CN111254215B

CN111254215B - Method for identifying purity of cucumber hybrid and SNP primer combination used by same

Info

Publication number: CN111254215B
Application number: CN202010263973.XA
Authority: CN
Inventors: 温常龙; 张建; 杨静静; 罗江; 毛爱军
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-04-07
Filing date: 2020-04-07
Publication date: 2021-05-11
Anticipated expiration: 2040-04-07
Also published as: CN111254215A

Abstract

The invention discloses a method for identifying purity of cucumber hybrid and an SNP primer combination used by the method. The SNP primer combination provided by the invention consists of 8 primer groups; each primer group consists of 3 primer sequences and is used for amplifying one SNP locus; the nucleotide sequence of each primer is shown as SEQ ID NO: 1 to SEQ ID NO: as shown at 24. The SNP primer combination can be used for early identification of seeds or seedling stage of cucumber hybrid seeds, ensures the purity of the hybrid seeds, practically protects rights and interests of producers and breeders, and provides technical support for seed quality management of cucumber varieties. 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.

Description

Method for identifying purity of cucumber hybrid and SNP primer combination used by same

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for identifying purity of cucumber hybrid and an SNP primer combination used by the method; hybrid refers to the first generation of hybrids, possibly with parental scrambling.

Background

The cucumber is one of the important vegetable crops in China, can be cultivated in four seasons and supplied all year round, and the cultivation area reaches 139.6 million hectares, which accounts for 6.2 percent of the vegetable cultivation area in China. With the acceleration of the breeding process, the number of new cucumber varieties is also increasing continuously. Because the cucumber breeding enterprises in China are small and dispersed, the quality management of cucumber seeds cannot be effectively followed up, and a lot of counterfeit and shoddy seeds with unqualified purity exist, the seed quality accidents caused by the counterfeit and shoddy seeds sometimes occur. In 4 months of 2017, since the implementation of the registration method for non-major crop varieties, 1607 cucumber varieties have been applied and registered nationwide, and more than 93% of the cucumber varieties are first-filial generation varieties (hereinafter, referred to as hybrid varieties). Before the hybrid seeds enter the market, purity identification is required. The purity of cucumber hybrids was not less than 98% as specified by the crop seed test protocol (GB/T3543.1-3543.7-1995). Compared with the traditional field purity identification, the DNA-based molecular detection technology has low cost, time saving and high efficiency. Therefore, it is important to screen and obtain molecular marker combinations for identifying the purity of cucumber hybrids based on DNA detection technology.

In recent years, the third generation molecular marker SNP has been widely regarded as having advantages such as a large number, a wide distribution, and genetic stability. With the development of high-throughput sequencing technology and the continuous reduction of sequencing cost, large-scale cucumber re-sequencing becomes possible. Based on analysis of cucumber mutation group information, more stable and efficient SNP sites can be mined. And developing a specific primer by adopting an allele competitive specific PCR method, and finally obtaining the genotype of the sample at the SNP site.

At present, the DNA molecular detection for identifying the purity of cucumber hybrid seeds in China mainly adopts an SSR molecular marking method, but the research and development of SSR primers do not refer to the information of cucumber genome variation groups, so that the unreal variation condition exists; the SSR primers are small in screening variety quantity and cannot represent the sales varieties in the current market in China; in addition, the method is limited by that unreal, false positive and false negative results are easily caused by an SSR detection mode; can not meet the requirements of automation, high flux and large scale. SNPs have several advantages over SSR markers: the variation is clear, stable and easy to detect, and the authenticity is high in accuracy; millions of SNPs are available for selection per crop; the method is suitable for high-throughput, low-cost and automatic rapid detection, and can reduce human errors; SNP typing does not require a control variety, and results are presented with accurate bases.

Disclosure of Invention

The invention aims to identify the purity of cucumber hybrid seeds.

The invention firstly protects the SNP locus combination, which can comprise 8 SNP loci of cucumber genome; the 8 SNP sites are as follows: the HGSNP01 site is the 16975775 th nucleotide on chromosome 4; the HGSNP02 site is the 9582207 th nucleotide on chromosome 5; the locus HGSNP03 is the 17508118 th nucleotide on chromosome 1; the locus HGSNP04 is the 1976800 th nucleotide on chromosome 1; the HGSNP05 site is the 4054461 th nucleotide on chromosome 5; the HGSNP06 site is the 20929474 th nucleotide on chromosome 6; the HGSNP07 site is the 8333363 th nucleotide on chromosome 3; the HGSNP08 site is the 2133319 th nucleotide on chromosome 5.

The SNP site combination can specifically consist of the 8 SNP sites.

The invention also protects SNP primer combinations, which can comprise a primer group 1 for amplifying the CuSNP01, a primer group 2 for amplifying the CuSNP02, a primer group 3 for amplifying the CuSNP03, a primer group 4 for amplifying the CuSNP04, a primer group 5 for amplifying the CuSNP05, a primer group 6 for amplifying the CuSNP06, a primer group 7 for amplifying the CuSNP07 and a primer group 8 for amplifying the CuSNP 08.

