CN116064911A - InDel molecular marker co-separated from cucumber dense thorn and less thorn genes, detection primer and application thereof - Google Patents

InDel molecular marker co-separated from cucumber dense thorn and less thorn genes, detection primer and application thereof Download PDF

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CN116064911A
CN116064911A CN202211404279.0A CN202211404279A CN116064911A CN 116064911 A CN116064911 A CN 116064911A CN 202211404279 A CN202211404279 A CN 202211404279A CN 116064911 A CN116064911 A CN 116064911A
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陈岳
张微微
佘为为
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Shanghai Vocational College Of Agriculture And Forestry
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Abstract

The invention relates to an InDel molecular marker co-separated from cucumber dense thorn and less thorn genes, a detection primer and application thereof. The InDel molecular marker coseparated with the cucumber dense thorn and the less thorn genes comprises a nucleotide sequence fragment shown as SEQ ID NO.1 and a nucleotide sequence fragment shown as SEQ ID NO.2, wherein the nucleotide sequence fragment shown as SEQ ID NO.1 is coseparated with the dense thorn genes, and the nucleotide sequence fragment shown as SEQ ID NO.2 is coseparated with the less thorn genes. The invention utilizes InDel molecular marker pair F which is co-separated with cucumber dense thorn and less thorn genes 2 Genotyping is carried out on 1600 single plants in the population, and the coincidence rate reaches 100%. The InDel molecular marker has high stability, is not only beneficial to identification and auxiliary breeding of cucumber thorn-less single plants, but also lays a foundation for pattern cloning and analysis of thorn-less genesThe theory basis is provided, and the method has wide popularization value.

Description

InDel molecular marker co-separated from cucumber dense thorn and less thorn genes, detection primer and application thereof
Technical Field
The invention relates to the technical field of agricultural biology, in particular to an InDel molecular marker co-separated from cucumber dense thorn and less thorn genes, a detection primer, an application method thereof and application thereof.
Background
Cucumber (Cucumis sativus l.) belongs to annual herbaceous plants of cucurbitaceae, is one of ten vegetable crops in the world, and has a cultivation history of over 2000 in our country. The cultivation area is inferior to that of tomatoes. Cucumber is an important cultivated vegetable in China, and statistics of food and agricultural organization data (https:// www.fao.org/fastat/zh/# home) in the United nations in 2020 show that the cultivated area of cucumber in China reaches 128 ten thousand hectares, and the total yield is 7283 ten thousand metric tons, which respectively account for 56.6% and 79.8% of the total world. As an important vegetable crop, the yield and quality of cucumber is significant for growers and consumers.
Cucumber epidermis hair belongs to multicellular type and is widely distributed on the surfaces of stems, branches, leaves, tendrils, petals, sepals and ovaries (fruits) of cucumber. The cucumber fruit has a highly specialized coat morphology, commonly called fruit thorns, consisting of 4-9 long cells, arranged longitudinally in needle-like fashion, keratinized cell walls, and hard and brittle fruit thorns. Cucumber thorns are important appearance quality characters of fruits, and directly affect commodity value of cucumbers. The types of cucumbers in China are mainly classified into North China type and south China type, wherein the North China type is mostly thorn-shaped. At present, the cucumber production has more pesticides, the dense thorn cucumber is inconvenient to clean, pesticide residues are easy to cause, and the dense thorn is also unfavorable for packaging and transportation. Therefore, the breeding of the thorn-less cucumber becomes an important direction of cucumber quality breeding. Studies have shown that replacement of an 812bp fragment of the Trichome-less (Tril) gene promoter region resulted in a few spin 1 (fs 1) type mutant exhibiting a less-thorny phenotype (FIG. 1C), and detailed results can be seen in: zhang et al, theoretical and Applied Genetics (theory of application genetics) volume 129, 7, 1289-1301, entitled A fragment substitution in the promoter of CsHDZIV/CsGL 3 is responsible for fruit spine density in cucumber (Cucumis sativus L.). However, fs1 type mutation is commonly found in south China type, european greenhouse type and American processed cucumber inbred lines, but rarely found in North China type cucumber inbred lines, and the breeding of North China type cucumber less thorn varieties is directly restricted.
