 245853 and 245870 of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome; an eleventh SSR locus located at chromosome 9 of the reference genome of watermelon at position 10388587 and 10388595, or an interspecies homologous genomic fragment thereof; the twelfth SSR locus is positioned at the No. 4024099-4024118 th chromosome 9 of the watermelon reference genome or an interspecies homologous genome fragment thereof; the thirteenth SSR locus is positioned at the No. 23367823-23367874 of chromosome 6 of the watermelon reference genome or a homologous genome segment among varieties thereof; a fourteenth SSR locus located at position 23078098-23078115 of chromosome 2 of the watermelon reference genome or an interspecies homologous genomic fragment thereof; a fifteenth SSR locus located at the 12518423-12518450 locus of the watermelon reference genome 5 or an interspecies homologous genome fragment thereof; a sixteenth SSR locus which is located at the No. 10534664 and No. 10534702 position of the 11 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome; a seventeenth SSR locus which is positioned at the 22668179-22668204 th chromosome 1 of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome; an eighteenth SSR locus which is located at the 15916747-15916782 th chromosome 10 of the watermelon reference genome or an interspecies homologous genome fragment thereof; the watermelon reference genome is watermelon 97103 reference genome version V1.

An SSR primer group for identifying the authenticity of a watermelon variety, wherein the SSR primer group is used for respectively amplifying the SSR loci, and comprises: a first SSR primer pair for amplifying said first SSR site; a second SSR primer pair for amplifying said second SSR site; a third SSR primer pair for amplifying said third SSR site; a fourth SSR primer pair for amplifying said fourth SSR site; a fifth SSR primer pair for amplifying said fifth SSR site; a sixth SSR primer pair for amplifying said sixth SSR site; a seventh SSR primer pair for amplifying said seventh SSR locus; an eighth SSR primer pair for amplifying said eighth SSR locus; a ninth SSR primer pair for amplifying said ninth SSR locus; a tenth SSR primer pair for amplifying said tenth SSR site; an eleventh SSR primer pair for amplifying said eleventh SSR site; a twelfth SSR primer pair for amplifying said twelfth SSR site; a thirteenth SSR primer pair for amplifying said thirteenth SSR site; a fourteenth SSR primer pair for amplifying said fourteenth SSR site; a fifteenth SSR primer pair for amplifying said fifteenth SSR site; a sixteenth SSR primer pair for amplifying the sixteenth SSR site; a seventeenth SSR primer pair for amplifying the seventeenth SSR site; and the eighteenth SSR primer pair is used for amplifying the eighteenth SSR locus.

In some embodiments, the SSR primer set: the first SSR primer pair is respectively matched with SEQID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the second SSR primer pair is respectively matched with SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the third SSR primer pair is respectively matched with SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fourth SSR primer pair is respectively matched with SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fifth SSR primer pair is respectively matched with SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the sixth SSR primer pair is respectively matched with SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the seventh SSR primer pair is respectively matched with SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the eighth SSR primer pair is respectively matched with SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the ninth SSR primer pair is respectively matched with SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the tenth SSR primer pair is respectively matched with SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; the eleventh SSR primer pair is respectively matched with SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the twelfth SSR primer pair is respectively matched with SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the thirteenth SSR primer pair is respectively matched with SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the fourteenth SSR primer pair is respectively matched with SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and the fifteenth SSR primer pair is respectively matched with SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; and the sixteenth SSR primer pair is respectively matched with SEQ ID NO: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%; the seventeenth SSR primer pair is respectively matched with SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; and the eighteenth SSR primer pair is respectively matched with SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%; preferably, one primer of each pair of said primers is linked to a fluorescent molecule, more preferably said fluorescent molecule is selected from the group consisting of ROX, TAMRA, FAM, HEX.

An SSR kit for identifying the authenticity of watermelon varieties, wherein reagents are used for being prepared into a PCR reaction system; the PCR reaction system comprises: the SSR primer group, preferably, the concentration ratio of the upstream primer and the downstream primer of each pair in the SSR primer group in the system is 1: 1; more preferably, the final concentration of the upstream primer and the final concentration of the downstream primer in the system are both preferably 0.25 mu mol/L; preferably, the system further comprises: dNTPs: final concentration in the system was 0.15mmol/L each, magnesium chloride: the final concentration in the system is 2.5mmol/L, DNA polymerase: the final concentration in the system was 0.05U/. mu.L, PCR buffer: is prepared from potassium chloride with final concentration of 10-50mmol/L in the system and Tris-HCL (pH7.5-9.0) with final concentration of 1-10mmol/L in the system.

A detection method for identifying the authenticity of a watermelon variety comprises the following steps: the method comprises the following steps: detecting the genotype of the SSR locus of claim 1 in a watermelon to be tested; step two: the variety judgment step of the watermelon to be detected comprises the following steps: if the number of the different loci of the genotype of the watermelon to be detected based on the 18 SSR loci and the genotype of a certain specified standard variety in a watermelon standard variety bank based on the 18 SSR loci is 0, the watermelon to be detected and the standard variety in the watermelon standard variety bank belong to the same variety; if the number of the difference loci of the genotypes of the to-be-detected watermelons based on the 18 SSR loci and the genotypes of a certain specified standard variety in a watermelon standard variety bank based on the 18 SSR loci is 1, the to-be-detected watermelons and the standard variety in the watermelon standard variety bank belong to similar varieties; and if the number of the different loci of the genotype of the watermelon to be detected based on the 18 SSR loci and the genotype of each variety in the watermelon standard variety bank based on the 18 SSR loci is more than or equal to 2, the variety of the watermelon to be detected is different from that of each watermelon standard variety in the watermelon standard variety bank.