In the SNP primer combination, the primer group 1 consists of SEQ ID NO: 1, forward primer 1F1, SEQ ID NO: 2 and the forward primer 1F2 shown in SEQ ID NO: 3, and a reverse primer 1R shown in the specification. The primer group 2 consists of SEQ ID NO: 4, forward primer 2F1, SEQ ID NO: 5 and the forward primer 2F2 shown in SEQ ID NO: 6, and a reverse primer 2R. The primer group 3 consists of SEQ ID NO: 7, forward primer 3F1, SEQ ID NO: 8 and the forward primer 3F2 shown in SEQ ID NO: 9, and a reverse primer 3R. The primer group 4 consists of SEQ ID NO: 10, forward primer 4F1, SEQ ID NO: 11 and the forward primer 4F2 shown in SEQ ID NO: 12, and a reverse primer 4R. The primer group 5 consists of SEQ ID NO: 13, forward primer 5F1, SEQ ID NO: 14 and the forward primer 5F2 shown in SEQ ID NO: 15, and a reverse primer 5R. The primer group 6 consists of SEQ ID NO: 16, forward primer 6F1 shown in SEQ ID NO: 17 and the forward primer 6F2 shown in SEQ ID NO: 18, and a reverse primer 6R. The primer group 7 consists of SEQ ID NO: 19, forward primer 7F1, SEQ ID NO: 20 and the forward primer 7F2 shown in SEQ ID NO: 21, and a reverse primer 7R shown in fig. 21. The primer group 8 consists of SEQ ID NO: 22, forward primer 8F1, SEQ ID NO: 23 and the forward primer 8F2 shown in SEQ ID NO: 24, and a reverse primer 8R.

In the SNP primer combination, the primer group 1 consists of SEQ ID NO: 1, forward primer 1F1 shown at positions 22 to 51 from the 5' end, SEQ ID NO: 2 from position 22 to 52 from the 5' end and the forward primer 1F2 shown in SEQ ID NO: 3, and a reverse primer 1R shown in the specification. The primer group 2 consists of SEQ ID NO: 4, forward primer 2F1 shown at positions 22 to 47 from the 5' end, SEQ ID NO: 5 forward primer 2F2 at positions 22 to 46 from the 5' end and SEQ ID NO: 6, and a reverse primer 2R. The primer group 3 consists of SEQ ID NO: 7, forward primer 3F1 shown at positions 22 to 52 from the 5' end, SEQ ID NO: 8 from position 22 to 51 from the 5' end and the forward primer 3F2 shown in SEQ ID NO: 9, and a reverse primer 3R. The primer group 4 consists of SEQ ID NO: 10 from position 22 to 46 from the 5' end, 4F1, SEQ ID NO: 11 from position 22 to 46 from the 5' end and the forward primer 4F2 shown in SEQ ID NO: 12, and a reverse primer 4R. The primer group 5 consists of SEQ ID NO: 13 from position 22 to 48 from the 5' end, 5F1, SEQ ID NO: 14 from the 5' end, the forward primer 5F2 shown at positions 22 to 47 and SEQ ID NO: 15, and a reverse primer 5R. The primer group 6 consists of SEQ ID NO: 16 from position 22 to 49 from the 5' end, a forward primer 6F1, SEQ ID NO: 17 from position 22 to 48 from the 5' end and a forward primer 6F2 shown in SEQ ID NO: 18, and a reverse primer 6R. The primer group 7 consists of SEQ ID NO: 19 from position 22 to 50 from the 5' end, 7F1, SEQ ID NO: 20 from position 22 to 48 from the 5' end and the forward primer 7F2 shown in SEQ ID NO: 21, and a reverse primer 7R shown in fig. 21. The primer group 8 consists of SEQ ID NO: 22 from position 22 to 48 from the 5' end, 8F1, SEQ ID NO: 23 from position 22 to 49 from the 5' end, 8F2 and SEQ ID NO: 24, and a reverse primer 8R.

In any of the above primer sets, the molar ratio of the primer named as "F1", the primer named as "F2" and the primer named as "R" may be specifically 2:2: 5.

Any one of the above-mentioned SNP primer combinations may specifically consist of the primer set 1, the primer set 2, the primer set 3, the primer set 4, the primer set 5, the primer set 6, the primer set 7, and the primer set 8.

In the above, the nucleotide sequence shown in 1 st to 21 st positions from the 5' end of the sequence 1 in the sequence table is a fluorescent tag sequence (i.e., FAM fluorescent tag sequence), and the fluorescent signal is specifically blue. The nucleotide sequence shown in 1 st to 21 th positions from the 5' end of the sequence 2 in the sequence table is also a fluorescent label sequence (namely a HEX fluorescent label sequence), and the fluorescent signal is red.

A kit containing any one of the SNP primer combinations also belongs to the protection scope of the invention. The kit is used for identifying the purity of cucumber hybrid seeds.

The preparation method of the kit also belongs to the protection scope of the invention. The preparation method of the kit comprises the step of packaging each primer in any one of the primer groups separately.

The invention also protects the application of any one of the SNP locus combinations or any one of the SNP primer combinations, which can be x1) or x 2): x1) preparing a kit for identifying the purity of cucumber hybrids; x2) identifying the purity of the cucumber hybrid.

The invention also discloses a method for identifying the purity of the cucumber hybrid to be detected.

