KASP marker closely linked with cucumber fruit length regulation gene CsERF1a and application of KASP marker

Abstract

The invention discloses a KASP marker closely linked with a cucumber fruit length regulation gene CsERF1a and application of the KASP marker. The SNP primer combination provided by the invention comprises a first primer group and / or a second primer group, each primer group consists of three primer sequences and is used for amplifying an SNP (Single Nucleotide Polymorphism) site; the nucleotide sequences of the primers are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 6. The SNP primer combination can be used for early identification in the seed or seedling stage of cucumbers, so that screening of materials of different fruit length types is accelerated, and technical support is provided for cucumber fruit length breeding. 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

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a gene that regulates cucumber fruit length. CsERF1a Closely linked KASP tags and their applications. Background Technology

[0002] Cucumber is one of the important cucurbit crops in my country, accounting for 6.2% of the national vegetable cultivation area. Fruit length is an important agronomical trait that directly affects cucumber yield and quality, and it is also a major reference indicator for classifying cucumber ecological types. During the long process of evolution and domestication, the fruit length of cucumbers in different ecological types has shown significant differences due to selection. Wild-type cucumbers have a fruit length of about 4 cm, European fruit-type cucumbers are about 10–18 cm long (Wang Caixia, High-Quality and High-Yield Cultivation Technology of Fruit Cucumbers in Solar Greenhouses, Modern Agriculture, 2020); South China-type cucumbers have a fruit length of about 18–25 cm, while North China-type densely spined cucumbers typically have a fruit length exceeding 25 cm (Zhou Xiuyan et al., Evaluation of the Commerciality of Cucumber Variety Resources, Journal of Northeast Agricultural University, 2005). Cucumber fruit length is easily affected by cultivation and environmental conditions. In addition to auxins, ethylene plays a key dosage-effect in regulating cucumber fruit length. Appropriate amounts of ethylene can promote fruit cell division and fruit elongation, while excessively high or low ethylene concentrations will inhibit cell division, leading to shorter fruits. Currently, the QTLs affecting cucumber fruit length vary among different genetic populations under different cultivation environments. The Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, constructed a recombinant inbred line population using wild cucumber PI183967 and Xintai Mici as parents, and identified four QTLs related to fruit length. Fl3.1 , Fl4.1 , Fl5.1 , Fl6.1 (Wang et al., 2013). The University of Wisconsin, using a population of recombinant inbred lines constructed from Gy14 and 9930, detected a total of 29 QTLs related to fruit length at three stages of fruit development. Among them, 12 QTLs had the same interval, including qFL3.1 (Weng et al., 2015). qFL3.1 It is a stable QTL located on cucumber chromosome 3 that affects fruit length. Currently, candidate genes within the QTL region have not been cloned and their functions verified, and research on the genetic mechanism controlling cucumber fruit length is still very weak.

[0003] Third-generation molecular markers (SNPs) have received widespread attention due to their advantages such as large number, wide distribution, and genetic stability. With the development of high-throughput sequencing technology and the continuous reduction of sequencing costs, whole-genome sequencing based on the DNA of individuals with mixed extreme traits (BSA, bulked segregant analysis) can rapidly locate major genes controlling cucumber fruit length, discover more stable and efficient SNP sites, and develop their specific KASP molecular markers, thus establishing a high-throughput molecular marker-assisted breeding technology for cucumber fruit length traits. Summary of the Invention

[0004] To address the problems of lack of markers for cucumber fruit length and difficulty in targeted improvement, this invention has identified a key gene that regulates cucumber fruit length. CsERF1a They also developed a gene specifically regulating fruit length. CsERF1a Tightly linked KASP molecular markers.

[0005] One objective of this invention is to provide an SNP primer combination for detecting genes that regulate cucumber fruit length. CsERF1a Primer combinations for tightly linked KASP marker genotypes.

[0006] The second objective of this invention is to provide a reagent or kit for detecting the genotype of the aforementioned molecular markers.

[0007] A third objective of this invention is to provide applications of the above-mentioned primer combinations or kits.

