CN114836563A - Method for identifying European fruit type cucumber, special SNP primer set and application thereof - Google Patents

Method for identifying European fruit type cucumber, special SNP primer set and application thereof Download PDF

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CN114836563A
CN114836563A CN202210474792.0A CN202210474792A CN114836563A CN 114836563 A CN114836563 A CN 114836563A CN 202210474792 A CN202210474792 A CN 202210474792A CN 114836563 A CN114836563 A CN 114836563A
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张建
温常龙
杨静静
张晓飞
夏昌选
赵泓
毛爱军
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention belongs to the technical field of biology, and provides a method for identifying European fruit cucumber, a special SNP primer set and application thereof. The SNP loci are the following second SNP loci and/or third SNP loci: the second SNP site SNP02 is located at 3924266 of chromosome 2, and the nucleotide base of the site is T or C; the third SNP site SNP03 is located at 1870398 of chromosome 3, and the nucleotide base of the site is C or A; second SNP site: t is the genotype of non-European fruit cucumber and C is the genotype of European fruit cucumber; third SNP site: c is the genotype of non-European fruit type cucumber, A is the genotype of European fruit type cucumber. By utilizing the SNP locus and the special primer group thereof, European fruit type cucumber can be identified efficiently and accurately, and the breeding efficiency and the population identification efficiency of variety cucumber varieties can be improved remarkably.

Description

Method for identifying European fruit type cucumber, special SNP primer set and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for identifying European fruit cucumber, a special SNP primer set and application thereof.
Background
Cucumber is an important vegetable crop in China, and annual sowing area and yield are the first place in the world. Cucumbers belong to annual cucurbitaceae vine plants, and the cucumbers cultivated in China can be classified into four types, namely north China type, south China type, European fruit type and Xishuangbanna type. The European fruit-type cucumber is also called mini cucumber because of small and exquisite appearance, and is originally a greenhouse cultivation type variety introduced from the Netherlands, Germany and other countries. The fruit type cucumber has the length of 14-18 cm and the diameter of about 3 cm, the fruit surface is smooth and has no thorns, the taste is crisp and tender, the melon flavor is strong, the content of nutrient components is obviously higher than that of the common cucumber, and the fruit type cucumber can be eaten as vegetables and fruits and is deeply favored by middle-high grade catering industry. In recent years, the cultivation area of European fruit type cucumbers is gradually enlarged, and the European fruit type cucumbers have great market development potential.
The cultivation environment and the commercial application of European fruit type cucumbers and other types of cucumbers are greatly different, and the European fruit type cucumbers are clearly distinguished in the agricultural production and variety breeding processes. However, with cucumber cross breeding and gene communication between different species, it has not been possible to identify European fruit cucumbers simply by seed and cotyledon. According to statistics, the cucumber varieties applied and registered in China exceed 1600 shares, the traditional field phenotype identification wastes time and labor, and the identification requirement of the current cucumber variety quantity surge cannot be met. Therefore, establishing a method for simply and quickly identifying European fruit type cucumber varieties is particularly important, and provides technical support for cucumber variety breeding and population identification.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide an SNP locus for identifying European fruit cucumber; the second purpose of the invention is to provide an SNP primer group for identifying European fruit type cucumber; it is a third object of the present application to provide a kit comprising the above primer set; the fourth purpose of the present application is to provide the use of the SNP site, or the SNP primer set, or the kit; the fifth purpose of the application is to provide a method for identifying European fruit type cucumber.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides a method for identifying SNP sites of european fruit cucumber, which are the second and/or third SNP sites: a second SNP site SNP02, the second SNP site located at chromosome 2 at 3924266 with the nucleotide base being T or C; a third SNP site SNP03, the third SNP site located at chromosome 3 at 1870398, the nucleotide base of the site being C or a. Genotype corresponding to the second SNP site: t is the genotype of non-European fruit type cucumber and C is the genotype of European fruit type cucumber; genotype corresponding to the third SNP site: c is the genotype of non-European fruit type cucumber, A is the genotype of European fruit type cucumber.
In a second aspect, the present invention provides an SNP primer set for identifying european fruit-type cucumber, the primer set being selected from a second KASP primer pair for amplifying a second SNP site for identifying european fruit-type cucumber, or determining the genotype corresponding to the second SNP site, and/or a third KASP primer pair; the third KASP primer pair is used for amplifying a third SNP locus for identifying European fruit cucumber or determining the genotype corresponding to the third SNP locus; each KASP primer pair comprises: a first forward primer, a second forward primer, and a universal reverse primer.