The method for identifying the purity of the cucumber hybrid to be detected, which is protected by the invention, can be specifically the first method and comprises the following steps:

(a1) obtaining genome DNA of N cucumber hybrid seeds to be detected; n is a natural number greater than 95;

(a2) respectively taking the genomic DNA of the cucumber hybrid to be detected of 8-12 strains (such as 8-10 strains, 10-12 strains, 8 strains, 10 strains or 12 strains) obtained in the step (a1) as a template, and respectively adopting 8 primer groups (each primer contains a fluorescent tag sequence) in the SNP primer combination to carry out PCR amplification to obtain corresponding PCR amplification products;

(a3) after the step (a2) is completed, detecting the fluorescent signal of each PCR amplification product by using an instrument, and counting the number of strains of which the green fluorescent signals are displayed by 8 primer groups; the primer group with the largest number of green fluorescent strains is the target primer group;

(a4) respectively taking the genome DNA of the N cucumber hybrid seeds to be detected obtained in the step (a1) as templates, and respectively adopting a target primer group to carry out PCR amplification to obtain corresponding PCR amplification products;

(a5) and (a4) after the step (a), detecting the fluorescent signal of each PCR amplification product by using an instrument, and obtaining the purity of the cucumber hybrid to be detected according to the color of the fluorescent signal.

In the first method, the method for obtaining the purity of the cucumber hybrid to be detected according to the color of the fluorescent signal may be: counting the number of strains showing green fluorescence and the number of strains without fluorescence of each target primer group, respectively calculating the purity, and then calculating the average value;

no fluorescence strain number ═ N-strain number showing green fluorescence-strain number showing red fluorescence-strain number showing blue fluorescence;

purity ═ number of strains showing green fluorescence of the target primer set/(N-number of strains showing no fluorescence of the target primer set) × 100%.

In the first method, the reaction procedure for performing PCR amplification may specifically be: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles. If the fluorescence signal is weak after the PCR amplification is finished and the data analysis is influenced, the cycle (denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min and 5 cycles) can be added until the result is satisfactory.

The method for identifying the purity of the cucumber hybrid to be detected, which is protected by the invention, can be specifically the method II, and can comprise the following steps of:

(b1) obtaining genome DNA of N cucumber hybrid seeds to be detected; n is a natural number greater than 95;

(b2) respectively taking the genome DNA of the cucumber hybrid to be detected of 8-12 strains (such as 8-10 strains, 10-12 strains, 8 strains, 10 strains or 12 strains) obtained in the step (1) as a template, and respectively adopting 8 primer groups in any one of the SNP primer combinations to carry out PCR amplification to obtain corresponding PCR amplification products;

(b3) sequencing each PCR amplification product obtained in the step (b 2); according to the sequencing result, obtaining the number of strains which are heterozygous based on the genotypes of the 8 SNP sites, wherein the primer group with the largest number of strains is the target primer group;

(b4) respectively taking the genome DNA of the N cucumber hybrid seeds to be detected obtained in the step (b1) as templates, and respectively adopting a target primer group to carry out PCR amplification to obtain corresponding PCR amplification products;

(b5) sequencing each PCR amplification product obtained in the step (b 4); and obtaining the purity of the cucumber hybrid to be detected according to the sequencing result.

In the second method, the primers of 8 primer groups in the SNP primer combination may or may not contain fluorescent tag sequences.

In the second method, the step of obtaining the purity of the cucumber hybrid to be detected according to the sequencing result may be: counting the number of strains of each target primer group which are heterozygous based on the genotype of the SNP locus and the number of strains of which PCR amplification products are not obtained, respectively calculating the purity, and then calculating the average value;

the purity was defined as the number of strains of the target primer set that were heterozygous based on the genotype at the SNP site/(N — the number of strains from which PCR amplification products were not obtained by the target primer set) × 100%.