[0008] The fourth objective of this invention is to provide a method for identifying the length type of a cucumber fruit to be tested.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of this invention provides an SNP primer set, comprising a first primer set and / or a second primer set. The first primer set is used to identify the genotype of the SNP01 locus on the cucumber genome, and the second primer set is used to identify the genotype of the SNP02 locus on the cucumber genome. The SNP01 locus is nucleotide 1972403 on chromosome 3 of the cucumber genome, with a base of T or A; the SNP02 locus is nucleotide 1972889 on chromosome 3 of the cucumber genome, with a base of T or G. The positions of the SNP01 and SNP02 loci in the genome are determined based on the cucumber reference genome sequence, the version number of which is 9930 V3.

[0010] In the SNP primer combination of the first aspect described above, the first primer set further comprises the first forward primer shown in SEQ ID NO: 1, the second forward primer shown in SEQ ID NO: 2, and the first reverse primer shown in SEQ ID NO: 3; the second primer set comprises the third forward primer shown in SEQ ID NO: 4, the fourth forward primer shown in SEQ ID NO: 5, and the second reverse primer shown in SEQ ID NO: 6.

[0011] In the SNP primer combination of the first aspect described above, the first primer set further comprises a first forward primer, a second forward primer, and a first reverse primer, wherein the first forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 1 from position 22 to 43 from the 5' end, the second forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 2 from position 22 to 43 from the 5' end, and the nucleotide sequence of the first reverse primer is shown in SEQ ID NO: 3; The second primer set consists of a third forward primer, a fourth forward primer, and a second reverse primer, wherein the third forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 4 from position 22 to 40 from the 5' end, the fourth forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 5 from position 22 to 39 from the 5' end, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO: 6.

[0012] Furthermore, the 5' ends of the first and second forward primers are respectively supplemented with fluorescent tag sequences emitting different fluorescent signals. The 5' ends of the third and fourth forward primers are also supplemented with fluorescent tag sequences emitting different fluorescent signals. The fluorescent tags can be selected from commonly used tags in the field, such as FAM and HEX.

[0013] The second aspect of this invention provides the application of the SNP primer combination of the first aspect described above in any of the following aspects (x1)-(x3): (x1) Prepare a kit for identifying whether cucumber fruits are short-fruited or long-fruited; (x2) Identify whether cucumber fruits are short-fruited or long-fruited; (x3) Select cucumber varieties with short or long fruit lengths; When identifying or selecting cucumber fruits of short or long fruit type, the following judgment is made based on the genotype at SNP01 and / or the genotype at SNP02: If the genotype of the cucumber to be tested is homozygous TT based on SNP01 and / or homozygous TT based on SNP02, then the cucumber to be tested is or is suspected to be a short fruit type; if the genotype of the cucumber to be tested is homozygous AA based on SNP01 and / or homozygous GG based on SNP02, then the cucumber to be tested is or is suspected to be a long fruit type.

[0014] A third aspect of the present invention provides a reagent or kit comprising the SNP primer combination described in the first aspect.

[0015] The fourth aspect of this invention provides the application of the kit described in the third aspect in identifying whether cucumber fruits are short-fruited or long-fruited, or in selecting cucumber varieties with short or long fruit lengths. When identifying or selecting cucumber fruits of short or long fruit type, the following judgment is made based on the genotype at the SNP01 locus and / or the genotype at the SNP02 locus: if the genotype of the cucumber to be tested is homozygous for TT based on the SNP01 locus and / or homozygous for TT based on the SNP02 locus, then the cucumber to be tested is or is suspected to be a short-fruited variety; if the genotype of the cucumber to be tested is homozygous for AA based on the SNP01 locus and / or homozygous for GG based on the SNP02 locus, then the cucumber to be tested is or is suspected to be a long-fruited variety.

[0016] The fifth aspect of this invention provides a method for identifying whether a cucumber to be tested is a long or short fruit, comprising the following steps: detecting the genotype of the cucumber to be tested based on the SNP01 locus and / or the SNP02 locus, and then making the following judgment: if the genotype of the cucumber to be tested based on the SNP01 locus is homozygous TT and / or the genotype based on the SNP02 locus is homozygous TT, then the cucumber to be tested is or is suspected to be a short fruit; if the genotype of the cucumber to be tested based on the SNP01 locus is homozygous AA and / or the genotype based on the SNP02 locus is homozygous GG, then the cucumber to be tested is or is suspected to be a long fruit; the length of the short fruit is less than or equal to 18 cm, and the length of the long fruit is greater than 25 cm.