According to the traditional cucumber breeding, time and labor are wasted, and the process of multi-generation backcrossing and thorn-less phenotype screening is needed. Due to the genetic rule of the recessive gene mediated by the less thorn gene, the introgression of recessive resistance can be confirmed by the need of selfing and screening to homozygote in each generation in the backcross breeding process. These factors all increase the period and difficulty of breeding. With the rapid development of modern biotechnology, the cultivation of new plant varieties by biotechnology has become a current hot spot. Through developing molecular markers closely linked with the target characters, the molecular markers are used for selecting the genotypes of the target characters, so that the breeding period can be effectively shortened, the breeding process can be accelerated, and the breeding efficiency can be improved. The InDel (Insertions and Deletion, inDel) polymorphism molecular marker is a marker for PCR amplification by designing specific primers based on sequences at two sides of insertion/deletion sites among different parents, and has the characteristics of strong universality, high accuracy, good stability and great cost saving.
The development of the molecular marker linked with the thorn-less gene provides important resources and theoretical support for the molecular marker-assisted breeding of thorn-less cucumber varieties (especially North China thorn-less varieties), and lays a foundation for the molecular mechanism of the thorn initiation development regulation of the cucumber.
Disclosure of Invention
The invention aims at providing an InDel molecular marker co-separated from a cucumber thorn-less gene, which can be used for screening cucumber thorn-less mutants and molecular assisted breeding, and can be simply, quickly and high-flux applied to breeding practice.
The above object of the present invention is achieved by the following means.
An InDel molecular marker co-separated from cucumber dense thorn and less thorn genes is named InDel-FS2 and comprises a nucleotide sequence fragment shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO.2, wherein the nucleotide sequence fragment shown in SEQ ID NO.1 is co-separated from the dense thorn genes, and the nucleotide sequence fragment shown in SEQ ID NO.2 is co-separated from the less thorn genes.
The invention also provides a preparation method of the InDel molecular marker coseparated from the cucumber dense thorn and the thorn less gene, which comprises the step of carrying out PCR amplification by using an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO.4 to obtain the InDel molecular marker.
In a further aspect, the invention provides the use of the InDel molecular marker co-isolated from the cucumber dense thorn and the less thorn genes in identifying cucumber dense thorn and less thorn varieties.
The InDel molecular marker co-separated from the cucumber dense thorn and the thorn-less gene can be used for molecular marker assisted selective breeding.
In a further aspect, the invention provides an InDel molecular marker detection primer co-separated from cucumber dense thorn and less thorn genes, which comprises an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO.4.
In still another aspect, the present invention provides a method for identifying cucumber thorn and thorn-less varieties using detection primers of InDel molecular markers co-isolated from cucumber thorn and thorn-less genes, comprising the steps of:
(1) Extracting genome total DNA of cucumber varieties;
(2) Carrying out PCR amplification on genome total DNA of the cucumber variety by using the detection primer to obtain a PCR product;
(3) Performing electrophoresis analysis on the PCR product obtained in the step (1);
(4) If the electrophoresis strip only has a DNA fragment with the length of 165bp, judging that the cucumber sample is homozygous with the dense thorn gene; if the electrophoresis strip only has a DNA fragment with the length of 125bp, judging that the cucumber sample is homozygous with less thorn genes; if the electrophoresis bands simultaneously generate DNA fragments with the lengths of 165bp and 125bp, the cucumber sample is judged to be heterozygous carrying the dense thorn gene and the less thorn gene.
Preferably, the conditions for PCR amplification in the step (1) are as follows: the total PCR amplification system was 12. Mu.L, wherein the Premix Taq was 6. Mu.L, the forward and reverse primers (10. Mu.M) were 0.5. Mu.L, respectively, the DNA (50 ng/. Mu.L) was 2. Mu.L, and ddH 2 O was 3. Mu.L.
The PCR amplification procedure was: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 30s,35 cycles; and at 72℃for 5min.
The electrophoresis analysis in the step (1) is as follows: the electrophoretic analysis is polyacrylamide gel electrophoresis.
Compared with the prior art, the invention has the main advantages that: the co-separated InDel molecular marker is co-dominant, can be directly used for identifying the cucumber less thorn character and the corresponding genotype, and can be used for genotype identification at any period of cucumber growth and development, thereby effectively solving the problem of time and labor waste in conventional breeding. The molecular marker is used for screening in the cucumber seedling stage, so that the required plants can be rapidly screened, and the selection efficiency and accuracy are improved. Meanwhile, the co-segregation mark can promote the segregation of cucumber spike genes, and lay a foundation for the deep research of molecular mechanisms of cucumber spike development.