In any of the above methods, the cucumber hybrid to be tested may be MC2065, jasper, bome 5032, ludwigsheng, meiestri 09, nanesten No. 1, nanesten No. 2, ningfeng 09, youjia full-female 09, 83-ms702, native cucumber, chunqiwen No. 3, chunxi F1, cuckoo brilliant star, dianna, derrit 723, derrit 727, derrit 7876, derrit cucumber, dongnong 804, cyanine big cucumber, gold embryo 98F1, gold embryo 98-1F1, gold embryo 99-1F1, gold embryo 99-2F1, jin you No. 108, jin you No. 36, jing mini No. 1, jing mini No. 3, jing mini No. 5, mini-minzumi No. 9, jing qinmei, zumi, qinqi, qinqing No. 2, qinqing No. 3, qinqing No. 1, maqinqing type pickled cucumber (kohling type) Qianghanjie, Ruiguang No. two, Xinjingchun No. four, Zhongnong No. 19, Zhongnong No. 50, Zhongnong No. 9, Zhongji No. 17F1, Zhuang Gua, 128 generation hybrid, 13AC230, AMATA765, Vlaspik, white horse prince, Beijing 204, Beijing white jade No. three, Bilv Cheng, Biyu No. 2, Bomei No. 6913, Bomei No. 8, Chaoshu F1, Culcita chinensis No. 3, Chunhua No. 1, Chunhong 9801, Cucumis sativus, Dadoxing (Deltatar), De Ruite 15-10, De Ruite No. 4, De Ruite D19, De Ruiz GZ1601, De Ruta L14-2, De Ruta L14-5, Dinghao oil-shinning continuous cropping, Dong nong Li 806, Guyu Mei Xinyu Xingying, Hai No. Yijie, Ji Zao Bao No. 2, Mi No. 3-30, Mi No. 3-7, De Luo jin No. 99, No. 3-7, De Luo-7, Jin Ming No. four, jin you No. 1, jin you No. 303, jin you No. 308, jin you No. 315, jin you No. 35, jin you No. 48, jin you No. one, jin Zheng A207, Jing AK18, Jing Bai Yu super white leaf three, Jing Ming 107, Jing Ming Chunmei, Jing Ming Mini No. 2, Jing Ming Yongsheng, exquisite white leaf three, military eighteen, Longquan Wang-Longquan Green crown, Lu Cucumis sativus No. 8, Lubao Zhu No. 188, Manguan crown, Mici Wang cucumber, Ningxing Chunfu F2, Qingmei, Qingshuang, Qing sweet, Shengfeng 908, Shushou vegetable HG No. 1, Shuo Feng Ba eight, Jifeng, Tian Yi, Wannong Sanhao No. 3966, Dai Cucumis Breynus cucumber No. 35, Shi Si Mei, Su Mei Te No. 01, Bai De Li Shi De Li No. 3, Bai De Li Shi De No. 8, Bode Rui No. 16, Bode Rui 8, Bode Rui Ruan, Bode Rui No. 3, Bode Rui No. 16, Shi De 3623, Jizai No. 16, jin you 20-11, jin za No. 2, Jing A008, Jingfeng No. 298, Jing grind No. 106, Jing grind 118, Jing grind drought treasure No. 5, Jing grind Green exquisite No. 2, Lv Bo eleven, Meifeng Gua King, Euro No. 1, Qiang Huo Jie 80, Shen Green 64, Shen Green 72, Suiying Laoliao, Tangchun 100, Tangshan Cuobao F1, specially selected Ji miscellaneous No. four, Tianjiao No. eight, Xin grind No. two, Xin grind No. four, Yi Wang fruit type cucumber, Zhongnong No. 16, Zhongnong 19F1, P02, Bei nong Jia Xiu, Bo Mei Cucumis 517, Bo Mei No. 74, Chun Mei, Cuilu Green, Ying Feng No. 15, Jin embryo No. 99F1, Chun Lu No. 4, Lu Wang jin No. 3, Shuo Wang jin 203, Shuo you No. 11, Jing you No. 35, Jing jin No. 38, Jing jin you No. 38, Jing jin No. 2, Jing jin Yinjin Shen jin No. 2, Ji jin No. 15, Ji jin No. 2, Ji, HH1-8-1-2, Korean cucumber 2-06-97-164, He Han, Black oil Bright 999, Hu miscellaneous 6, Huanai H1104, Ji miscellaneous 16, jin you 12, jin you 2, jin you 401, jin you 4, Ningjia 3, Qiade 117, Tianjiao No. 7, Huanan type, Yuan 6, Cuili, Jiza 9, jin you 10, jin you 31 and Bright Liang.

In any of the above methods, the greater the value of N, the higher the accuracy of identifying the purity of the cucumber hybrid to be tested.

It should be noted that the cucumber hybrid of the present invention refers to the first generation hybrid, and only parents may be mixed; is not mechanically mixed, namely, is formed by mixing a plurality of cucumber hybrid seeds.

The SNP primer combination provided by the invention can be used for early identification in the seed or seedling stage of cucumber hybrid, ensures the purity of the hybrid, practically protects the rights and interests of producers and breeders, and provides technical support for seed quality management of cucumber varieties. 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 shows the SNP typing effect of 8 primer sets in a part of cucumber hybrids tested.

FIG. 2 shows the number distribution of heterozygous sites for 8 primer sets in a portion of cucumber hybrids tested.

FIG. 3 shows the SNP typing effects of primer set 1 and primer set 6 in 96 Kyoto research elite hybrids.

Detailed Description

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.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 obtaining of SNP primer combination for identifying purity of cucumber hybrid

Discovery of one or 8 SNP sites

The invention obtains 8 SNP loci based on the resequencing data of 49 cucumber representative resources. The 49 cucumbers are rich in resource types, include North China (7), Indian (18), Japanese (3), south China (2), European fruit (5), American processed (4), Xishuangbanna (5) and intermediate (5), basically comprise the main ecological types and agronomic traits of the cucumbers, reflect germplasm representativeness as much as possible, and have higher genetic diversity.

Specifically, the screening criteria for SNP sites are as follows: SNP sites with uniform positions, good polymorphism, small heterozygosity, MAF >0.3, good PCA clustering effect, high discrimination and two-wing 50bp sequence conservation (no InDel, no SSR and no other SNP) are selected in the whole genome range.

The basic information of the 8 SNP sites is detailed in columns 1 to 4 of Table 1. The position of the SNP locus on the chromosome is determined based on the comparison of cucumber 9930 reference genome sequences, and the version number of the cucumber 9930 reference genome sequence is V2 (download address: http:// cucurbitangencies. org/organissm/2).

TABLE 1.8 basic information of SNP sites

Name of SNP site	Chromosome of	Location on chromosome	Base type (alt)	Base type (ref)
					HGSNP01	Chr4	16975775	C	T
HGSNP02	Chr5	9582207	A	G
					HGSNP03	Chr1	17508118	G	T
HGSNP04	Chr1	1976800	A	T
					HGSNP05	Chr5	4054461	A	G
HGSNP06	Chr6	20929474	T	C
					HGSNP07	Chr3	8333363	A	C
HGSNP08	Chr5	2133319	A	G

II, obtaining of SNP primer combination for identifying purity of cucumber hybrid

Based on the 8 SNP sites found in the step one, the inventor of the invention develops an SNP primer combination with higher polymorphism for identifying the purity of cucumber hybrids.