[0017] In the above method, the method for detecting the genotype of the cucumber to be tested based on the SNP01 locus and / or the SNP02 locus is as follows: (a1) Using the genomic DNA of the cucumber to be tested as a template, PCR amplification was performed using the primer sets in the above SNP primer combinations to obtain PCR amplification products; (a2) The fluorescence signal of the PCR amplification product obtained in step (a1) is detected by an instrument, and the genotype of the cucumber to be tested based on the SNP01 site and / or the SNP02 site is obtained according to the color of the fluorescence signal. For the PCR amplification product obtained with the first primer set, when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the first forward primer, the genotype of the SNP01 site is homozygous for the 3' terminal nucleotide of the first forward primer; when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the second forward primer, the genotype of the SNP01 site is homozygous for the 3' terminal nucleotide of the second forward primer. For the PCR amplification product obtained with the second primer set, when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the third forward primer, the genotype of the SNP02 site is homozygous for the 3' terminal nucleotide of the third forward primer; when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the fourth forward primer, the genotype of the SNP02 site is homozygous for the 3' terminal nucleotide of the fourth forward primer.

[0018] In the above method, the method for detecting the genotype of the cucumber to be tested based on the SNP01 locus and / or the SNP02 locus is as follows: (b1) Using the genomic DNA of the cucumber to be tested as a template, PCR amplification was performed using the primer sets in the above SNP primer combinations to obtain PCR amplification products; (b2) Take the PCR amplification product obtained in step (b1) and sequence it; (b3) Based on the sequencing results obtained in step (b2), obtain the genotype of the cucumber to be tested based on the SNP01 site and / or the SNP02 site.

[0019] In this invention, preferably, the length of short-fruited cucumber is less than or equal to 18cm, and the length of long-fruited cucumber is greater than 25cm.

[0020] The cucumber varieties tested in this invention are selected from Sasha No. 1, Nanshui No. 2, Jingyan No. 4, Heishui No. 1, Biyu No. 3, Qingxiu No. 3, Qiande 1217, Jinmei No. 6, Hayan No. 1, Liyuan 966, Xinxiu No. 1, Lvwang, Tony 106, Jingyan Mini No. 6, Biyu No. 2, Yanxiu, Bicui 19, Hayan No. 4, Zhexiu No. 3, Tony 102, Jingyan Mini No. 8, Jingyan Mini No. 9, Ouman, Jenny 102, Yijia Xinxiu, Lvjingling No. 4, Guangliang 1618, Junke No. 11, Jingyan Mini No. 5, Mingxing No. 8, Liangshen Duanzun, Deruit 345, Shengfeng No. 50, Jinlv No. 17, Jinyue 301, Lushu No. 551, Deruit 298, Sheng Meifuman 16, Jinyou 501, Bomei 805, Deruit D81, Shengmei 909, Liangyou M162, Jinyou 316, Bonai 808, Bojie 16, Liangyou B05, Jinyou No. 8, Jinyou 636, Chunguang, Jinyou 358, Nongjiale, Lvfeicui, Tianwei No. 1, Boxin L73, Bomei 618, Bomei 22, Deruit F7, Deruit 17, Bomei No. 9, Jinchun 19, Liangyou H06, Liangyou B12, Jinsheng No. 2, Jinxiu 1601, Best 908, Senongfeng Liangjian, Jinyou 601, Huanong 402, Deruit 351, Liangyou H08, Zhexiu 302, Lvcui You No. 1, Jinyou No. 109, Qianchun 858.

[0021] Compared with existing technologies, the present invention has the following advantages: The SNP primer combination provided by the present invention can be used for early identification of cucumber seeds or seedlings, accelerate the directional improvement of fruit length, and provide technical support for the breeding of cucumber fruit length traits. The method provided by the present invention has the advantages of high throughput, accuracy, low cost, simple operation, and saving manpower and material resources, and has a very broad application prospect. Attached Figure Description

[0022] Figure 1 Genes regulating cucumber fruit length were constructed and sequenced using mixed-pool sequencing to identify them in the F2 population. CsERF1a In image a, the left image shows photos of the parent cucumbers, and the right image shows photos of some cucumbers from the F2 population; image b shows mixed-pool sequencing to locate genes regulating cucumber fruit length. CsERF1a The result.