In the above SNP primer set for identifying european fruit type cucumber, as a preferred embodiment, the second KASP primer pair: the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.1, and specifically comprises the following steps: 5'-GTATGGATATGTGAAGGATATGGTGT-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.2, and specifically comprises the following steps: 5'-TATGGATATGTGAAGGATATGGTGC-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.3, and specifically comprises the following steps: 5'-GAATAATTATATCACGGTGAATGTAGAAGTTTG-3'; the third KASP primer pair: the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.4, and specifically comprises the following steps: 5'-TCTTTCAATCAAATCCATTTCCTATGATC-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.5, and specifically comprises the following steps: 5'-CTCTTTCAATCAAATCCATTTCCTATGATA-3'; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.6, and specifically comprises the following steps: 5'-GCAACGGTTTAGGTCAAGAAGATCAAG-3' are provided.
In the above SNP primer set for identification of fruit-type cucumis sativus, as a preferred embodiment, the 5' ends of the first and second forward primers in each KASP primer pair are linked to different fluorescent linker sequences, respectively.
In the above SNP primer set for identifying european fruit type cucumber, as a preferred embodiment, the fluorescent linker sequence is one selected from FAM, HEX, FITC, RED, TET, JOE, R110.
In a third aspect, the present invention provides a kit comprising the above primer set and PCR reaction reagents.
In the above kit, as a preferred embodiment, the PCR reaction reagent is a reagent having a competitive allele-specific PCR detection ability; preferably, the PCR reaction reagent is Master mix.
In a fourth aspect, the present invention provides the use of the SNP sites, or the SNP primer sets, or the kits described above, in any one of the following: (a) auxiliary breeding of European fruit type cucumber varieties; (b) and (4) identifying whether the cucumber variety is European fruit type cucumber.
In a fifth aspect, the present invention provides a method for identifying european fruit-type cucumbers, comprising the steps of:
(1) extracting the genome DNA of a cucumber sample to be detected; (2) taking the genome DNA of a cucumber sample to be detected as a template, and respectively carrying out PCR amplification by using each primer pair in the primer group; (3) performing fluorescence detection and analysis on the amplification product to obtain the genotype of the SNP locus corresponding to the cucumber sample to be detected, thereby determining whether the cucumber variety to be detected is European fruit type cucumber; wherein the condition for determining whether the cucumber sample to be detected is European fruit type cucumber is selected from one or more of the following conditions: when the genotype based on the second SNP locus SNP02 is T, namely T is homozygous, the cucumber sample to be detected is non-European fruit type cucumber; when the genotype of the SNP02 based on the second SNP locus is C, namely C is homozygous, the cucumber sample to be detected is European fruit type cucumber; or/and when the genotype based on the third SNP locus SNP03 is C: C pure, the cucumber sample to be detected is non-European fruit type cucumber; and when the genotype based on the third SNP locus SNP03 is A, namely A is homozygous, the cucumber sample to be detected is European fruit type cucumber.
In the above method for identifying a fruit-type european cucumber, as a preferred embodiment, the PCR amplification conditions are, in order: pre-denaturation at 94 ℃ for 15min and denaturation at 94 ℃ for 20 s; annealing at 61-55 deg.C for 1min for 10 cycles, with 61 deg.C being the annealing temperature of the first cycle, and then reducing the annealing temperature by 0.6 deg.C for each cycle; denaturation at 94 ℃ for 20s, renaturation/elongation at 55 ℃ for 1min, for 26 cycles.
In the above method for identifying a fruit-type european cucumber, as a preferred embodiment, the concentration ratio of the first forward primer, the second forward primer and the universal reverse primer of each primer pair in the PCR system is 2:2: 5.
In the above method for identifying a European fruit type cucumber, as a preferred embodiment, the concentration of the genomic DNA of the cucumber sample to be tested is 10-30 ng/. mu.L.
Template for PCR of the present invention: the agarose electrophoresis band of the genome DNA of a sample to be detected is single and has no obvious dispersion, the concentration of the agarose electrophoresis band meets 10-30 ng/mu L, and the ratio of A260/A280 in the detection of an ultraviolet spectrophotometry is more than 1.8.