The SNP primer set consists of 8 primer sets, and the name of each primer set is shown in the 2 nd column in Table 2. Each primer set consists of 3 primer sequences and is used for amplifying one SNP site. The nucleotide sequences of the individual primers in the 8 primer sets are shown in column 4 of Table 2.

TABLE 2

Note: single underlined is FAM fluorescent tag sequence and double underlined is HEX fluorescent tag sequence.

Example 2 and example 1 validation of the SNP primer combinations developed

The basic information of the 212 cucumber hybrids tested in this example is shown in table 3. 212 cucumber hybrids are all common excellent hybrids or foreign introduced hybrids.

TABLE 3.212 basic information of the cucumber hybrids tested

1. Acquisition of genomic DNA of cucumber hybrids to be tested

Genomic DNAs of leaves (leaves of 30 seeds mixed) of 212 cucumber hybrids to be tested were extracted by the CTAB method, respectively, to obtain the genomic DNAs of the cucumber hybrids to be tested.

The quality and concentration of the genome DNA of the cucumber hybrid to be tested both need to meet the PCR requirement, and the standard of standard is as follows: agarose electrophoresis showed that the DNA band was single and not dispersed significantly; 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 genomic DNA of the cucumber hybrids tested was between 10 and 30 ng/. mu.L.

2. And respectively taking the genomic DNA of 212 cucumber hybrids to be tested as templates, and respectively adopting 8 primer groups to carry out PCR amplification to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer named "F1", the primer named "F2" and the primer named "R" was 2:2: 5.

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, denaturation at 61-55 ℃ (touch down program is selected, reduction of 0.6 ℃ per cycle) is carried out, 1min is carried out, and amplification is carried out for 10 cycles; denaturation at 94 ℃ for 20s, renaturation at 55 ℃ and extension for 1min, and amplification is continued for 26 cycles.

3. And (3) after the step 2 is completed, when the temperature of each PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading the reading value of a FAM fluorescence label sequence under the wavelength of 485nm of exciting light and 520nm of emitted light, reading the reading value of an HEX fluorescence label sequence under the wavelength of 528nm of exciting light and 560nm of emitted light), and judging the genotype of 212 cucumber hybrids to be tested based on each SNP site according to the color of a fluorescence signal. The specific judgment principle is as follows: if a certain cucumber hybrid to be tested shows a blue fluorescent signal based on a certain SNP locus, the cucumber hybrid to be tested is homozygote based on the genotype of the SNP locus, wherein the complementary base is ' the complementary base of the 1 st base at the 3 ' end of the primer which amplifies the SNP locus and contains ' F1 ' in the name '; if a certain test cucumber hybrid shows a red fluorescent signal based on a certain SNP locus, the test cucumber hybrid is homozygotic based on the genotype of the SNP locus, namely the complementary base of the 1 st base at the 3' end of the primer which amplifies the SNP locus and contains F2 in the name; if a cucumber hybrid to be tested shows a green fluorescent signal based on a SNP site, the cucumber hybrid to be tested is a hybrid type based on the genotype of the SNP site, one base is a complementary base of the 1 st base at the 3 'end of the primer which amplifies the SNP site and contains F1 in the name, and the other base is a complementary base of the 1 st base at the 3' end of the primer which amplifies the SNP site and contains F2 in the name.

If the fluorescence signal is weak after the PCR amplification is finished and affects data analysis, cycles (denaturation at 94 ℃ for 20s, renaturation and extension at 55 ℃ for 1min and 5 cycles) can be added until the result is satisfactory.

Partial results are shown in FIG. 1. The results show that each primer group can obtain good parting effect in the tested cucumber hybrid.

4. Heterozygous site number distribution and efficiency assessment

(1) And (4) counting the number of heterozygous loci of each cucumber hybrid to be tested according to the genotypes of the 212 cucumber hybrids to be tested based on the 8 SNP loci.

The results of the distribution of the number of heterozygous sites of 212 cucumber hybrids tested, established on 8 primer sets, are shown in FIG. 2. The results show that 8 primer sets enable each cucumber hybrid tested to have at least one heterozygous site.

(2) The purity identification of the hybrid seeds can reduce the workload by adopting a sequential analysis mode.

The results show that the heterozygous site coverage of the 8 primer groups in 212 cucumber hybrids tested reaches 100%.

Therefore, the SNP primer combination developed in example 1 can be applied to purity identification of cucumber hybrids.

Example 3 detection of the purity of the Jingyan super-win hybrid Using the SNP primer combination developed in example 1

First, the SNP primer combination developed in example 1 is used to detect the purity of Jingyan super-win hybrid

1. Acquisition of genomic DNA of Jing-Ming-shou hybrid

(1) 200 commercially available Jingyan super hybrid seeds are planted to obtain Jingyan super hybrid seedlings.

(2) Randomly taking 96 leaves or roots of the seedlings of the Jing-Ming super hybrid, respectively extracting genome DNA by adopting a CTAB method, and sequentially obtaining 96 parts of the genome DNA of the Jing-Ming super hybrid.

2. Screening of primer set

(1) The genome DNA of 8 portions of the Jing research elite hybrid is used as a template, and 8 primer groups in the SNP primer combination developed in example 1 are respectively adopted for PCR amplification to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer named "F1", the primer named "F2" and the primer named "R" was 2:2: 5.