[0023] Figure 2 To and CsERF1a A schematic diagram of the locations of two closely linked SNP molecular markers and the changes in haplotypes and fruit length in 50 cucumber variants.

[0024] Figure 3 The SNP typing results of 96 tested cucumber varieties were obtained using primer group 1.

[0025] Figure 4 The SNP typing results of 96 tested cucumber varieties were obtained using primer group 2.

[0026] Figure 5 The SNP typing results of 12 cucumber varieties were obtained using primer group 1.

[0027] Figure 6 The SNP typing results of 12 cucumber varieties to be tested were obtained using primer group 2. Detailed Implementation

[0028] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the scope of the invention.

[0029] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0030] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0031] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0032] Example 1: Related to cucumber fruit length regulating genes CsERF1a Obtaining closely linked SNP sites I. Genes regulating cucumber fruit length CsERF1a The discovery This invention utilizes the male parent (13-c43) and female parent (12-c23-13) of the Jingyan Lulinglong 6 cucumber variety to construct a segregating F2 population of 300 plants. The two parents exhibit significant differences in fruit length: the male parent has an average fruit length of 28.5 cm, while the female parent has an average fruit length of 10.3 cm. Significant segregation in fruit length is observed in the F2 population (e.g., ...). Figure 1(As shown on the left side of image a). Further, 20 individual plants with extreme fruit lengths were selected from the F2 population for pooled sequencing analysis. Both parents yielded 10G of sequencing data, and each of the two extreme pools yielded 20G of sequencing data. SNPs with fewer than 5 read support and multiple alleles were filtered out, and homozygous inconsistent SNPs in the parents were screened for subsequent analysis. The deletion rate (NA_rate), homozygous mutant genotype ratio (Hom_alt_rate), heterozygous mutant genotype ratio (Het_alt_rate), and non-mutated genotype ratio (Ref_rate) of each core SNP in all tested samples were calculated. Using the paternal parent (extremely long fruit length trait) as a reference, the SNP-index (i.e., the frequency of SNPs) of the marker sites (i.e., homozygous inconsistent SNP sites in the parents) after filtering between the parents in the two offspring pools were analyzed and calculated. SNPs completely identical to the reference parent had an SNP-index of 0, and SNPs completely different from the reference parent had an SNP-index of 1. The difference in SNP-index between the two progeny pools, Δ(SNP-index), was calculated. The stronger the association between the marked SNP and the trait, the closer Δ(SNP-index) was to 1. At a 95% confidence level, windows exceeding the positive threshold were identified as candidate regions, located near 1.9 Mb on chromosome 3. Further analysis of variant sites and gene annotation information identified the candidate gene in this region as an ethylene-responsive ERF transcription factor. CsERF1a ( Figure 1 b). The inventors of this invention further combined 50 cucumber resequencing data (data source: www.vegsnpdb.cn) and found that there were 2 functional SNP sites (as shown in Table 1). CsERF1a The two SNP loci can form three haplotypes in cucumber, and the different haplotypes show significant differences among cucumber populations. Haplotype 1 is mainly found in European cucumbers, haplotype 2 is mainly found in South China cucumbers, and haplotype 3 is mainly found in North China spiny cucumbers. Figure 2 ).

[0033] The location of the aforementioned SNP sites on the chromosome was determined based on the alignment of the cucumber reference genome sequence, version number 9930V3 (downloadable from: http: / / cucurbitgenomics.org / v2 / ftp / genome / cucumber / Chinese_long / v3 / ).

[0034] Table 1. Genes regulating cucumber fruit length CsERF1a Basic information of the two SNP sites

[0035] II. Obtaining SNP primer combinations for identifying cucumber fruit length 1. After completing step one, the inventors of this invention, based on CsERF1a Two SNP variant sites on the gene were used to develop a combination of SNP primers for identifying cucumber fruit length.

[0036] 2. The SNP primer set consists of two primer sets, the names of which are shown in column 2 of Table 2. Each primer set consists of three primer sequences used to amplify one SNP site. The nucleotide sequences of each primer in the two primer sets are shown in column 4 of Table 2.

[0037] Table 2. SNP primer combinations used to identify cucumber fruit length

[0038] Note: A single underscore indicates a FAM fluorescent tag sequence, and a double underscore indicates a HEX fluorescent tag sequence.