Compared with the prior art, the invention has the beneficial effects that: the invention obtains 110492 cucumber perfect SNPs by screening based on the re-sequencing data of 68 cucumber representative resources representing extensive cucumber genetic diversity, further screens 8 SNP loci from the cucumber perfect SNPs, wherein the SNP typing result shows that the homozygous genotype proportion is higher and is SNP02 and SNP03 which are closely linked with the European fruit type cucumber, a second KASP primer pair used for genotyping of a second SNP locus or a third KASP primer pair used for genotyping of a third SNP locus are used for identifying whether 12 cucumbers to be detected are the European fruit type cucumbers, the detected homozygous genotypes are completely consistent with the phenotypes, and powerful technical support is provided for identifying whether the cucumbers are the European fruit type cucumbers. The specific amplification primer developed by the SNP locus can be used for efficiently and accurately identifying European fruit type cucumbers, and the variety breeding and population identification efficiency of cucumber varieties can be obviously improved.
Drawings
Fig. 1 shows PCA clustering results of 68 cucumbers representing resource resequencing data.
FIG. 2 is a graph showing the results of genotyping or KASP amplifying 144 cucumber species tested in example 2 using the second KASP primer set of the present invention, wherein the genotype at A is European fruit cucumber (C: C genotype, red); the genotype at B is non-European fruit cucumber (T: T genotype, blue); at C is a sample with no genotype detected (.
FIG. 3 is a graph showing the results of genotyping or KASP amplifying 144 cucumber species tested in example 2 using the third KASP primer set of the present invention, wherein the genotype at A is European fruit cucumber (A: A genotype, red); the genotype at B is non-European fruit cucumber (C: C genotype, blue); sample with no genotype detected at C (; at D is a sample with heterozygous genotype (A: C genotype, green).
FIG. 4 is a diagram showing the results of genotyping or KASP amplifying the 12 cucumber varieties to be tested in example 3 using the second KASP primer set of the present invention, wherein the genotype at A is European fruit cucumber (C: C genotype, red); the genotype at B is non-European fruit cucumber (T: T genotype, blue); at C is a sample with heterozygous genotype (C: T genotype, green).
FIG. 5 is a diagram showing the results of genotyping or KASP amplifying the 12 cucumber varieties to be tested in example 3 using the third KASP primer set of the present invention, wherein the genotype at A is European fruit cucumber (A: A genotype, red); the genotype at B is non-European fruit cucumber (C: C genotype, blue); at C is a sample with heterozygous genotype (A: C genotype, green).
Detailed Description
The method utilizes the resequencing data of the cucumber germplasm resources to screen specific genetic loci of European fruit type populations from the whole genome, and further develops the specific molecular markers for identifying European fruit type cucumber varieties. The SNP is taken as a third-generation molecular marker, is widely distributed in a genome, has one SNP variation per 1000bp on average, is stable in heredity and is easy to detect automatically. KASP (Kompetitive Allelele Specific PCR), a competitive allele-Specific PCR technology, is a common method for SNP typing and has the characteristics of high stability, accuracy and low cost.
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 conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
In order to make the objects, technical solutions and advantages of the present invention more clearly understood, the method for identifying european fruit type cucumber, and the special SNP primer set and application thereof of the present invention are further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1 obtaining of SNP primer combination for identifying cucumber species as belonging to European fruit type cucumber
Discovery of one or 8 SNP sites
The invention is based on the re-sequencing data of 68 parts of cucumber representative resources, the cucumber germplasm represents wide cucumber genetic diversity, including 21 east Asia type (North China type and south China type) cucumbers, 28 European type cucumbers and 19 Xishuangbanna type cucumbers (figure 1), and Perfect SNP locus with 50bp of SNP wings having no other variation and specific genome is screened, and the specific screening standard of the SNP locus is as follows: SNP loci with MAF >0.3, heterozygosity less than 0.1, deletion rate less than 0.1 and conserved flanking 50bp sequences (no InDel, no SSR, no other SNP) are selected in the whole genome. 110492 cucumber perfect SNPs are obtained in total, non-synonymous SNPs are further screened for calculating the SNP variation frequency between European fruit type cucumbers and other types of cucumbers, and finally the difference value of the SNP frequency between the European fruit type cucumbers and other types of cucumber populations of 8 SNP loci is 1.