In some embodiments, the step of detecting the SSR locus genotype of the watermelon to be detected comprises the following substeps: the method comprises the following steps: respectively taking the genome DNA of the watermelon to be detected and the genome DNA of the standard variety of the watermelon as templates, and respectively adopting the primer groups in the SSR primer combination to carry out PCR amplification to obtain PCR amplification products; step two is carried out: and (3) carrying out length detection on the PCR amplification product to obtain the genotypes of the to-be-detected watermelon and the watermelon standard variety based on 18 SSR loci.

In some embodiments, the detection method of the second step comprises: and (3) fluorescent signal detection: detecting a fluorescent signal of the PCR amplification product to obtain genotypes of the to-be-detected watermelon and the standard watermelon varieties based on the 18 SSR loci; or: detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the watermelon to be detected and the standard watermelon variety based on the 18 SSR loci.

In some embodiments, in the second step, the determination result is obtained according to a clustering analysis.

In some embodiments, the standard watermelon seed bank is selected from any number of 1-196 of the following 196 watermelon species:

jingpin Xin, Kangkang Zaohuang, Xinyun, Jinlan seedless, Zhengkang seedless No. four, Lufenghuang, Luyuan 095, Hualei No. one, Jiangyi 1584, Meixin 101, Shenmi 968, Wanyue No. one, RX1, Chunlei, Xiyue No. 6, Zaochun jade, Longsheng No. 8, Hongxiaoyu, Meihui, Xicheng orange, Jinyu linglong seedless No. 1, Yidong No. 2, Tianwang No. three, Chuanjia Xuanxuan, Chuanxuanxuanli Hongmu, Chuanqi No. 6K, Jingjia No. 2, Jingmei, Longshengjia sweet, Yongmi No. 3, Yongmi No. 2, Longshengjiamei, RX2, sweet honey, Linzida tablet 88708, Yunan Baxing, Jinchun Miao, Miao, Xiyun Hongyao, Xiyu No. 1, Xiyun Yiyue No. 6, Yiyue Feng Yuyue, Yiyue No. 1, Yiyun Feng Yuyun Yuyue No. 6, Yiyun Yuyun, Epoch, Xinxing, Sumiqi No. 6, Shenkang 988, black pearl, Zaojia, Longkang No. 9, Jiakangbao, Jingxin No. one, Fengshan corolla, Zhejiang honey No. 3, Xinjinlan, Xiaolan, Jiaxin, Xinxin No. 99K, Shuangxing 37, Jingxin father, Shouxicai XG1, Ruhong, Shamijiayi No. one, Jingxin No. two, century, Xiujin No. 24, Xiuqi, Dujing Xinxin, Jingjia No. 6, Jinmamu No. nine, Meumei, Yu kylin, Su honey No. 8, Zao Xixiu No. two, Xiuya, Gemei, Zheng Kangkang No. three, Yuyue No. 5, Texiaofeng, Huanghuanghe, Boujin No. 1, Qifuxin No. 1, Qingfa red, Jinbao, Miao selection, Miao No. 4, Yingyun No. 2, Qiangyuanyuanyue No. 2, Qiangyuannan No. 2, Qiangmei No. 2, Qiangxin No. 3, Qiangfeng, Qiangmei No. 2, Qiangxin No. 3, Qiangxin No. 2, Qiangfeng Yingxin No. 2, Qiangxin, Beinongji Jia, boat Xin No. 1, yellow jade, happy spring, Jingxiu, good luck, Zhongchong No. 11, Zhongchong No. 12, boat Xin No. 3, Jingxin No. 2, Jinglongbao, Heimeren, Jiahua, Yongfeng No. 1, spring bud, Jingxin No. 3, Jingxin No. 2, Jingxin No. 4, Qingfa No. twelve, black boy, Red Xiaoshuai, jin Yu Ling, Tianhai Feng, Tianbao Xiaoseedless, Tianquan, Henbao, Huangjingbao No. 1, Shinong Zhen Mei, Xiangxiu, Fangke No. 3, Xuefeng Xiaoyu No. 7, Meixiao, Luhong hong No. 2, Guo, Jingkang No. 2, Jingxi No. 8, Bao Mi Zi Shuyu, Fu Yu Xin, Dong Tong Tian nong Mi, Xin Mi Ju No. 3, Feng Bao No. 1, Xin Ju Bao, Xin Ju No. 6, Xin No. 1, Xin No. 1, Xin No. 2, Xin No. 2, jiamei, beinong shijia, suzuo 8, angel, black crystal, jinshuai 2, middle alling 58, middle alling black general, agricultural 10, taike Cuibao 2, Xinrui, transcending, morning glory, Jing appendices, Jing glu, Huaxin 2, jin bao, hong Xiaoshuai 6, beinong brocade, bei nong Tianjiao 2, danong, rake, curiosity, Chuanqi 1, and zhongtian red.