Drawings
The following drawings are illustrative of particular embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 is a view of cucumber thorn-less mutants fs1, fs2 and wild type 06-1 thorn phenotype. Panel A in FIG. 1 shows a photograph of a spinless mutant fs1 cucumber (fs 1 refers to a reported spinless mutant of few spines1, replacement of a 812bp fragment of the promoter region of the Trichome-less gene results in a phenotype of fs 1), panel B in FIG. 1 shows a photograph of a spinless mutant fs2 cucumber, and panel C in FIG. 1 shows a photograph of a wild type spiny 06-1 cucumber, scale bar is 1cm. The graph D in FIG. 1 shows statistics of the number of the fruit thorns fs1, fs2 and 06-1. The lower case letters at the top of the error bars represent significant differences (P < 0.05) resulting from the base multiple comparisons.
FIG. 2 shows an electrophoretogram of PCR amplification products in two parents of the molecular marker InDel-FS 2; wherein, P1 represents the parent 06-1 of the cucumber with the dense thorn (the polyacrylamide gel electrophoresis band only presents the DNA fragment with the length of 165 bp), and P2 represents the parent 2-358 of the cucumber with the dense thorn (the polyacrylamide gel electrophoresis band only presents the DNA fragment with the length of 125 bp).
FIG. 3 shows an electrophoretogram of the PCR amplification product of the molecular marker InDel-FS 2; wherein P1 represents the closely-spaced cucumber parent 06-1 (only a DNA fragment with the length of 165bp appears in the polyacrylamide gel electrophoresis band), and P2 represents the less-spaced cucumber parent 2-358 (only a DNA fragment with the length of 125bp appears in the polyacrylamide gel electrophoresis band); f1 represents the filial generation of the two parents; the F2 dense thorn single plant and the F2 less thorn single plant represent 15 dense thorn and 15 less thorn cucumber plants selected randomly in the F2 group respectively. The heterozygote F2 dense thorn single plant simultaneously presents DNA fragments with the lengths of 165bp and 125bp.
Detailed Description
The inventor of the invention is subjected to extensive and intensive research, and firstly obtains a chromosome region closely linked with the cucumber thorn-less and thorn-dense genes by utilizing a method of separating body grouping mixed analysis (bulked segregant analysis, BSA) positioning, and develops an InDel molecular marker which is named as InDel-FS2 in a candidate region. F is constructed by using wild type dense thorn cucumber and mutant type less thorn cucumber material 2 Population, pair F using the InDel molecular marker 2 Genotyping is carried out on 1600 single plants in the population, and the coincidence rate reaches 100%. The InDel molecular marker has high stability, is not only beneficial to identification and auxiliary breeding of cucumber thorn-less single plants, but also lays a theoretical foundation for map cloning and analysis of thorn-less genes, and has wide popularization value. On this basis, the present invention has been completed.
SEQ ID NO.1 is a sequence amplified by InDel-FS2 in a wild type dense thorn parent, and consists of 165 nucleotides, specifically as follows:
TCTTTACCATCCAGATCAGCCACCTCCCTCTGATCTCCGACCTCTATCTACCAACGCCATTGTACCGTATACCGGTGGGAGATATCGACATTCAGGTCGACGGCACCGGCGGAGCCGGAGAAAGGGAGACCCGAATCATCCAAAACCGAATAGAAGTGGGTACAA。
SEQ ID NO.2 is the sequence of InDel-FS2 amplified in the less thorn parent, consisting of 125 nucleotides, specifically as follows:
TCTTTACCATCCAGATCAGCCATCTCCCTCTGATGTGGGAGATATCGACATTCAGGTCGACGGCACCGGCGGAGCCGGAGAAAGGGAGACCCGAATCATCCAAAACCGAATAGAAGTGGGTACAA。
the nucleotide sequence of the upstream primer shown in SEQ ID NO.3 is as follows: TCTTTACCATCCAGATCAGCC.
The nucleotide sequence of the downstream primer shown in SEQ ID NO.4 is as follows: TTGTACCCACTTCTATTCGG.
In a preferred embodiment of the invention, the InDel molecular marker co-isolated from the cucumber dense thorn and the thorn less gene consists of a nucleotide sequence fragment shown in SEQ ID NO.1 and a nucleotide sequence shown in SEQ ID NO. 2.