[0039] Example 2: Validation of the SNP primer combinations developed in Example 1 To verify CsERF1a The effect of different haplotypes on cucumber fruit length was investigated in this example. SNP genotyping and phenotypic analysis of cucumber fruit length were performed on 114 commercial cucumber varieties. Basic information for the 114 tested cucumber varieties is shown in columns 1-4 of Table 3. All 114 tested cucumber varieties were common commercial varieties. Based on phenotype, cucumbers with a fruit length greater than 25 cm were classified as long cucumbers, those with a fruit length less than or equal to 18 cm as short cucumbers, and those with a fruit length greater than 18 cm and less than or equal to 25 cm as medium cucumbers. The classification of cucumber fruit length was based on the lengths of European fruit-type cucumbers, South China-type cucumbers, and North China densely spined cucumbers. Of the 114 tested cucumber varieties, 57 were long cucumbers, 13 were medium cucumbers, and 44 were short cucumbers (Table 3).

[0040] Table 3.1 Fruit length and genetic information of 114 tested cucumber varieties CsERF1a haplotype

[0041]

[0042]

[0043] 1. Obtaining genomic DNA from the tested cucumber varieties Genomic DNA was extracted from the leaves of 114 tested cucumber varieties using the SDS method.

[0044] The quality and concentration of genomic DNA from the tested cucumber varieties must meet the requirements for PCR. The standards are as follows: agarose gel electrophoresis shows a single DNA band without obvious diffusion; the A260 / A280 ratio detected by a Nanodrop2000 (Thermo) UV spectrophotometer is around 1.8, and the A260 / A230 ratio is greater than 1.8; the concentration of genomic DNA from the tested cucumber varieties is 10-30 ng / μL.

[0045] 2. Using genomic DNA from 114 tested cucumber varieties as templates, PCR amplification was performed using two primer sets to obtain the corresponding PCR products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2", and primers containing "R" was 2:2:5.

[0046] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0047] 3. After completing step 2, when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning with the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of each SNP locus of the 114 tested cucumber varieties is determined based on the fluorescence signal color. The specific judgment principles are as follows: If a tested cucumber variety shows a blue fluorescent signal based on a certain SNP locus, then the genotype of the tested cucumber variety based on that SNP locus is homozygous for the 3' terminal first base of the primer that amplifies the SNP locus and whose name contains "F1"; if a tested cucumber variety shows a red fluorescent signal based on a certain SNP locus, then the genotype of the tested cucumber variety based on that SNP locus is homozygous for the 3' terminal first base of the primer that amplifies the SNP locus and whose name contains "F2"; if a tested cucumber variety shows a green fluorescent signal based on a certain SNP locus, then the genotype of the tested cucumber variety based on that SNP locus is heterozygous, with one base being the 3' terminal first base of the primer that amplifies the SNP locus and whose name contains "F1", and the other base being the 3' terminal first base of the primer that amplifies the SNP locus and whose name contains "F2". In this invention, when the genotypes of two SNP loci are heterozygous, they cannot be used to determine the cucumber fruit length type.

[0048] It should be noted that if the fluorescence signal is weak after PCR amplification, affecting data analysis, additional cycles can be added (94℃ denaturation for 20s, 55℃ annealing and extension for 1min, 5 cycles) until the results are satisfactory.

[0049] Partial results of primer set 1 are shown below Figure 3 The statistical results are shown in column 5 of Table 3.

[0050] Partial results of primer set 2 are shown below Figure 4 The statistical results are shown in column 6 of Table 3.

[0051] The results showed that the two primer sets could achieve good typing results in 114 tested cucumber varieties, and the typing results were highly consistent with the phenotypes.

[0052] 4. Evaluation of fruit length types of 114 tested cucumber varieties using two primer pairs. (1) The genotypes of 114 tested cucumber varieties based on SNP01 were statistically analyzed.

[0053] The results showed that 41 cucumber varieties tested, based on the SNP01 genotype being homozygous for TT, were identified as short-fruited cucumbers; among them, 29 varieties were indeed short-fruited cucumbers, with a uniformity of 70.7% (29 / 41 × 100% = 70.7%). 73 cucumber varieties tested, based on the SNP01 genotype being homozygous for AA, were identified as long-fruited cucumbers; 51 varieties were indeed long-fruited cucumbers, with a uniformity of 69.9% (51 / 73 × 100% = 69.9%).