The SSR locus, or the SSR primer combination, or the SSR kit, or the detection method, is applied to the following X1 or X2: x1: identifying whether the variety of the watermelon to be detected belongs to one of standard watermelon varieties; x2: and identifying the variety of the watermelon to be detected as the standard variety of the watermelon.

Compared with the prior art, the invention has the following beneficial effects:

1. the SSR primer combination provided by the invention can be used for early identification of watermelon varieties in seed or seedling stages, so that the authenticity of the varieties is ensured, the rights and interests of producers and breeders are practically protected, and technical support is provided for the protection of watermelon germplasm resources and new varieties.

2. The method provided by the invention can be used for identifying that: whether the watermelon variety to be detected belongs to one of the standard watermelon varieties or not and which watermelon variety is to be detected. Therefore, the method can be used for identifying the unknown watermelon varieties and also identifying the authenticity of the known varieties.

3. The method provided by the invention has the advantages of high throughput, accuracy, low cost, simplicity in operation, manpower and material resource saving and the like, and has a very wide application prospect.

Drawings

FIG. 1 is a cluster plot of the 196 watermelon varieties tested, built on the 18 primer sets in example 1.

FIG. 2 is the difference marker chart of example 2, which shows the number of SSR markers (i.e., the number of SSR loci) and the differentiation among 196 varieties of watermelon to be tested.

FIGS. 3-20 are graphs showing the SSR typing effect of the 18 primer sets of example 2 on part of the watermelon varieties to be tested. Wherein, fig. 3: SSR typing effect profile using BVWS062 primer, fig. 4: SSR typing effect profile using BVWS00177 primer, FIG. 5: SSR typing effect profile using BVWS00233 primer, fig. 6: SSR typing effect profile using BVWS00314 primer, fig. 7: SSR typing effect profile using BVWS00441 primer, FIG. 8: SSR typing effect profile using BVWS0089 primer, fig. 9: SSR typing effect profile using BVWS00948 primer, fig. 10: SSR typing effect profile using BVWS02048 primer, FIG. 11: SSR typing effect profile using the wmsr 037 primer, fig. 12: SSR typing effect profile using the WmSSr044 primer, fig. 13: SSR typing effect plots using the WmSSr069 primer, fig. 14: SSR typing effect plots using the wmsr 081 primer, fig. 15: SSR typing effect profile using the wmsr 084 primer, fig. 16: SSR typing effect profile using wmsr 090 primer, fig. 17: SSR typing effect plots using the wmsr 103 primer, fig. 18: SSR typing effect plots using the wmsr 106 primer, fig. 19: SSR typing effect plots using the wmsr 115 primer, fig. 20: SSR typing effect map using WmSSR121 primer.

Detailed Description

The definition is as follows:

authenticity of watermelon varieties: refers to the real correspondence of a watermelon variety and its genetic background.

Interspecies homologous genomic fragments: the gene is a gene fragment which is homologous with a specific gene fragment sequence in the reference gene sequence of the watermelon 97103 in other varieties except that the reference gene sequence of the watermelon 97103 has the version number of V1. For example, with respect to a specific genome segment, a genome segment having homology with the version number V1 of the reference genome sequence of watermelon 97103 in the variety of Table 3 of the present invention.

For a particular SSR sequence of watermelon, the interspecies homologous genomic fragment refers to: the version number of the specific watermelon variety and the reference genome sequence of the watermelon 97103 is the same or similar with the flanking sequence of the SSR sequence in V1, and the SSR sequences in the two are the same or similar.

In a first aspect, the invention provides SSR loci for identifying the authenticity of watermelon varieties, wherein the SSR loci are respectively located in watermelon genomes, the number of the SSR loci is 18, and 1 or more of the SSR loci can be selected, and specific information is shown in Table 1.

A first SSR locus which is located at the No. 1 chromosome 30509464-30509481 of the watermelon reference genome or an interspecies homologous genome fragment thereof;

a second SSR locus which is positioned at the 13593981-13593992 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome;

a third SSR locus which is positioned at the 3380588-3380608 site of the 4 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the third SSR locus;

the fourth SSR locus is positioned at the No. 4 chromosome 3380588-3380608 of the watermelon reference genome or the homologous genome segment among varieties of the watermelon reference genome;

a fifth SSR locus which is located at the 19330086-19330093 th chromosome 5 of the watermelon reference genome or an interspecies homologous genome segment thereof;

the sixth SSR locus is positioned at the No. 5 chromosome 27924990-27925007 of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome;

the seventh SSR locus is positioned at the 13260487-13260494 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome;

the eighth SSR locus is positioned at the 16630830-16630839 th chromosome of the watermelon reference genome or an interspecies homologous genome segment thereof;

a ninth SSR locus located at position 29257614-29257639 of chromosome 5 of the reference genome of watermelon, or an interspecies homologous genome fragment thereof;

the tenth SSR locus is positioned at the No. 8 chromosome 245853 and 245870 of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome;

an eleventh SSR locus located at chromosome 9 of the reference genome of watermelon at position 10388587 and 10388595, or an interspecies homologous genomic fragment thereof;

the twelfth SSR locus is positioned at the No. 4024099-4024118 th chromosome 9 of the watermelon reference genome or an interspecies homologous genome fragment thereof;

the thirteenth SSR locus is positioned at the No. 23367823-23367874 of chromosome 6 of the watermelon reference genome or a homologous genome segment among varieties thereof;