(2) And (2) after the step (1) is completed, when the temperature of each PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading values of FAM fluorescent label sequences are observed under the wavelength of 485nm exciting light and 520nm emitting light, reading values of HEX fluorescent label sequences are observed under the wavelength of 528nm exciting light and 560nm emitting light), and obtaining the color of a fluorescence signal. Comparing the strains of the 8 primer groups which show green fluorescent signals, wherein the primer group which shows the most green fluorescent strains is the screened primer group.

The results showed that the number of strains showing green fluorescence signals in the primer set 1 and the primer set 6 was the largest and 8 strains were both present. Therefore, the primer group 1 and the primer group 6 are the screened primer groups, and subsequent experiments are carried out.

3. Obtaining the purity of Jingyangyousheng hybrid

(1) And (3) performing PCR amplification by respectively adopting a primer group 1 and a primer group 6 by taking 96 parts of genome DNA of the Jing research elite hybrid as a template to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the primer named "F1", the primer named "F2" and the primer named "R" was 2:2: 5.

(2) And (2) after the step (1) is completed, when the temperature of each PCR amplification product is reduced to be below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading values of FAM fluorescent label sequences are observed under the wavelength of 485nm exciting light and 520nm emitting light, reading values of HEX fluorescent label sequences are observed under the wavelength of 528nm exciting light and 560nm emitting light), and obtaining the color of a fluorescence signal.

The SNP typing results are shown in FIG. 3 (the left panel shows primer set 1, and the right panel shows primer set 6).

(3) After completion of step (2), the number of strains showing green fluorescence and the number of strains showing no fluorescence (96-number of strains showing green fluorescence-number of strains showing red fluorescence-number of strains showing blue fluorescence) were counted for each of the primer set 1 and the primer set 6; calculating the purity of the Jing-Ming super-win hybrid according to the following formula; the average was further calculated to obtain the average purity.

The purity is the number of strains showing green fluorescence/(96-number of strains with no fluorescence of the primer set) × 100%.

The results showed that the number of strains showing green fluorescence in the primer set 1 was 93, the number of strains showing no fluorescence was 2, and the purity was 93/(96-2) ═ 98.94%; the number of strains showing green fluorescence of the primer group 6 was 95, the number of strains showing no fluorescence was 0, and the purity was 95/96-98.96%; the average purity of Jing Yun hybrid is (98.94% + 98.96%)/2 ═ 98.95%.

<110> agriculture and forestry academy of sciences of Beijing City

<120> a method for identifying purity of cucumber hybrid and SNP primer combination used therein