On the basis of fine positioning of the thorn-less gene, the invention utilizes a BSA method to screen a molecular marker linked with the thorn-less gene of the cucumber, finally positions the thorn-less gene in a chromosome region of cucumber No.3, develops a molecular marker linked with the thorn-less trait in the region, wherein InDel-FS2 is a co-dominant InDel molecular marker co-separated from the thorn-less trait. The marker can be applied to auxiliary screening judgment of cucumber seedling stage dense thorn single plants and less thorn single plants.
According to the invention, through F2 separation population analysis, the nucleotide sequence fragment shown in SEQ ID NO.1 in the InDel marker is utilized to be coseparated with the dense thorn gene, the nucleotide sequence fragment shown in SEQ ID NO.2 is coseparated with the less thorn gene, and cucumber less thorn is identified and screened, so that the method can be simply, quickly and high-flux applied to breeding of less thorn cucumber varieties.
The invention is further illustrated below in connection with examples, but the invention is not limited to these examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
InDel-FS2 molecular marker development and identification
1.1 cucumber population construction
For this trait of cucumber thorn density, a north China cucumber thorn-less mutant was found (see, panel B in FIG. 1), and the analysis found that the mutation site was different from the reported thorn-less mutant, few spin 1 (fs 1) (see, panel A in FIG. 1), and thus was named few spin 2 (fs 2). The North China type less thorn mutant fs2 and the dense thorn wild type cucumber 06-1 (see graph C in figure 1) are selected, fruits (at least 6) of one week after pollination are taken, the number of all thorns on the fruits is counted (see graph D in figure 1), and obvious difference in the number of thorns is found. Next, an F2 (06-1 Xfs 2) population and a BC1P2 ((06-1 Xfs 2). Times.fs 2) backcross population were constructed using the two parents for localization.
1.2 statistics of cucumber thorn number
And taking the fruits after the flowering and pollination of cucumber fruits for one week, counting the number of all the thorns, counting 6 fruits by an selfing system, and counting 2-3 fruits by a thorn-less positioning group plant system. In the population of 123 cucumber F2 (06-1 xfs 2), the number of the dense thorn single plants is 90, and the number of the thin thorn single plants is 33; in the 104 cucumber BC1P2 population, the number of the dense thorn single plants is 53, and the number of the thin thorn single plants is 51. Chi-square test shows that the plant coefficient mesh of the dense-thorn type and the less-thorn type in the F2 population are in accordance with the separation ratio of 3:1, and the plant coefficient mesh of the dense-thorn type and the less-thorn type in the BC1P2 population are in accordance with the separation ratio of 1:1. These results indicate that the spinless phenotype of fs2 mutants is controlled by a recessive single gene.
1.3 molecular marker development
Because fs2 mutant and wild type parent 06-1 belong to North China type cucumber, the genetic background is similar, and the markers for gene positioning are fewer. According to the resequencing data, the mutation site of insertion deletion (InDel) with larger difference between two parents is found out, inDel primers are designed, and genotyping is carried out through polyacrylamide gel electrophoresis. Simultaneously finding out single nucleotide difference (SNP) loci between two parents, uniformly selecting the SNP loci in a preliminary positioning interval, and developing SNP markers; genotyping was performed on fs2 mutants and 06-1 offspring segregating populations.
1.4 preliminary positioning
Preliminary localization of fs2 sites was performed using the BSA method, and in F2 populations of 06-1 Xfs 2, a low-thorn pool and a high-thorn pool were constructed according to the phenotype of thorns, each pool containing 10 lines of DNA. Screening the mixed pool DNA by using 50 pairs of polymorphic markers among parents, and finding a plurality of polymorphic InDel molecular markers on chromosome 4, wherein the polymorphic InDel molecular markers indicate that the thorn-free genes are located on chromosome 4.
Based on the BSA analysis results, the obtained pool-to-pool polymorphic markers were analyzed by BSA, and the fs2 site was initially located within the 944kb region between markers InDel4-13-1 (upstream sequence: GATATCGTCTGTTGTTTAGT; downstream sequence: GATCTTTAATTTGGTCGAAC) and InDel4-14-1 (upstream sequence: CTGTCATAAATTATTCGAATTCG; downstream sequence: GCGTTAATCATCAAACTTGC) using 123 individuals of the 06-1 Xfs 2F 2 population and 104 individuals of BC 1.