a fourteenth SSR locus located at position 23078098-23078115 of chromosome 2 of the watermelon reference genome or an interspecies homologous genomic fragment thereof;

a fifteenth SSR locus located at the 12518423-12518450 locus of the watermelon reference genome 5 or an interspecies homologous genome fragment thereof;

a sixteenth SSR locus which is located at the No. 10534664 and No. 10534702 position of the 11 th chromosome of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome;

a seventeenth SSR locus which is positioned at the 22668179-22668204 th chromosome 1 of the watermelon reference genome or a homologous genome segment among varieties of the watermelon reference genome;

an eighteenth SSR locus which is located at the 15916747-15916782 th chromosome 10 of the watermelon reference genome or an interspecies homologous genome fragment thereof;

the watermelon reference genome is watermelon 97103 reference genome version V1.

In a second aspect, the invention provides an SSR primer group for identifying the authenticity of a watermelon variety, and a PCR amplification product based on the SSR locus can be obtained through PCR amplification reaction.

The SSR primer combination is selected from:

the first SSR primer pair is respectively matched with SEQ ID NO: 1 and SEQ ID NO: 2 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the second SSR primer pair is respectively matched with SEQ ID NO: 3 and SEQ ID NO: 4 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the third SSR primer pair is respectively matched with SEQ ID NO: 5 and SEQ ID NO: 6 is more than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the fourth SSR primer pair is respectively matched with SEQ ID NO: 7 and SEQ ID NO: 8 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the fifth SSR primer pair is respectively matched with SEQ ID NO: 9 and SEQ ID NO: 10 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the sixth SSR primer pair is respectively matched with SEQ ID NO: 11 and SEQ ID NO: 12 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the seventh SSR primer pair is respectively matched with SEQ ID NO: 13 and SEQ ID NO: 14 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the eighth SSR primer pair is respectively matched with SEQ ID NO: 15 and SEQ ID NO: 16 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the ninth SSR primer pair is respectively matched with SEQ ID NO: 17 and SEQ ID NO: 18, the homology is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the tenth SSR primer pair is respectively matched with SEQ ID NO: 19 and SEQ ID NO: 20 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

the eleventh SSR primer pair is respectively matched with SEQ ID NO: 21 and SEQ ID NO: 22 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the twelfth SSR primer pair is respectively matched with SEQ ID NO: 23 and SEQ ID NO: 24 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the thirteenth SSR primer pair is respectively matched with SEQ ID NO: 25 and SEQ ID NO: 26 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the fourteenth SSR primer pair is respectively matched with SEQ ID NO: 27 and SEQ ID NO: 28, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;

and the fifteenth SSR primer pair is respectively matched with SEQ ID NO: 29 and SEQ ID NO: 30, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;

and the sixteenth SSR primer pair is respectively matched with SEQ ID NO: 31 and SEQ ID NO: 32, is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98%, or 99%, preferably 100%;

the seventeenth SSR primer pair is respectively matched with SEQ ID NO: 33 and SEQ ID NO: 34 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

and the eighteenth SSR primer pair is respectively matched with SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%.

In a preferred embodiment, the SSR primer combination is selected from one or more of primer sets 01-18; the DNA sequence information of the primer groups 01-18 is shown in a sequence table SEQ ID: 1-36, see table 2.

In the primer set, the 5' end of the upstream primer can be provided with a fluorescent tag sequence for fluorescent PCR detection, for example, the fluorescent signal of the FAM fluorescent tag sequence is blue, and the fluorescent signal of the HEX fluorescent tag sequence is red.

In a third aspect, the invention provides an SSR kit for identifying the authenticity of watermelon varieties,

SSR reagents are formulated into a PCR reaction system, which preferably includes:

in the SSR primer group, the ratio of the final concentration of the upstream primer to the final concentration of the downstream primer is preferably 1: 1.

In a fourth aspect, the invention provides a detection method for identifying the authenticity of a watermelon variety, which comprises the following steps:

the method comprises the following steps: detecting the SSR locus genotype of the watermelon to be detected.

The method comprises the following steps: respectively taking the genomic DNA of the watermelon to be detected and the genomic DNA of the standard variety of the watermelon as templates, and respectively adopting the primer groups in the SSR primer combination to carry out PCR amplification reaction to obtain PCR amplification products;

step two is carried out: and (3) carrying out length detection on the PCR amplification product to obtain genotypes of the watermelon to be detected and the watermelon standard variety based on 18 SSR loci.

The detection may be a fluorescence signal detection: detecting the fluorescent signal of the PCR amplification product to obtain the genotypes of the watermelon to be detected and the standard watermelon variety based on the 18 SSR loci;

the detection can also be the detection of amplified product fragments: and (3) detecting the fragment size of the PCR amplification product by utilizing capillary electrophoresis to obtain the genotypes of the watermelon to be detected and the standard watermelon variety based on the 18 SSR loci.