The basic information of the 8 SNP sites is detailed in Table 1. The position of the SNP locus on the chromosome is determined based on the comparison of cucumber Chinese Long 9930 reference genome sequences, and the version number of the cucumber Chinese Long 9930 reference genome sequence is V2 (download address: http:// cucurbitangenomics. org/org anasm/2).
TABLE 1.8 basic information of specific non-synonymous SNP loci of European fruit-type Cucumis sativus
Figure BDA0003624882180000061
Secondly, obtaining a specific SNP primer for identifying European fruit type cucumbers
Based on the 8 SNP sites found in the first step, the inventors of the present invention designed 8 pairs of KASP primers and performed SNP typing on 24 cucumber varieties of known population attributes. According to the SNP genotype result, 2 SNP locus combinations which have higher homozygous genotype proportion and are closely linked with European fruit type cucumber, namely SNP02 and SNP03, are obtained. Therefore, the present invention preferably selects a second KASP primer pair based on the second SNP site SNP02 and/or a third KASP primer pair based on the third SNP site SNP03, each primer pair comprising a first forward primer, a second forward primer and a universal reverse primer, the sequences of which are shown in table 2 below, as specific SNP primer combinations for identifying european fruit cucumber.
TABLE 2 specific SNP primer combinations for the identification of European fruit-type cucumber
Figure BDA0003624882180000062
Note: single underlined is FAM fluorescent tag sequence and double underlined is HEX fluorescent tag sequence.
KASP primer pairs designed based on other six SNP sites are shown as follows,
the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.11, and specifically comprises the following steps: 5'-CATCTTCCATTTCCAACAACTCCG-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.12, and specifically comprises the following components: 5'-TTCATCTTCCATTTCCAACAACTCCA-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.13, and specifically comprises the following steps: 5'-GAGATGGTTGTCGTTTTGAAAGGTGG-3' are provided.
Fourth KASP primer pair:
the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.14, and specifically comprises the following steps: 5'-CCTTCTCTCAGTCTCTAACTGGTT-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.15, and specifically comprises the following steps: 5'-CTTCTCTCAGTCTCTAACTGGTG-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.16, and specifically comprises the following components: 5'-ATCCGATTGGGAAGGTATGAGAGGT-3' are provided.
Fifth KASP primer pair:
the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.17, and specifically comprises the following steps: 5'-TCGCCATTACTGCTGACTTGGAA-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.18, and specifically comprises the following steps: 5'-TCGCCATTACTGCTGACTTGGAT-3', respectively; the base sequence of the universal reverse primer is shown in a sequence table SEQ ID NO.19, and specifically comprises the following components: 5'-GAGTCAGAGATGAATGGAGGTAGAAAC-3' are provided.
Sixth KASP primer pair:
the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.20, and specifically comprises the following steps: 5'-CCTTCCTTGACATCTCTTTAAGGAAA-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.21, and specifically comprises the following steps: 5'-CTTCCTTGACATCTCTTTAAGGAAG-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.22, and specifically comprises the following steps: 5'-GCTTGACGTTAGAAAATAGAAATAGCAGTG-3' are provided.
Seventh KASP primer pair:
the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.23, and specifically comprises the following components: 5'-CCGAACTCTTTGTTTCGGTTCATG-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.24, and specifically comprises the following steps: 5'-AACCGAACTCTTTGTTTCGGTTCATT-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.25, and specifically comprises the following steps: 5'-CAATCAAAAGTATTACAGAAAGACCGACTG-3' is added.
Eighth KASP primer pair:
the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.26, and specifically comprises the following steps: 5'-GTGGGAAATCCTTGGAGTTTAGAAG-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.27, and specifically comprises: 5'-GTGGGAAATCCTTGGAGTTTAGAAA-3', respectively; the base sequence of the universal reverse primer is shown in a sequence table SEQ ID NO.28, and specifically comprises the following components: 5'-AGCAAGTACTGAAGATGAGATCTCAAAG-3' are provided.
The 5' ends of the first forward primer and the second forward primer in each KASP primer pair are respectively connected with different fluorescent adaptor sequences. The fluorescent linker sequence is selected from one of FAM, HEX, FITC, RED, TET, JOE and R110.