[0054] (2) The genotypes of 114 tested cucumber varieties based on SNP02 were statistically analyzed.

[0055] The results showed that 56 cucumber varieties tested, based on the SNP02 genotype being homozygous for TT, were identified as short-fruited cucumbers; among them, 36 varieties were indeed short-fruited cucumbers, with a uniformity of 64.3% (36 / 56 × 100% = 64.3%). 58 cucumber varieties tested, based on the SNP02 genotype being homozygous for GG, were identified as long-fruited cucumbers; 46 varieties were indeed long-fruited cucumbers, with a uniformity of 79.3% (46 / 58 × 100% = 79.3%).

[0056] (3) Forty-one cucumber varieties were tested based on both the TT homozygous genotype of SNP01 and the TT homozygous genotype of SNP02, and were identified as short-fruited cucumbers; among them, 29 varieties exhibited the short-fruited cucumber phenotype, with a uniformity of 70.7% (29 / 41×100%=70.7%). Fifty-eight cucumber varieties were tested based on both the AA homozygous genotype of SNP01 and the GG homozygous genotype of SNP02, and were identified as long-fruited cucumbers; among them, 46 varieties exhibited the long-fruited cucumber phenotype, with a uniformity of 79.3% (46 / 58×100%=79.3%).

[0057] Therefore, the SNP primer combination developed in Example 1 can identify the length of cucumber fruits. The accuracy of identifying cucumber fruit length is higher when using the combination of primer set 1 and primer set 2. Specifically, the method for determining the length of the cucumber fruit to be tested based on the SNP01 and SNP02 genotypes is as follows: When both the SNP01 and SNP02 genotypes are homozygous (TT), the length of the cucumber fruit to be tested is determined to be short, and the haplotype is Hap1. When both the SNP01 and SNP02 genotypes are homozygous (AA), the length of the cucumber fruit to be tested is determined to be long, and the haplotype is Hap3. When both the SNP01 and SNP02 genotypes are homozygous (TT), the haplotype is Hap2. In this case, the phenotypic differences in cucumber fruit length are large and unstable; therefore, the length type of the cucumber fruit to be tested cannot be determined when the haplotype is Hap2.

[0058] Example 3: Detection of fruit length type in cucumber varieties using the SNP primer combination developed in Example 1. The cucumber varieties to be tested are cucumber variety 1 to cucumber variety 12.

[0059] 1. The fruit length type of the cucumber variety under test was detected using the SNP primer combination developed in Example 1. (1) Obtaining genomic DNA from the cucumber variety to be tested Seeds of the cucumber variety to be tested were planted to obtain seedlings; leaves or roots of the cucumber variety to be tested were taken, and genomic DNA was extracted using the SDS method to obtain the genomic DNA of the cucumber variety to be tested.

[0060] (2) Using the genomic DNA of the cucumber variety to be tested as a template, PCR amplification was performed using primer set 1 and primer pair 2 respectively to obtain the corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of primers containing "F1", primers containing "F2" and primers containing "R" in the name was 2:2:5.

[0061] The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (using the touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ annealing & extension for 1 min, and continued amplification for 26 cycles.

[0062] (3) After completing step (2), when the temperature of each PCR amplification product drops below 40℃, the fluorescence value is read by scanning the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is observed at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is observed at an excitation wavelength of 528nm and an emission wavelength of 560nm). The fluorescence signal color is obtained, and the following judgment is made: If the tested cucumber variety shows a blue fluorescence signal based on a certain SNP site, then the genotype of the tested cucumber variety based on the SNP site is homozygous for the primer that amplifies the SNP site and whose name contains "F1" at the 3' end; if a tested cucumber variety shows a red fluorescence signal based on a certain SNP site, then the genotype of the tested cucumber variety based on the SNP site is homozygous for the primer that amplifies the SNP site and whose name contains "F2" at the 3' end.

[0063] The SNP typing results of 12 cucumber varieties tested using primer group 1 are shown below. Figure 5 .

[0064] The SNP typing results of 12 cucumber varieties tested using primer group 2 are shown below. Figure 6 .

[0065] The statistical results are shown in columns 2-3 of Table 4. The tested cucumber varieties with homozygous TT genotypes for both SNP01 and SNP02 were classified as short-fruited cucumbers. The tested cucumber varieties with homozygous AA genotypes for both SNP01 and SNP02 were classified as long-fruited cucumbers.