1.5 Fine positioning
According to the initial positioning result, performing genotyping on the other 1802F 2 generation single plants and 420 BC1 generation single plants of 06-1 xfs 2 by using markers InDel4-13-1 and InDel4-14-1 to find 185 lines with exchange between the two markers; these individuals were subjected to field planting and their fruit-thorn phenotype was observed. Meanwhile, according to retest data of the two parents, SNP markers are designed between the markers InDel4-13-1 and InDel 4-14-1. Genotyping 185 crossover individuals, combined with the fruit-bearing phenotype of these individuals, finely mapped the fs2 locus within an 11kb interval between SNP11 (upstream sequence: CCAACAACCAGTGAGTATTGTAC; downstream sequence: CCATTCTTCACTTTCTTATCTTCG) and SNP12 (upstream sequence: CCCACCACCCAAATCTCCTG; downstream sequence: GGATTCTATTTCCATCGGTTAG). There is a 40bp InDel in the FS2 location interval, for which the molecular marker InDel-FS2 was developed.
Forward and reverse primers were designed on both sides of the mutation using primer 5.0 primer design software. The upstream primer is shown as SEQ ID NO.3, the downstream primer is shown as SEQ ID NO.4, and the primers are used for amplifying genome DNA of fs2 mutant and wild parent 06-1 by PCR, so that the amplified band of the cucumber with the dense thorns is 165bp, and the amplified band of the cucumber with the less thorns is 125bp. The invention obtains InDel molecular markers which are co-separated with cucumber dense thorn and less thorn genes through the amplification of an upstream primer and a downstream primer, the InDel molecular markers are named as InDel-FS2, and the InDel-FS2 consists of a nucleotide sequence fragment shown by SEQ ID NO.1 and a nucleotide sequence fragment shown by SEQ ID NO. 2; wherein the nucleotide sequence fragment shown in SEQ ID NO.1 is coseparated with the dense thorn gene, and the nucleotide sequence fragment shown in SEQ ID NO.2 is coseparated with the dense thorn gene.
Genomic DNA of fs2 mutant and wild type parent 06-1 can be extracted by methods commonly used in the art, see 1.6 below. The PCR amplification condition is 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 30s,35 cycles; and at 72℃for 5min. The total amplification system was 12. Mu.L, wherein the Premix Taq was 6. Mu.L, the forward and reverse primers (10. Mu.M) were 0.5. Mu.L, respectively, the DNA (50 ng/. Mu.L) was 2. Mu.L, and ddH 2 O was 3. Mu.L. Detecting amplified product by 8% polyacrylamide gel electrophoresis, and electrophoresis buffer solution of 0.5 XTBE, voltage of 180V, electrophoresisTime 2h. After the electrophoresis was completed, silver nitrate (0.5 g AgNO) 3 +500mL ddH 2 O) solution followed by dyeing with sodium hydroxide solution (10 g NaOH+2mL formaldehyde+500 mL ddH) 2 O) color development, the result is shown in figure 2, only DNA fragments with the length of 165bp appear after the amplification of the dense thorn parent 06-1, and only DNA fragments with the length of 125bp appear in the small thorn parent 2-358, so that the electrophoresis band is clear and stable, and the InDel-FS2 can be used for the subsequent population co-segregation verification.
1.6 population Co-segregation verification
To demonstrate the linkage relationship of the marker InDel-FS2 and the rare thorn gene, the marker was validated by population linkage analysis using a 1600 strain F2 (06-1 Xfs 2) population.
CTAB method is adopted to extract genome DNA of cucumber parents and isolated population. Placing young leaves into a 2mL centrifuge tube, adding steel balls and 500 mu L of CTAB extracting solution, placing the centrifuge tube into a box special for a high-flux tissue grinder, balancing two sides of the centrifuge tube, adjusting the amplitude to 50Hz, and grinding for 2 min; after finishing grinding, putting the centrifuge tube into a 65 ℃ oven for 30-60 min, taking out the centrifuge tube every 10min, and shaking the centrifuge tube up and down for several times; adding 500 mu L of chloroform, mixing the mixture upside down, and centrifuging the mixture for 10min at 12000 r/min; 200 mu L of supernatant was transferred to a 1.5mL centrifuge tube; adding 2.5 times of pre-cooled absolute ethyl alcohol into the supernatant, fully mixing, centrifuging for 10min at 12000r/min, pouring out the supernatant, opening the cover of a centrifuge tube, air drying, and adding 200 μl ddH 2 O, storing at-20 ℃ for standby.