In the invention, the first KASP primer pair is used for amplifying the first SNP site SNP01 or determining the genotype of the first SNP site; the second KASP primer pair is for amplifying the second SNP site SNP02 or for determining the second SNP site genotype; the third KASP primer pair is used for amplifying the third SNP site SNP03 or determining the third SNP site genotype; the fourth KASP primer pair is used for amplifying the fourth SNP site SNP04 or determining the fourth SNP site genotype; the fifth KASP primer pair is used for amplifying the fifth SNP site SNP05 or determining the fifth SNP site genotype; the sixth KASP primer pair is for amplifying the sixth SNP locus SNP06 or for determining the genotype of the sixth SNP locus; the seventh KASP primer pair is used for amplifying the seventh SNP site SNP07 or determining the seventh SNP site genotype; the eighth KASP primer pair is for amplifying the eighth SNP site SNP08 or for determining the eighth SNP site genotype.
Example 2 and example 1 validation of the SNP primer combinations developed
The basic information of 144 cucumber cultivars tested in this example is given in table 3 below in columns 1 to 3. 144 cucumber varieties to be tested are common market varieties. Depending on the phenotype, 36 tested cucumber varieties were european fruit cucumbers and 108 tested cucumber varieties were non-european fruit cucumbers.
1. Acquisition of genomic DNA of cucumber varieties to be tested
Genomic DNAs of the rootlets (rootlets of mixed 10 seeds) of 144 cucumber varieties to be tested were extracted by the CTAB method, respectively, to obtain the genomic DNAs of the cucumber varieties to be tested.
The quality and concentration of the genome DNA of the cucumber variety to be tested both need to meet the PCR requirement, and the standard of reaching the 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 variety to be tested was 10-30 ng/. mu.L.
2. And respectively taking the genome DNA of 144 cucumber varieties to be tested as templates, and respectively adopting a second KASP primer pair and a third KASP primer pair to carry out PCR amplification to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the first forward primer, the second forward primer and the reverse universal primer is 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.
The PCR reagent is: a kit with competitive allele-specific PCR detection capability. In this example, Mastermix from LGC (96 or 384 well plates, PartNo. KBS-1016-
PCR reaction system of table 4384 pore plate or 96 pore plate
Figure BDA0003624882180000091
3. After the step 2 is completed, when the temperature of each PCR amplification product is reduced to below 40 ℃, scanning and reading fluorescence values through FAM and HEX light beams of a microplate reader (reading values of FAM fluorescent label sequences are observed under the wavelength of 485nm of exciting light and 520nm of emitted light, reading values of HEX fluorescent label sequences are observed under the wavelength of 528nm of exciting light and 560nm of emitted light), and judging the genotypes of 144 cucumber varieties to be tested based on each SNP locus according to the colors of fluorescence signals. The specific judgment principle is as follows:
the sample to be tested is based on the PCR result of the second SNP site SNP 02: if the blue fluorescent signal is displayed, the genotype of the locus is T (the same as the last basic group at the 3' end of the first forward primer of the second KASP primer pair), and the cucumber sample to be detected is judged to be non-European fruit type cucumber; c (the same as the last base at the 3' end of the second forward primer of the second KASP primer pair) is obtained, and the cucumber sample to be detected is judged to be European fruit type cucumber; and a green fluorescent signal is displayed, the genotype of the site is heterozygous, one base is T (the last base at the 3 'end of the first forward primer of the second KASP primer pair is the same), the other base is C (the last base at the 3' end of the second forward primer of the second KASP primer pair is the same), and T: C is judged according to the result. The genotype statistics for 144 test samples based on SNP02 in this example are shown in table 3, column 4 below (see figure 2 for some results).
When the sample to be detected is based on the PCR result of the third SNP site SNP 03: c (the same as the last base at the 3' end of the first forward primer of the third KASP primer pair), and judging that the cucumber sample to be detected is non-European fruit type cucumber; if the red fluorescent signal is displayed, the genotype of the locus is A: A (the last basic group of the 3' end of the second forward primer of the third KASP primer pair is the same), and the cucumber sample to be detected is judged to be European fruit type cucumber; the locus genotype is heterozygote when a green fluorescence signal is displayed, one base is C (the last base at the 3 'end of the first forward primer of the third KASP primer pair), the other base is A (the last base at the 3' end of the second forward primer of the third KASP primer pair), and the A: C is judged according to the result. The genotype statistics for 144 test samples based on SNP03 in this example are shown in table 3, column 5 below (see figure 3 for some results).
It should be noted that 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, 5 cycles) can be added until the fluorescence signal is clustered compactly, and the relative fluorescence value of FAM and HEX is between 1 and 2.