<160> 24

<170> PatentIn version 3.5

<210> 1

<211> 51

<212> DNA

<213> Artificial sequence

<400> 1

gaaggtgacc aagttcatgc tataaatata aggctagact agtagcaaaa g 51

<210> 2

<211> 52

<212> DNA

<213> Artificial sequence

<400> 2

gaaggtcgga gtcaacggat tgataaatat aaggctagac tagtagcaaa aa 52

<210> 3

<211> 31

<212> DNA

<213> Artificial sequence

<400> 3

gaagaagtct atgttttccc tctatctaaa a 31

<210> 4

<211> 47

<212> DNA

<213> Artificial sequence

<400> 4

gaaggtgacc aagttcatgc tcaacgagtt gttaatgctt tggagct 47

<210> 5

<211> 46

<212> DNA

<213> Artificial sequence

<400> 5

gaaggtcgga gtcaacggat taacgagttg ttaatgcttt ggagcc 46

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence

<400> 6

actgacactc caggagcaat ctcaa 25

<210> 7

<211> 52

<212> DNA

<213> Artificial sequence

<400> 7

gaaggtgacc aagttcatgc tgaatgaaag tttaatcaag gatgcaacta tt 52

<210> 8

<211> 51

<212> DNA

<213> Artificial sequence

<400> 8

gaaggtcgga gtcaacggat taatgaaagt ttaatcaagg atgcaactat g 51

<210> 9

<211> 31

<212> DNA

<213> Artificial sequence

<400> 9

agattttgtg ccaatagagc aaagtcatat a 31

<210> 10

<211> 46

<212> DNA

<213> Artificial sequence

<400> 10

gaaggtgacc aagttcatgc tttatcaaac aactcgacgg cttcga 46

<210> 11

<211> 46

<212> DNA

<213> Artificial sequence

<400> 11

gaaggtcgga gtcaacggat tttatcaaac aactcgacgg cttcgt 46

<210> 12

<211> 30

<212> DNA

<213> Artificial sequence

<400> 12

gattgcactt tatgggaaat gtgggttttt 30

<210> 13

<211> 48

<212> DNA

<213> Artificial sequence

<400> 13

gaaggtgacc aagttcatgc tcggcataat atgaatcaac ctggtaaa 48

<210> 14

<211> 47

<212> DNA

<213> Artificial sequence

<400> 14

gaaggtcgga gtcaacggat tggcataata tgaatcaacc tggtaag 47

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<400> 15

gaccaccaag gcctatcact ccaa 24

<210> 16

<211> 49

<212> DNA

<213> Artificial sequence

<400> 16

gaaggtgacc aagttcatgc tcatatacac acaatacata gctgttgga 49

<210> 17

<211> 48

<212> DNA

<213> Artificial sequence

<400> 17

gaaggtcgga gtcaacggat tatatacaca caatacatag ctgttggg 48

<210> 18

<211> 31

<212> DNA

<213> Artificial sequence

<400> 18

ctctgccaga aacaaacaat aacttttgta a 31

<210> 19

<211> 50

<212> DNA

<213> Artificial sequence

<400> 19

gaaggtgacc aagttcatgc ttacaataat tagaggcctt aaacagagta 50

<210> 20

<211> 48

<212> DNA

<213> Artificial sequence

<400> 20

gaaggtcgga gtcaacggat tcaataatta gaggccttaa acagagtc 48

<210> 21

<211> 35

<212> DNA

<213> Artificial sequence

<400> 21

aaatctttaa tcctatattt tcttaagcat tgcaa 35

<210> 22

<211> 48

<212> DNA

<213> Artificial sequence

<400> 22

gaaggtgacc aagttcatgc tagactagca aagagtaata tggttgac 48

<210> 23

<211> 49

<212> DNA

<213> Artificial sequence

<400> 23

gaaggtcgga gtcaacggat tgagactagc aaagagtaat atggttgat 49

<210> 24

<211> 31

<212> DNA

<213> Artificial sequence

<400> 24

tacatccgtt atggaatttt gagttacact a 31

Claims

The SNP primer combination comprises a primer group 1 for amplifying an HGSNP01 locus, a primer group 2 for amplifying an HGSNP02 locus, a primer group 3 for amplifying an HGSNP03 locus, a primer group 4 for amplifying an HGSNP04 locus, a primer group 5 for amplifying an HGSNP05 locus, a primer group 6 for amplifying an HGSNP06 locus, a primer group 7 for amplifying an HGSNP07 locus and a primer group 8 for amplifying an HGSNP08 locus;

site HGSNP 01-site HGSNP08 is 8 SNP sites of cucumber genome;

the HGSNP01 site is the 16975775 th nucleotide on chromosome 4; the HGSNP02 site is the 9582207 th nucleotide on chromosome 5; the locus HGSNP03 is the 17508118 th nucleotide on chromosome 1; the locus HGSNP04 is the 1976800 th nucleotide on chromosome 1; the HGSNP05 site is the 4054461 th nucleotide on chromosome 5; the HGSNP06 site is the 20929474 th nucleotide on chromosome 6; the HGSNP07 site is the 8333363 th nucleotide on chromosome 3; the HGSNP08 site is the 2133319 th nucleotide on chromosome 5;

the positions of the 8 SNP loci on the chromosome are determined based on the alignment of the cucumber 9930 reference genome sequence, and the version number of the cucumber 9930 reference genome sequence is V2.
2. A SNP primer set according to claim 1 characterised in that:

the primer group 1 consists of SEQ ID NO: 1, forward primer 1F1, SEQ ID NO: 2 and the forward primer 1F2 shown in SEQ ID NO: 3, and a reverse primer 1R;

the primer group 2 consists of SEQ ID NO: 4, forward primer 2F1, SEQ ID NO: 5 and the forward primer 2F2 shown in SEQ ID NO: 6 is shown as a reverse primer 2R;

the primer group 3 consists of SEQ ID NO: 7, forward primer 3F1, SEQ ID NO: 8 and the forward primer 3F2 shown in SEQ ID NO: 9, and a reverse primer 3R;

the primer group 4 consists of SEQ ID NO: 10, forward primer 4F1, SEQ ID NO: 11 and the forward primer 4F2 shown in SEQ ID NO: 12, and a reverse primer 4R;

the primer group 5 consists of SEQ ID NO: 13, forward primer 5F1, SEQ ID NO: 14 and the forward primer 5F2 shown in SEQ ID NO: 15, and a reverse primer 5R;

the primer group 6 consists of SEQ ID NO: 16, forward primer 6F1 shown in SEQ ID NO: 17 and the forward primer 6F2 shown in SEQ ID NO: 18, and a reverse primer 6R;

the primer group 7 consists of SEQ ID NO: 19, forward primer 7F1, SEQ ID NO: 20 and the forward primer 7F2 shown in SEQ ID NO: 21, and a reverse primer 7R;

the primer group 8 consists of SEQ ID NO: 22, forward primer 8F1, SEQ ID NO: 23 and the forward primer 8F2 shown in SEQ ID NO: 24, and a reverse primer 8R.
3. A SNP primer set according to claim 1 characterised in that:

the primer group 1 consists of SEQ ID NO: 1, forward primer 1F1 shown at positions 22 to 51 from the 5' end, SEQ ID NO: 2 from position 22 to 52 from the 5' end and the forward primer 1F2 shown in SEQ ID NO: 3, and a reverse primer 1R;

the primer group 2 consists of SEQ ID NO: 4, forward primer 2F1 shown at positions 22 to 47 from the 5' end, SEQ ID NO: 5 forward primer 2F2 at positions 22 to 46 from the 5' end and SEQ ID NO: 6 is shown as a reverse primer 2R;

the primer group 3 consists of SEQ ID NO: 7, forward primer 3F1 shown at positions 22 to 52 from the 5' end, SEQ ID NO: 8 from position 22 to 51 from the 5' end and the forward primer 3F2 shown in SEQ ID NO: 9, and a reverse primer 3R;

the primer group 4 consists of SEQ ID NO: 10 from position 22 to 46 from the 5' end, 4F1, SEQ ID NO: 11 from position 22 to 46 from the 5' end and the forward primer 4F2 shown in SEQ ID NO: 12, and a reverse primer 4R;