The total PCR amplification system was 12. Mu.L, wherein the Premix Taq was 6. Mu.L, the forward and reverse primers (10. Mu.M) were 0.5. Mu.L, respectively, the DNA (50 ng/. Mu.L) was 2. Mu.L, and ddH 2 O was 3. Mu.L.
Wherein the nucleotide sequence of the forward primer is SEQ ID NO.3, and the nucleotide sequence of the reverse primer is SEQ ID NO.4.
The PCR amplification procedure was: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 30s,35 cycles; and at 72℃for 5min. The amplified product was detected by 8% polyacrylamide gel electrophoresis with an electrophoresis buffer of 0.5 XTBE at a voltage of 180V for 2h. After the electrophoresis was completed, silver nitrate (0.5 g AgNO) 3 +500mL ddH 2 O) solution feedingThe rows were stained for 20min and then washed with sodium hydroxide solution (10 g NaOH+2mL formaldehyde+500 mL ddH) 2 O) developing, and counting the bands after developing. As shown in FIG. 3, the electrophoresis band was clear and stable. The results in FIG. 3 show that the single plants with the same banding pattern as the parent 06-1 in the population are densely entangled, the plants with the same banding pattern as the diluted-spine parent fs2 are less entangled, the plants with the mixed banding pattern are densely entangled, and the coincidence rate reaches 100%. The results fully show that the InDel-FS2 marker has universality and accuracy and can be applied to prediction, identification and screening of cucumber rare-thorn plants.
All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Claims (8)

1. An InDel molecular marker coseparated from cucumber dense thorn and less thorn genes, which is characterized by comprising a nucleotide sequence fragment shown in SEQ ID NO.1 and a nucleotide sequence fragment shown in SEQ ID NO.2, wherein the nucleotide sequence fragment shown in SEQ ID NO.1 is coseparated from the dense thorn genes, and the nucleotide sequence fragment shown in SEQ ID NO.2 is coseparated from the less thorn genes.
2. The method for preparing the InDel molecular marker co-separated from the cucumber dense thorn and the thorn less gene according to claim 1, which is characterized by comprising the step of carrying out PCR amplification by using an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO.4 to obtain the InDel molecular marker.
3. The use of the InDel molecular marker co-isolated from cucumber dense thorn and less thorn genes of claim 1 for identifying cucumber dense thorn and less thorn varieties.
4. Use according to claim 3, characterized in that the InDel molecular marker co-isolated with cucumber dense and spiny genes is used for molecular marker assisted selection breeding.
5. The detection primer of the InDel molecular marker co-separated from the cucumber dense thorn and the less thorn genes is characterized by comprising an upstream primer shown as SEQ ID NO.3 and a downstream primer shown as SEQ ID NO.4.
6. A method for identifying cucumber thorn and thorn-less varieties using the InDel molecular marker detection primer co-isolated with cucumber thorn and thorn-less genes of claim 5, characterized in that the method comprises the steps of:
(1) Extracting genome total DNA of cucumber varieties;
(2) Carrying out PCR amplification on genome total DNA of the cucumber variety by using the detection primer to obtain a PCR product;
(3) Performing electrophoresis analysis on the PCR product obtained in the step (1);
(4) If the electrophoresis strip only has a DNA fragment with the length of 165bp, judging that the cucumber sample is homozygous with the dense thorn gene; if the electrophoresis strip only has a DNA fragment with the length of 125bp, judging that the cucumber sample is homozygous with less thorn genes; if the electrophoresis bands simultaneously generate DNA fragments with the lengths of 165bp and 125bp, the cucumber sample is judged to be heterozygous carrying the dense thorn gene and the less thorn gene.
7. The method for identifying cucumber dense thorn and thorn-less varieties according to claim 6, characterized in that in the step (1),
the total PCR amplification system was 12. Mu.L, wherein the Premix Taq was 6. Mu.L, the forward and reverse primers (10. Mu.M) were 0.5. Mu.L, respectively, the DNA (50 ng/. Mu.L) was 2. Mu.L, and ddH 2 O is 3 mu L of the total amount of the components,
the PCR amplification procedure was: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 30s,35 cycles; and at 72℃for 5min.
8. The method for identifying cucumber dense thorn and thorn-less varieties according to claim 6, characterized in that the electrophoresis analysis in the step (2) is polyacrylamide gel electrophoresis.
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