[0066] Table 4. Identification of Fruit Length Types of 12 Cucumber Varieties Tested

[0067] 2. Based on the phenotype, determine the fruit length type of the cucumber variety to be tested.

[0068] The statistical results are shown in column 4 of Table 3.

[0069] The results showed that the consistency between the SNP primer combination developed in Example 1 and the phenotypic identification of cucumber fruit length was 83.3% (10 / 12 × 100% = 83.3%). Therefore, the SNP primer combination developed in Example 1 can identify the fruit length type of the tested cucumber variety.

Claims

1. An SNP primer combination, characterized in that, The SNP primer set includes a first primer set and / or a second primer set. The first primer set is used to identify the genotype of the SNP01 locus on the cucumber genome, and the second primer set is used to identify the genotype of the SNP02 locus on the cucumber genome. The SNP01 site is the 1972403rd nucleotide on chromosome 3 of the cucumber genome, with a base of T or A. The SNP02 site is the 1972889th nucleotide on chromosome 3 of the cucumber genome, with a base of T or G. The positions of the SNP01 and SNP02 sites in the genome were determined based on the cucumber reference genome sequence, version number 9930V3.

2. The SNP primer combination as described in claim 1, characterized in that: The first primer set consists of the first forward primer shown in SEQ ID NO: 1, the second forward primer shown in SEQ ID NO: 2, and the first reverse primer shown in SEQ ID NO: 3; The second primer set consists of the third forward primer shown in SEQ ID NO:4, the fourth forward primer shown in SEQ ID NO:5, and the second reverse primer shown in SEQ ID NO:

6.

3. The SNP primer combination as described in claim 1, characterized in that: The first primer set consists of a first forward primer, a second forward primer, and a first reverse primer, wherein the first forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 1 from position 22 to 43 from the 5' end, the second forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 2 from position 22 to 43 from the 5' end, and the nucleotide sequence of the first reverse primer is shown in SEQ ID NO: 3; The second primer set consists of a third forward primer, a fourth forward primer, and a second reverse primer, wherein the third forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 4 from position 22 to 40 from the 5' end, the fourth forward primer contains the specific nucleotide sequence shown in SEQ ID NO: 5 from position 22 to 39 from the 5' end, and the nucleotide sequence of the second reverse primer is shown in SEQ ID NO:

6.

4. The SNP primer combination as described in claim 3, characterized in that: The first and second forward primers each have fluorescent tag sequences that emit different fluorescent signals added to their 5' ends; The third and fourth forward primers each have fluorescent tag sequences that emit different fluorescent signals added to their 5' ends.

5. The application of any of the SNP primer combinations described in claims 1 to 4 in any of the following aspects (x1)-(x3): (x1) Prepare a kit for identifying whether cucumber fruits are short-fruited or long-fruited; (x2) Identify whether cucumber fruits are short-fruited or long-fruited; (x3) Select cucumber varieties with short or long fruit lengths; When identifying or selecting cucumber fruits of short or long fruit type, the following judgment is made based on the genotype at SNP01 and / or the genotype at SNP02: If the genotype of the cucumber to be tested is homozygous TT based on SNP01 and / or homozygous TT based on SNP02, then the cucumber to be tested is or is suspected to be a short fruit type; if the genotype of the cucumber to be tested is homozygous AA based on SNP01 and / or homozygous GG based on SNP02, then the cucumber to be tested is or is suspected to be a long fruit type.

6. A kit comprising any one of the SNP primer combinations of claims 1 to 4.

7. The application of the kit described in claim 6 in identifying whether cucumber fruits are short-fruited or long-fruited, or in selecting cucumber varieties with short or long fruit lengths; when identifying or selecting cucumber fruits of short or long fruit type, the following judgment is made based on the genotype at the SNP01 locus and / or the genotype at the SNP02 locus: if the genotype of the cucumber to be tested is TT homozygous based on the SNP01 locus and / or TT homozygous based on the SNP02 locus, then the cucumber to be tested is or is suspected to be a short-fruited variety; if the genotype of the cucumber to be tested is AA homozygous based on the SNP01 locus and / or GG homozygous based on the SNP02 locus, then the cucumber to be tested is or is suspected to be a long-fruited variety.