Step two: the variety judgment step of the watermelon to be detected comprises the following steps:

the following results are obtained by cluster analysis of the watermelon to be tested and the standard watermelon variety library based on the genotypes of the 18 SSR loci:

if the genotype of the watermelon to be detected based on the 18 SSR loci and the number of different loci of a certain variety in the standard watermelon variety base based on the genotypes of the 18 SSR loci are 0, namely the difference loci are completely consistent, the watermelon to be detected and the certain variety in the standard watermelon variety base belong to the same variety;

if the number of the difference loci of the watermelon to be detected based on the genotypes of the 18 SSR loci and a certain variety in the watermelon standard variety library based on the genotypes of the 18 SSR loci is 1, the watermelon to be detected and the certain variety in the watermelon standard variety library belong to similar varieties;

if the genotypes of the to-be-detected watermelons based on the 18 SSR sites and the number of the different sites of the genotypes of the to-be-detected watermelons based on the 18 SSR sites in each variety in the standard watermelon seed bank are more than or equal to 2, the varieties of the to-be-detected watermelons and the varieties of each standard watermelon seed bank are different.

The procedure of the PCR amplification reaction is preferably:

pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and reducing the temperature by 0.8 ℃ per cycle for 12 cycles; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 25 cycles; final extension at 72 ℃ for 10 min. The amplification products were stored at-20 ℃ or on ice prior to electrophoresis.

The standard watermelon variety bank comprises the following 196 watermelon varieties:

In a fifth aspect, the invention provides the above SSR locus, SSR primer combination, SSR kit, and the detection method, and uses thereof in x1 or x 2:

x1: identifying whether the variety of the watermelon to be detected belongs to one of standard watermelon varieties;

x2: and identifying the variety of the watermelon to be detected as the standard variety of the watermelon.

Both X1 and X2 belong to the application of identifying the authenticity of watermelon varieties.

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example 1

Acquisition of SSR primer combination for identifying authenticity of watermelon varieties

Discovery of one and 19 SSR loci

The invention obtains 18 SSR sites based on the re-sequencing data of 20 watermelon representative resources (NCBI: SRA052158, https:// www.ncbi.nlm.nih.gov/SRA/. The 20 parts of watermelon are rich in resource types, cover east subtype (6 parts), American type (6 parts), sticky-seed watermelon (4 parts) and wild type (4 parts), basically comprise the main ecological types of watermelon, have higher genetic diversity in the aspects of agronomic characters such as growth period, fruit shape, peel thickness, flesh color, seed size, sugar content and the like, and have representativeness of watermelon germplasm.

Specifically, the screening criteria for SSR sites are as follows: the SSR sites of each chromosome are uniform in quantity, the spacing is more than 1Mb, the minimum allele frequency MAF is more than 0.1, the heterozygosity He is less than 0.1, PIC is more than 0.3, and the two wings of the SSR sites are 50bp sequence conservative (no InDel, no SSR, no SNP and no other SSR).

The basic information for the 18 SSR sites is detailed in columns 1 to 4 of Table 1. The position of the SSR locus on the chromosome is determined based on the alignment of a watermelon 97103 reference genome sequence, and the version number of the watermelon 97103 reference genome sequence is V1 (download addresses: ftp:// curbitangenetics. org/pub/curbitat/genome/watermelonlon/97103/, and the website https:// www.ncbi.nlm.nih.gov/genome/.

Table 1: basic information of 18 SSR loci

Second, obtaining SSR primer combination for identifying watermelon variety authenticity

Based on the 18 SSR sites found in step one, the inventors of the present invention developed SSR primer combinations for identifying the authenticity of watermelon varieties that have a higher amount of polymorphism information (i.e., PIC value, which refers to the value of a marker for detecting polymorphisms in a population; PIC value depends on the number of detected alleles and their frequency distribution of alleles; PIC value is equal to 1 minus the sum of the squares of all allele frequencies; see column 5 in Table 4). Primers are designed on the basis that the version number of the SSR locus in a reference genome sequence of the watermelon 97103 is an upstream sequence and a downstream sequence in V1, an SSR primer combination consists of 18 primer groups, and the name of each primer group is shown in a column 2 in a table 2. Each primer group consists of 2 primer sequences and is used for amplifying one SSR locus. The nucleotide sequences of the individual primers in the 18 primer sets are shown in column 4 of Table 2.

Table 2: 18 pairs of primer sequences of watermelon SSR sites and fluorescent modifying groups

Example 2

This example is a validation test of the SSR primer combination developed in example 1.

The basic information of the 196 watermelon varieties tested in this example is shown in Table 3. The 196 watermelon varieties to be tested are all common excellent varieties or partially foreign introduced varieties.

Table 3: basic information of 196 watermelon varieties to be tested

1. Acquisition of genomic DNA of watermelon species tested

The genome DNA of 196 leaves of the watermelon species to be tested (the true leaves of 30 seeds are mixed, namely the true leaves of 30 seeds of each species are mixed, namely the true leaves of 30 different plants of the same species are mixed) is respectively extracted by adopting a CTAB method, so that the genome DNA of the watermelon species to be tested is obtained.

The CTAB method is specifically operated as follows:

respectively picking 196 leaves of the above variety in seedling stage, dewatering in freeze dryer (CoolSafe55-4), breaking the leaves with high throughput grinder (Geno/Grind6875), adding 20 μ g of dry leaf powder, adding 800 μ L CTAB extract (2% CTAB, 1.4mM NaCl, 100mM Tris-HCl pH8.0, 20mM EDTA pH8.0, 1% PVP-40, 0.2% β -mercaptoethanol), mixing, centrifuging in 65 deg.C water bath for 30min, adding equal volume chloroform/isoamyl alcohol (24: 1), centrifuging at 10000rpm/min for 10min, transferring supernatant to new centrifuge tube, adding 0.8 times volume of precooled isopropanol, slightly inverting, standing at-20 deg.C for 30min, centrifuging at 4 deg.C, 12,000r/min, discarding supernatant, adding 70% ethanol solution, washing for 2 times, drying under natural condition, and adding 100 μ L ddH₂And dissolving the DNA by O to obtain the genome DNA of the watermelon variety to be tested, and detecting the concentration and then keeping the temperature at 4 ℃ for later use.