Table 3.144 basic information of cucumber varieties tested
Figure BDA0003624882180000101
Figure BDA0003624882180000111
Figure BDA0003624882180000121
Figure BDA0003624882180000131
Note: NA is a deletion of genotype data.
The results show that 2 primer groups can obtain good typing effect in 144 cucumber varieties to be tested, the typing results are highly consistent, and the specific consistency data are shown in the 4 th part of the embodiment.
4. Evaluation of efficiency of two primer pairs to identify 144 cucumber varieties to be tested as fruit type cucumbers in Europe
(1) 144 cucumber varieties tested were counted based on the genotype of SNP 02.
The results show that 39 tested cucumber varieties, which are homozygous C based on SNP02 genotype in table 3 above, were judged as fruit-type cucumbers in europe, and the statistical results of the phenotypes of the tested cucumbers in column 3 in table 3 above show that 35 of them are fruit-type cucumbers in europe and the consistency is 89.7%. In the above table 3, based on the SNP02, the genotype is T, 104 tested cucumber varieties which are homozygous for T are judged to be non-European fruit cucumbers; as can be seen from the phenotypic statistics of the tested cucumbers in column 3 of table 3 above, all of these 104 cucumbers are non-european fruit cucumbers with a consistency of 100%.
As can be seen from table 3 above, in the present example, based on the genotype of the second SNP site, of the 144 cucumber varieties tested, a total of 5 cucumber varieties whose genotypes did not match the phenotype (in which the genotype detection result of HH45 was heterozygous) were identified by using the second primer pair, and thus, the identity of the phenotype to the european fruit cucumber in the 144 samples tested was 96.53%.
(2) 144 cucumber varieties tested were counted based on the genotype of SNP 03.
The results show that 32 tested cucumber varieties of A homozygous type are judged to be European fruit type cucumbers based on that the SNP03 genotype is A in the above table 3, and the statistical results of the phenotypes of the tested cucumbers in the 3 rd row in the above table 3 show that 32 of the tested cucumber varieties are all European fruit type cucumbers and the consistency is 100%. In table 3 above, 108 tested cucumber varieties are judged to be non-european fruit type cucumbers based on the fact that the SNP03 genotype is C, C homozygous type cucumbers are determined, and according to the statistical results of the phenotypes of the tested cucumbers in the 3 rd row in table 3 above, 105 of the tested cucumbers are non-european fruit type cucumbers, and the consistency is 97.2%.
As can be seen from table 3 above, in the present example, out of the results of identifying 144 cucumber varieties tested by using the third primer pair based on the genotype at the third SNP site, there were 6 varieties with genotypes not matching the phenotype (in which the results of the genotype detection for HH16, HH29 and HH45 were heterozygous), and 1 variety with data loss (NA), and therefore, the third KASP primer pair identified the european fruit cucumber among the 144 samples tested with a phenotype matching 95.14%.
(3) The genotype of 144 cucumber varieties to be tested based on SNP02 and SNP03 is counted
The results show that 32 tested cucumber varieties are determined to be European fruit type cucumbers based on the genotype C: C, A: A homozygous for SNP02 and SNP03 in the above table 3, and the statistical results of the phenotypes of the tested cucumbers in the 3 rd row in the above table 3 show that 32 of the tested cucumbers are all European fruit type cucumbers and the consistency is 100%. In table 3 above, 101 cucumber varieties tested, which are homozygous for T: T, C: C based on SNP02 and SNP03 genotypes, are determined to be non-european fruit cucumbers, and according to the statistical results of the phenotypes of the tested cucumbers in column 3 above, 101 cucumber varieties are all non-european fruit cucumbers, and the consistency is 100%.
Therefore, it can be seen that the SNP loci and the special primer pairs thereof developed in example 1 can efficiently and accurately identify whether the cucumber to be detected is a european fruit cucumber, and the linkage degree of the genotypes of the second and third SNP loci is very high (except that the genotypes of the two SNP loci of HH45 are both heterozygous, the result of identifying whether the cucumber to be detected is a european fruit cucumber based on the genotypes of the two SNP loci is completely consistent with the phenotype), and in identifying whether the cucumber is a european fruit cucumber, the second KASP primer pair and the third KASP primer pair can be used as a primer group to be commonly used for identifying the european fruit cucumber and are complementary to each other, thereby further improving the accuracy of identifying the european fruit cucumber.