8. A method for identifying whether a cucumber to be tested is a long or short fruit, characterized in that, The method includes the following steps: detecting the genotype of the cucumber to be tested based on the SNP01 locus and / or the SNP02 locus as described in claim 1, and then making the following judgment: if the genotype of the cucumber to be tested based on the SNP01 locus is homozygous for TT and / or the genotype based on the SNP02 locus is homozygous for TT, then the cucumber to be tested is or is suspected to be a short-type fruit; if the genotype of the cucumber to be tested based on the SNP01 locus is homozygous for AA and / or the genotype based on the SNP02 locus is homozygous for GG, then the cucumber to be tested is or is suspected to be a long-type fruit; the length of the short-type fruit is less than or equal to 18 cm, and the length of the long-type fruit is greater than 25 cm.

9. The method as described in claim 8, characterized in that, The method for detecting the genotype of the cucumber to be tested based on the SNP01 locus and / or the SNP02 locus as described in claim 1 is as follows: (a1) Using the genomic DNA of the cucumber to be tested as a template, PCR amplification was performed using the primer sets in the SNP primer combinations described in claim 2 or 4 to obtain PCR amplification products; (a2) The fluorescence signal of the PCR amplification product obtained in step (a1) is detected by an instrument, and the genotype of the cucumber to be tested based on the SNP01 site and / or the SNP02 site is obtained according to the color of the fluorescence signal. For the PCR amplification product obtained with the first primer set, when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the first forward primer, the genotype of the SNP01 site is homozygous for the 3' terminal nucleotide of the first forward primer; when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the second forward primer, the genotype of the SNP01 site is homozygous for the 3' terminal nucleotide of the second forward primer. For the PCR amplification product obtained with the second primer set, when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the third forward primer, the genotype of the SNP02 site is homozygous for the 3' terminal nucleotide of the third forward primer; when the color of the fluorescence signal is consistent with the color displayed by the fluorescent tag of the fourth forward primer, the genotype of the SNP02 site is homozygous for the 3' terminal nucleotide of the fourth forward primer. Alternatively, the method for detecting the genotype of the cucumber to be tested based on the SNP01 locus and / or the SNP02 locus as described in claim 1 is as follows: (b1) Using the genomic DNA of the cucumber to be tested as a template, PCR amplification was performed using the primer sets in the SNP primer combination described in claim 3 to obtain PCR amplification products; (b2) Take the PCR amplification product obtained in step (b1) and sequence it; (b3) Based on the sequencing results obtained in step (b2), obtain the genotype of the cucumber to be tested based on the SNP01 site and / or the SNP02 site.

10. The method as described in claim 8, characterized in that, The cucumber varieties tested were selected from Sasha No. 1, Nanshui No. 2, Jingyan No. 4, Heishui No. 1, Biyu No. 3, Qingxiu No. 3, Qiande 1217, Jinmei No. 6, Hayan No. 1, Liyuan 966, Xinxiu No. 1, Lvwang, Tony 106, Jingyan Mini No. 6, Biyu No. 2, Yanxiu, Bicui 19, Hayan No. 4, Zhexiu No. 3, Tony 102, Jingyan Mini No. 8, Jingyan Mini No. 9, Ouman, Jenny 102, Yijia Xinxiu, Lvjingling No. 4, Guangliang 1618, Junke No. 11, Jingyan Mini No. 5, Mingxing No. 8, Liangshen Duanzun, Deruit 345, Shengfeng No. 50, Jinlv No. 17, Jinyue 301, Lushu No. 551, Deruit 298, and Shengmei. Fuman 16, Jinyou 501, Bomei 805, Deruit D81, Shengmei 909, Liangyou M162, Jinyou 316, Bonai 808, Bojie 16, Liangyou B05, Jinyou No. 8, Jinyou 636, Chunguang, Jinyou 358, Nongjiale, Lvfeicui, Tianwei No. 1, Boxin L73, Bomei 618, Bomei 22, Deruit F7, Deruit 17, Bomei No. 9, Jinchun 19, Liangyou H06, Liangyou B12, Jinsheng No. 2, Jinxiu 1601, Best 908, Senongfeng Liangjian, Jinyou 601, Huanong 402, Deruit 351, Liangyou H08, Zhexiu 302, Lvcui You No. 1, Jinyou No. 109, Qianchun 858.

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