The quality and the concentration of the genome DNA of the watermelon variety to be tested both need to meet the PCR requirement, and the standard of reaching the standard is as follows: detecting that the ratio of A260 to A280 is about 1.8 and the ratio of A260 to A230 is more than 1.8 by using an ultraviolet spectrophotometer Nanodrop2000 (Thermo); the concentration of the genome DNA of the watermelon variety to be tested is 10-30 ng/. mu.L.

2. And respectively adopting genome DNAs of 196 watermelon varieties to be tested as templates and respectively adopting 18 primer pairs to carry out PCR amplification to obtain PCR amplification products. In each PCR reaction system, the concentration ratio of the primer containing "F" in the name and the primer containing "R" in the name was 1: 1.

Each reaction system comprises:

the ratio of the concentration of the forward primer (named as "F") to the concentration of the reverse primer (named as "R") in the system was 1: 1.

The reaction procedure is as follows: pre-denaturation: 5min at 94 ℃; amplification: denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 45s, and extension at 72 ℃ for 45s, and reducing the temperature by 0.8 ℃ per cycle for 12 cycles; denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 45s, and extension at 72 ℃ for 45s for 25 cycles; final extension: 10min at 72 ℃. The resulting amplification product was stored at 4 ℃ before electrophoresis.

3. Fluorescence capillary electrophoresis

After step 2 is completed, a plurality of primer combinations can be selected for electrophoresis according to different instruments according to different sizes of the SSR molecular marker amplified fragments. According to the predetermined combined primer, the amplification products of different fluorescence labels of the same combined primer with the same volume are respectively taken, the TAMRA fluorescence product is diluted by 50 times, and other fluorescence products are fully and uniformly mixed after being diluted by 100 times. Pipette 2. mu.L of the mixture and add to a well dedicated to the sample loading plate of the DNA analyzer. Adding 0.1 μ L molecular weight internal standard and 8.9 μ L deionized formamide into each well, denaturing at 95 deg.C for 5min in PCR instrument, taking out, immediately placing in-20 deg.C refrigerator or ice, and cooling for 5 min. After being instantaneously centrifuged for 10s, the mixture was placed on a DNA analyzer. The DNA analyzer is turned on, and the working state and the reagent state of the instrument are checked. The loading plate with the sample is placed on the sample holder base, the buffer plate with the electrode buffer solution is placed on the buffer plate holder base, the data collection software is opened, and the operation is carried out according to the instruction manual of the DNA analyzer. The DNA analyzer will run the parameters automatically and save the raw data for electrophoresis. The excitation wavelength and color reference instrument default values (the maximum excitation wavelength of FAM is 494nm, the maximum excitation wavelength of HEX is 535nm, the maximum excitation wavelength of TAMRA is 560nm, and the maximum excitation wavelength of ROX is 587nm) for detecting the fluorescent primers need to perform spectrum correction on the capillary electrophoresis equipment regularly.

Some of the results are shown in FIGS. 3-20. The result shows that each primer group can obtain good typing effect in the watermelon variety (Jingxin No. two) to be tested.

4. Cluster analysis

And (3) carrying out cluster analysis on 196 watermelon varieties to be tested by utilizing MEGA7 software according to the genotypes of the 196 watermelon varieties based on 18 SSR loci.

The clustering chart of 196 watermelon species tested, which were established on 18 primer sets, is shown in FIG. 1. The results showed that 18 primer sets could completely distinguish the 196 watermelon species tested in Table 3. Therefore, the SSR primer combination developed in the embodiment 1 can be applied to the construction of the DNA fingerprint database of the watermelon variety and the authenticity identification of the variety.

5. Evaluation of efficiency

The variety authenticity identification can reduce the workload by adopting a sequential analysis mode. The inventor of the invention compares the relationship between the number of SSR markers (namely the number of primer groups) and the distinguishing rate of 196 varieties of watermelon to be tested.

The number of the difference markers is C2, which is the number of results of comparison between 196 varieties and is counted in pairs as shown in FIG. 2₁ ₉₆196 × 195/2/19110, in 19110, the number of differential sites is about 15% of the total number, 9% of the total number, 10% of the total number, 11% of the total number, 12% of the total number, 3% of the total number, 16-18% of the total number, so that the results for more than 8 differential sites account for about 49% of the total number, which indicates that the polymorphism in 196 varieties is good with these markers, and the discrimination rate in 196 varieties of watermelon tested with 18 primer sets (i.e. 18 markers) reaches 100%.

Table 4: basic information of 18 SSR loci of 196 varieties

Wherein, the main allelic variation is obtained according to the statistics of 196 watermelon varieties to be tested in the table 3. For example: if only 1 allelic variation occurs in a certain position of a sample, and the size is 162bp, the genotype of the main allelic variation at the position is written as 162/162; if a sample has two allelic variations at a certain locus, the sizes of which are 162bp and 168bp respectively, the genotype of the main allelic variation at the locus is written as 162/168.