Example 3, detecting whether the cucumber to be detected is European fruit type cucumber by adopting the SNP primer combination developed in example 1
The cucumber varieties to be detected are 1 to 12.
1. Detection of whether cucumber variety to be detected belongs to European fruit type cucumber by using SNP primer combination developed in example 1
(1) Obtaining of genome DNA of cucumber variety to be tested
Planting seeds of a cucumber variety to be detected to obtain seedlings; taking leaves or roots of the cucumber variety to be detected, and respectively extracting genome DNA by adopting a CTAB method to obtain the genome DNA of the cucumber variety to be detected.
(2) And (3) taking the genome DNA of the cucumber variety to be detected as a template, and respectively adopting the second KASP primer pair and the third KASP primer pair to carry out PCR amplification to obtain corresponding PCR amplification products. In each PCR reaction system, the concentration ratio of the first forward primer, the second forward primer and the universal reverse primer is 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. The PCR reagent is: mastermix from LGC corporation.
(3) After the step (2) is completed, when the temperature of each PCR amplification product is reduced to below 40 ℃, scanning and reading a fluorescence value through FAM and HEX light beams of a microplate reader (reading value of FAM fluorescence label sequence is observed under the wavelength of 485nm exciting light and 520nm emitting light, reading value of HEX fluorescence label sequence is observed under the wavelength of 528nm exciting light and 560nm emitting light) to obtain the color of a fluorescence signal, and judging the genotype of a corresponding SNP site according to the method in the embodiment 2, thereby judging whether the variety to be detected is European fruit type cucumber.
The SNP typing results of 12 cucumber varieties to be detected by adopting the second KASP primer pair are shown in figure 4;
the SNP typing results of the third KASP primer pair for 12 cucumber varieties to be tested are shown in figure 5.
The statistical results are shown in Table 5, columns 2-4. The cucumber variety to be tested is judged to be the European fruit cucumber based on the fact that the SNP02 genotype is C: C homozygous type and the SNP03 genotype is A: A homozygous type. The cucumber variety to be tested which is T: T homozygous based on the SNP02 genotype and C: C homozygous based on the SNP03 genotype is judged to be non-European fruit cucumber.
Table 512 cucumber varieties to be tested belong to the European fruit cucumber types
Cucumber variety to be detected SNP 02-based genotype SNP 03-based genotype European fruit type cucumber
Cucumber variety 1 to be tested C is C homozygote A is homozygote Is that
Cucumber variety 2 to be tested C: T heterozygote C is C homozygote Whether or not
Cucumber variety to be tested 3 T is T homozygote type C is C homozygote Whether or not
Cucumber variety to be tested 4 T is homozygote type C is C homozygote Whether or not
Cucumber variety 5 to be detected T is homozygote type C is C homozygote Whether or not
Cucumber variety 6 to be detected C is C homozygote A is homozygote Is that
Variety 7 of cucumber to be tested T is homozygote type C is C homozygote Whether or not
Cucumber variety 8 to be detected C: T heterozygote A: C heterozygote Is that
Cucumber variety 9 to be detected T is homozygote type C is C homozygote Whether or not
Cucumber variety to be tested 10 C is C homozygote A is homozygote Is that
Cucumber variety to be detected 11 C is C homozygote A is homozygote Is that
Cucumber variety to be tested 12 T is T homozygote type A: C heterozygote Whether or not
2. And judging whether the cucumber variety to be detected is a European fruit type cucumber type according to the phenotype.
The statistical results are shown in column 4 of Table 3.
The result shows that the SNP primer combination developed in the example 1 can identify European fruit type cucumber and has completely consistent phenotype identification result. Therefore, the SNP primer combination developed based on the embodiment 1 can detect whether the cucumber variety to be detected belongs to European fruit type cucumber.
The above embodiments are merely examples for clearly illustrating the present invention and do not limit the present invention. Other variants and modifications will be apparent to those skilled in the art in light of the foregoing description, which are not necessarily exhaustive of all embodiments and are therefore intended to be within the scope of the invention.
Sequence listing
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Claims (10)

1. An SNP site for identifying European fruit cucumber, wherein the SNP site is a second SNP site and/or a third SNP site as follows: a second SNP site SNP02, located at 3924266 of chromosome 2, at nucleotide base T or C;
a third SNP site SNP03, wherein the third SNP site is located at 1870398 of chromosome 3, and the nucleotide base of the third SNP site is C or A; genotype corresponding to the second SNP site: t is the genotype of non-European fruit cucumber and C is the genotype of European fruit cucumber;
genotype corresponding to the third SNP site: c is the genotype of non-European fruit type cucumber, A is the genotype of European fruit type cucumber.