the primer group 5 consists of SEQ ID NO: 13 from position 22 to 48 from the 5' end, 5F1, SEQ ID NO: 14 from the 5' end, the forward primer 5F2 shown at positions 22 to 47 and SEQ ID NO: 15, and a reverse primer 5R;

the primer group 6 consists of SEQ ID NO: 16 from position 22 to 49 from the 5' end, a forward primer 6F1, SEQ ID NO: 17 from position 22 to 48 from the 5' end and a forward primer 6F2 shown in SEQ ID NO: 18, and a reverse primer 6R;

the primer group 7 consists of SEQ ID NO: 19 from position 22 to 50 from the 5' end, 7F1, SEQ ID NO: 20 from position 22 to 48 from the 5' end and the forward primer 7F2 shown in SEQ ID NO: 21, and a reverse primer 7R;

the primer group 8 consists of SEQ ID NO: 22 from position 22 to 48 from the 5' end, 8F1, SEQ ID NO: 23 from position 22 to 49 from the 5' end, 8F2 and SEQ ID NO: 24, and a reverse primer 8R.
4. Use of the SNP primer set according to any one of claims 1 to 3, being x1) or x 2):

x1) preparing a kit for identifying the purity of cucumber hybrids;

x2) identifying the purity of the cucumber hybrid.
5. A method for identifying the purity of cucumber hybrid seeds to be detected comprises the following steps:

(a1) obtaining genome DNA of N cucumber hybrid seeds to be detected; n is a natural number greater than 95;

(a2) respectively taking the genomic DNA of 8-12 cucumber hybrids to be detected obtained in the step (a1) as templates, and respectively carrying out PCR amplification by using 8 primer groups in the SNP primer combination of claim 2 to obtain corresponding PCR amplification products;

(a3) after the step (a2) is completed, detecting the fluorescent signal of each PCR amplification product by using an instrument, and counting the number of strains of which the green fluorescent signals are displayed by 8 primer groups; the primer group with the largest number of green fluorescent strains is the target primer group;

SEQ ID NO: 1, the nucleotide sequence from 1 st to 21 st from the 5' end is a fluorescent label sequence, and the fluorescent signal is blue; SEQ ID NO: 2, the nucleotide sequence from 1 st to 21 st from the 5' end is a fluorescent label sequence, and the fluorescent signal is red;

if a blue fluorescent signal is displayed, the primer that amplifies the SNP site and contains F1 in the name is homozygous for the complementary base of the 1 st base at the 3' end based on the genotype of the SNP site; if a red fluorescent signal is displayed, the primer that amplifies the SNP site and contains F2 in the name is homozygous for the complementary base of the 1 st base at the 3' end based on the genotype of the SNP site; if a green fluorescent signal is displayed, the genotype based on the SNP site is heterozygous, one base is a complementary base of the 1 st base at the 3 'end of the primer which amplifies the SNP site and contains F1 in the name, and the other base is a complementary base of the 1 st base at the 3' end of the primer which amplifies the SNP site and contains F2 in the name;

(a4) respectively taking the genome DNA of the N cucumber hybrid seeds to be detected obtained in the step (a1) as templates, and respectively adopting a target primer group to carry out PCR amplification to obtain corresponding PCR amplification products;

(a5) and (a4) after the step (a), detecting the fluorescent signal of each PCR amplification product by using an instrument, and obtaining the purity of the cucumber hybrid to be detected according to the color of the fluorescent signal.
6. The method of claim 5, wherein: the method for obtaining the purity of the cucumber hybrid to be detected according to the color of the fluorescent signal comprises the following steps: counting the number of strains showing green fluorescence and the number of strains without fluorescence of each target primer group, respectively calculating the purity, and then calculating the average value;

number of non-fluorescent plants = N-number of plants showing green fluorescence-number of plants showing red fluorescence-number of plants showing blue fluorescence;

purity = number of strains showing green fluorescence/(N-number of strains showing no fluorescence of the target primer set) × 100%.
7. A method for identifying the purity of cucumber hybrid seeds to be detected comprises the following steps:

(b1) obtaining genome DNA of N cucumber hybrid seeds to be detected; n is a natural number greater than 95;

(b2) respectively taking the genomic DNA of 8-12 cucumber hybrids to be detected obtained in the step (b1) as templates, and respectively adopting 8 primer groups in the SNP primer combination of claim 2 or 3 to carry out PCR amplification to obtain corresponding PCR amplification products;

(b3) sequencing each PCR amplification product obtained in the step (b 2); according to the sequencing result, obtaining the number of strains which are heterozygous based on the genotypes of the 8 SNP sites, wherein the primer group with the largest number of strains is the target primer group;

(b4) respectively taking the genome DNA of the N cucumber hybrid seeds to be detected obtained in the step (b1) as templates, and respectively adopting a target primer group to carry out PCR amplification to obtain corresponding PCR amplification products;

(b5) sequencing each PCR amplification product obtained in the step (b 4); and obtaining the purity of the cucumber hybrid to be detected according to the sequencing result.
8. The method of claim 7, wherein: the method for obtaining the purity of the cucumber hybrid to be detected according to the sequencing result comprises the following steps: counting the number of strains of each target primer group which are heterozygous based on the genotype of the SNP locus and the number of strains of which PCR amplification products are not obtained, respectively calculating the purity, and then calculating the average value;

purity = number of strains with the target primer set heterozygous based on the genotype of the SNP site/(N-number of strains with no PCR amplification product obtained by the target primer set) × 100%.