Example 3

Method for detecting whether watermelon variety to be detected belongs to variety among 196 watermelon varieties to be detected

1. Obtaining of genomic DNA of watermelon species to be tested

The leaves of the watermelon species to be tested (actually Jingcai No. one) are taken from the test base of vegetable research center of agriculture and forestry academy of sciences of Beijing.

According to the method of the step 1 in the embodiment 2, the 'leaves of the watermelon variety to be tested' are replaced by the 'leaves of the watermelon variety to be tested', and other steps are not changed, so that the genome DNA of the watermelon variety to be tested is obtained.

2. SSR primer and configuration of PCR reaction system

According to the method of the step 2 in the embodiment 2, the 'genomic DNA of the watermelon variety to be tested' is replaced by the 'genomic DNA of the watermelon variety to be tested', and other steps are not changed, so that the PCR product of the watermelon variety to be tested is obtained.

3. Fluorescence capillary electrophoresis detection

Taking a PCR product of the watermelon variety to be detected.

Comparing the fragment sizes of the 18 SSR amplification products of the watermelon varieties to be detected with 18 SSR loci of 196 watermelon varieties to be detected (also standard watermelon varieties) (shown in Table 3), counting the number of difference loci of the watermelon varieties to be detected and the 196 standard watermelon varieties, and then judging as follows:

if the number of the ectopic difference points between the watermelon variety to be detected and a standard watermelon variety is 2 or more, the watermelon variety to be detected and the standard watermelon variety belong to different watermelon varieties; the greater the number of differential sites, the more distant the genetic relationship.

If the number of the ectopic difference points between the watermelon variety to be detected and a standard watermelon variety is 1 or 0, the watermelon variety to be detected and the standard watermelon variety are or are suspected to be the same watermelon variety.

The result shows that the number of the different sites of the watermelon variety to be detected and the 196 watermelon varieties to be tested on the 18 SSR sites is more than 4, so that the watermelon variety to be detected does not belong to any one of the 196 watermelon varieties to be tested, namely the watermelon variety to be detected is different from any one of the 196 watermelon varieties to be tested.

Example 4

In the embodiment, the watermelon variety is judged by comparing the sizes of the fragments through capillary electrophoresis instead of adopting a fluorescent signal.

In this case, the ABI 3730 fluorescent capillary detection platform is used as a reference, and if other platforms are used, corresponding adjustment is performed according to the operation requirements of the equipment.

According to the different sizes of the SSR molecular marker amplified fragments, a plurality of primer combinations can be selected for electrophoresis according to different instruments.

S1: according to the predetermined combined primer, the amplification products of different fluorescence labels of the same combined primer with the same volume are respectively taken, the TAMRA labeled product is diluted by 50 times, and other fluorescence labeled products are fully and uniformly mixed after being diluted by 100 times. Pipette 1. mu.L of the mixture and add to a well dedicated to the sample loading plate of the DNA analyzer. Adding 0.1 μ L molecular weight internal standard and 8.9 μ L deionized formamide into each well, denaturing at 95 deg.C for 1min in PCR instrument, taking out, immediately placing on ice, and cooling for 5 min. After being instantaneously centrifuged for 10s, the mixture was placed on a DNA analyzer.

S2: the ABI 3730DNA analyzer is opened and the instrument operating status and reagent status are checked. The loading plate with the sample is placed on the sample holder base, the buffer plate with the electrode buffer solution is placed on the buffer plate holder base, the data collection software is opened, and the operation is carried out according to the instruction manual of the DNA analyzer. The DNA analyzer will run the parameters automatically and save the raw data for electrophoresis. The excitation wavelength and color used by the fluorescent primers are detected by referring to default values of an instrument.

S3: exporting an electrophoresis original data file, and adopting data analysis software to perform data discrimination according to the following steps: presetting SSR primer names, fluorescence categories, molecular weight internal standards and amplification fragment sizes of corresponding primers in data analysis software; importing the electrophoresis original data file into analysis software, and selecting panel, molecular weight internal standard, Bin, quality control parameters and the like for analysis; the analysis software assigns a color mark to the detection quality for scoring, green indicates that the quality is reliable without intervention, red indicates that the quality is not over or does not fall within a specified segment size range, and yellow indicates that the original image needs to be checked for confirmation in question.

S4: the amplified fragment size was read after calibrating the data deviation between different electrophoresis plates by using a standard sample and a reference sample (a small amount of control was selected according to the primers) which were tested simultaneously. If the screened specific peak falls into the specified fragment size range, directly reading the size of the amplified fragment; if the peaks are not within the predetermined range, the data can be read by shifting the whole of the peak as far as possible within the peak setting range.

S5: comparing the fragment sizes of the 18 SSR amplification products of the watermelon varieties to be detected with 18 SSR loci of 196 watermelon varieties to be detected (shown in Table 3), counting the number of difference loci of the watermelon varieties to be detected and 18 standard watermelon varieties, and then judging as follows:

Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.