2. An SNP primer set for identifying a European fruit cucumber, wherein the primer set is selected from a second KASP primer pair and/or a third KASP primer pair, and the second KASP primer pair is used for amplifying a second SNP site for identifying the European fruit cucumber or determining the genotype corresponding to the second SNP site; the third KASP primer pair is used for amplifying a third SNP locus for identifying European fruit cucumber or determining the genotype corresponding to the third SNP locus; each KASP primer pair comprises: a first forward primer, a second forward primer, and a universal reverse primer.
3. The SNP primer set of claim 2, wherein the second KASP primer pair: the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.1, and specifically comprises the following steps: 5'-GTATGGATATGTGAAGGATATGGTGT-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.2, and specifically comprises the following steps: 5'-TATGGATATGTGAAGGATATGGTGC-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.3, and specifically comprises the following steps: 5'-GAATAATTATATCACGGTGAATGTAGAAGTTTG-3', respectively; the third KASP primer pair: the base sequence of the first forward primer is shown as a sequence table SEQ ID NO.4, and specifically comprises the following steps: 5'-TCTTTCAATCAAATCCATTTCCTATGATC-3', respectively; the base sequence of the second forward primer is shown as a sequence table SEQ ID NO.5, and specifically comprises the following steps: 5'-CTCTTTCAATCAAATCCATTTCCTATGATA-3', respectively; the base sequence of the universal reverse primer is shown as a sequence table SEQ ID NO.6, and specifically comprises the following steps: 5'-GCAACGGTTTAGGTCAAGAAGATCAAG-3' are provided.
4. The SNP primer set of claim 2, wherein the first and second forward primers in each KASP primer pair have different fluorescent linker sequences attached to their 5' ends.
5. A SNP primer set according to claim 4 wherein the fluorescent linker sequence is selected from one of FAM, HEX, FITC, RED, TET, JOE, R110.
6. A kit comprises the primer group and a PCR reaction reagent.
7. The kit of claim 7, wherein the PCR reaction reagents are reagents with competitive allele-specific PCR detection capability; preferably, the PCR reaction reagent is Master mix.
8. Use of a SNP site according to claim 1 or a SNP primer set according to claims 2-5 or a kit according to claim 6 or 7 in any of the following: (a) auxiliary breeding of European fruit type cucumber varieties; (b) and (4) identifying whether the cucumber variety is European fruit type cucumber.
9. A method for identifying European fruit type cucumber is characterized by comprising the following steps:
(1) extracting the genome DNA of a cucumber sample to be detected; (2) taking the genome DNA of a cucumber sample to be detected as a template, and respectively carrying out PCR amplification by using each primer pair in the primer group; (3) performing fluorescence detection and analysis on the amplification product to obtain the genotype of the SNP locus corresponding to the cucumber sample to be detected, thereby determining whether the cucumber variety to be detected is European fruit type cucumber; wherein the condition for determining whether the cucumber sample to be detected is European fruit type cucumber is selected from one or more of the following conditions:
when the genotype based on the second SNP locus SNP02 is T, namely T homozygote type, the cucumber sample to be detected is non-European fruit type cucumber; when the genotype of the SNP02 based on the second SNP locus is C, namely C is homozygous, the cucumber sample to be detected is European fruit type cucumber;
or/and when the genotype based on the third SNP locus SNP03 is C: C pure, the cucumber sample to be detected is non-European fruit type cucumber; and when the genotype based on the third SNP locus SNP03 is A, namely A is homozygous, the cucumber sample to be detected is European fruit type cucumber.
10. The method of claim 9, wherein the conditions for PCR amplification are, in order: pre-denaturation at 94 ℃ for 15 min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 1min for 10 cycles, 61 ℃ being the annealing temperature of the first cycle, and then reducing the annealing temperature by 0.6 ℃ per cycle; denaturation at 94 ℃ for 20s, renaturation/elongation at 55 ℃ for 1min, and 26 cycles; preferably, in the primer group, the concentration ratio of the first forward primer, the second forward primer and the universal reverse primer of each primer pair in the PCR system is 2:2: 5; preferably, the concentration of the genome DNA of the cucumber sample to be detected is 10-30 ng/muL.
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