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Claims

1. An SSR locus for use in identifying the authenticity of a watermelon variety, said SSR locus being selected from any 1 to 18 of the following first to eighteenth SSR loci:

2. An SSR primer set for identifying the authenticity of a watermelon variety, said SSR primer set for separately amplifying the SSR sites of claim 1, said SSR primer set comprising:

a first SSR primer pair for amplifying said first SSR site;

a second SSR primer pair for amplifying said second SSR site;

a third SSR primer pair for amplifying said third SSR site;

a fourth SSR primer pair for amplifying said fourth SSR site;

a fifth SSR primer pair for amplifying said fifth SSR site;

a sixth SSR primer pair for amplifying said sixth SSR site;

a seventh SSR primer pair for amplifying said seventh SSR locus;

an eighth SSR primer pair for amplifying said eighth SSR locus;

a ninth SSR primer pair for amplifying said ninth SSR locus;

a tenth SSR primer pair for amplifying said tenth SSR site;

an eleventh SSR primer pair for amplifying said eleventh SSR site;

a twelfth SSR primer pair for amplifying said twelfth SSR site;

a thirteenth SSR primer pair for amplifying said thirteenth SSR site;

a fourteenth SSR primer pair for amplifying said fourteenth SSR site;

a fifteenth SSR primer pair for amplifying said fifteenth SSR site;

a sixteenth SSR primer pair for amplifying the sixteenth SSR site;

a seventeenth SSR primer pair for amplifying the seventeenth SSR site;

and the eighteenth SSR primer pair is used for amplifying the eighteenth SSR locus.

3. The SSR primer set according to claim 2 characterized in that:

and the eighteenth SSR primer pair is respectively matched with SEQ ID NO: 35 and SEQ ID NO: 36 is greater than or equal to 85%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100%;

preferably, one primer of each pair of said primers is linked to a fluorescent molecule, more preferably said fluorescent molecule is selected from the group consisting of ROX, TAMRA, FAM, HEX.

4. A SSR kit for appraising watermelon variety authenticity, which is characterized in that: the reagent in the SSR kit is used for preparing a PCR reaction system; the PCR reaction system comprises:

the SSR primer set according to claim 2 or 3,

preferably, the concentration ratio of the upstream primer and the downstream primer of each pair in the SSR primer group in the system is 1: 1; more preferably, the final concentration of the upstream primer and the final concentration of the downstream primer in the system are both preferably 0.25 mu mol/L;

preferably, the system further comprises:

dNTPs: the final concentration in the system was 0.15mmol/L each,

magnesium chloride: the final concentration in the system is 2.5mmol/L,

DNA polymerase: the final concentration in the system is 0.05U/. mu.L,

PCR buffer solution: is prepared from potassium chloride with final concentration of 10-50mmol/L in the system and Tris-HCL (pH7.5-9.0) with final concentration of 1-10mmol/L in the system.

5. A detection method for identifying the authenticity of a watermelon variety is characterized by comprising the following steps: the detection method comprises the following steps:

the method comprises the following steps: detecting the genotype of the SSR locus of claim 1 in a watermelon to be tested;

if the number of the different loci of the genotype of the watermelon to be detected based on the 18 SSR loci and the genotype of a certain specified standard variety in a watermelon standard variety bank based on the 18 SSR loci is 0, the watermelon to be detected and the standard variety in the watermelon standard variety bank belong to the same variety;

if the number of the difference loci of the genotypes of the to-be-detected watermelons based on the 18 SSR loci and the genotypes of a certain specified standard variety in a watermelon standard variety bank based on the 18 SSR loci is 1, the to-be-detected watermelons and the standard variety in the watermelon standard variety bank belong to similar varieties;

and if the number of the different loci of the genotype of the watermelon to be detected based on the 18 SSR loci and the genotype of each variety in the watermelon standard variety bank based on the 18 SSR loci is more than or equal to 2, the variety of the watermelon to be detected is different from that of each watermelon standard variety in the watermelon standard variety bank.

6. The detection method of claim 5: the method is characterized in that:

the step of detecting the SSR locus genotype of the watermelon to be detected comprises the following sub-steps:

the method comprises the following steps: respectively carrying out PCR amplification by using the genomic DNA of the watermelon to be detected and the genomic DNA of a standard variety of the watermelon as templates and respectively adopting the primer group in the SSR primer combination according to claim 2 or 3 to obtain PCR amplification products;

step two is carried out: and (3) carrying out length detection on the PCR amplification product to obtain the genotypes of the to-be-detected watermelon and the watermelon standard variety based on 18 SSR loci.

7. The detection method of claim 6: the method is characterized in that:

the detection method of the substep two comprises the following steps:

and (3) fluorescent signal detection: detecting a fluorescent signal of the PCR amplification product to obtain genotypes of the to-be-detected watermelon and the standard watermelon varieties based on the 18 SSR loci; or:

detection of amplified product fragments: and detecting the fragment size of the PCR amplification product to obtain the genotypes of the watermelon to be detected and the standard watermelon variety based on the 18 SSR loci.

8. The detection method of claim 5: the method is characterized in that: in the second step, the judgment result is obtained according to cluster analysis.

9. The detection method according to any one of claims 5 to 8, wherein: the standard watermelon variety bank is selected from any one variety of 1-196 in the following 196 watermelon varieties:

10. The SSR site of claim 1, or the SSR primer combination of claim 2 or 3, or the SSR kit of claim 4, or the detection method of any one of claims 5 to 9, for use at X1 or X2: