dCAPS molecular marker co-segregating with cucumber fruit length gene and its application

Abstract

The invention discloses a dCAPS molecular marker co-segregated with a cucumber fruit length gene and its application, and belongs to the field of vegetable molecular marker assisted breeding. The dCAPS molecular marker includes a forward primer whose sequence is shown in SEQ ID NO:1 and a reverse primer shown in SEQ ID NO:2, or further includes a PstI endonuclease. The dCAPS molecular marker co-segregates with the cucumber fruit length gene during the cucumber molecular marker assisted breeding process, that is, the phenotypes of long-fruit and short-fruit individual plants in the population are respectively consistent with the long-fruit and short-fruit parent band types amplified by the dCAPS molecular marker, and the heterozygous band type amplified by the marker corresponds to a long-fruit type plant. The dCAPS molecular marker is used to perform genotyping of the plant at the seedling stage, and the length of the fruit of each individual plant is predicted based on the typing result, so that molecular marker assisted breeding can be performed at the seedling stage to improve breeding efficiency.

Description

Technical Field

[0001] The invention belongs to the field of vegetable molecular marker assisted breeding, and particularly relates to a dCAPS molecular marker co-segregated with a cucumber fruit length gene and an application thereof. Background Art

[0002] Cucumber (Cucumis sativus L) is an annual herbaceous plant of the Cucurbitaceae family. As one of the main vegetable crops cultivated in facilities in my country, it has important economic and production value. The size and shape of the fruit are important appearance qualities of vegetable horticultural crops such as cucumbers and tomatoes, and there are great differences among varieties. At the cellular level, cell division and cell expansion play a key role in the final fruit length, diameter and yield. The size and shape of cucumber fruit are important agronomic traits that affect the overall yield and market economic value. Due to different consumption habits in different regions, consumers' preferences for fruit length also vary.

[0003] At the same time, cucumber fruit is a gourd, which grows and develops rapidly and has diverse morphologies, providing convenient conditions for studying fruit morphological development. Studying the dCAPS molecular marker co-segregated with the cucumber fruit length gene is of great significance for exploring the function of the cucumber fruit length gene and applying the gene to molecular breeding practice. Summary of the invention

[0004] One of the purposes of the present invention is to provide a dCAPS molecular marker co-segregating with a cucumber fruit length gene.

[0005] The second object of the present invention is to provide the use of the dCAPS molecular marker co-segregated with the cucumber fruit length gene in cucumber molecular marker-assisted breeding.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The dCAPS molecular marker co-segregated with the cucumber fruit length gene provided by the present invention comprises a forward primer whose sequence is shown in SEQ ID NO: 1 and a reverse primer shown in SEQ ID NO: 2, which are specifically as follows:

[0008] Forward primer (5′-TACTGAACTTATTTTTCTGCCTGCA-3′) (SEQ ID NO: 1);

[0009] Reverse primer (5'-GGTAACCCATAATCTGAACGTTTAT-3') (SEQ ID NO: 2).

[0010] Preferably, the dCAPS molecular marker co-segregated with the cucumber fruit length gene also includes PstI endonuclease.

[0011] The present invention also provides the use of the dCAPS molecular marker co-segregated with the cucumber fruit length gene in cucumber molecular marker-assisted breeding.

[0012] Preferably, the dCAPS molecular marker co-segregating with the cucumber fruit length gene is used for the co-segregation of the cucumber fruit length gene, that is, the long-fruit and short-fruit individual plant phenotypes of the population are respectively consistent with the long-fruit and short-fruit parent band types amplified by the above-mentioned dCAPS molecular marker, and the heterozygous band type amplified by the marker corresponds to the long-fruit type plant, among which the short-fruit trait has been proved to be controlled by a single recessive gene.

[0013] Compared with the prior art, the present invention has the following beneficial effects: by using the dCAPS molecular marker provided by the present invention and cotyledon-extracted with the cucumber fruit length gene, DNA is extracted from the cotyledons of cucumber seedlings at the cotyledon stage for genotyping of different individual plants, the fruit length of the plant is predicted based on the genotyping result, the fruit length is measured 40 days after flowering, and analysis is performed combining the genotyping and fruit length phenotype data to find that the dCAPS molecular marker is co-segregated with the fruit length phenotype in the F2 population, indicating that the dCAPS molecular marker can be used to perform genotyping on the plants at the seedling stage, and the fruit length of each individual plant can be predicted based on the typing result, that is, molecular marker assistance can be performed at the seedling stage to improve breeding efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The phenotypic characteristics of cucumber sf1.1, 9930 and WD1 fruit length in the embodiment (bar=1 cm); wherein: a) the phenotypic characteristics of male and female flowers of WD1, 9930 and sf1.1 on the day of flowering; b) the comparison of the length of female flowers of WD1, 9930 and sf1.1 on the day of flowering; c) the phenotypic characteristics of the fruits of WD1, 9930 and sf1.1 when fully mature 40 days after flowering; d) the comparison of the length of the fruits of WD1, 9930 and sf1.1 40 days after flowering.

[0015] Figure 2 These are the one-leaf-one-heart phenotypic characteristics of cucumber sf1.1 and WD1 in the example; wherein: a and b are the hypocotyl and true leaf in the one-leaf-one-heart period.

[0016] Figure 3 The longitudinal sections of mesocarp cells of WD1 and sf1.1 on the day of flowering in the embodiment (bar = 100 μm); wherein: a) the longitudinal section of WD1 fruit on the day of flowering; b) the longitudinal section of sf1.1 fruit on the day of flowering; c) the average cell area of ​​WD1 and sf1.1 female flowers on the day of flowering (μm 2 ) comparison; d) On the day of flowering, the per unit area (1×10 6 μm2 )Comparison of average cell numbers.

[0017] Figure 4 It is the gene structure of the candidate gene Csa1G665390 in the embodiment; wherein: a) the Csa1G665390 gene in WD1; b) the Csa1G665390 gene in sf1.1; c) the coding region of the Csa1G665390 gene in WD1; d) the coding region of the Csa1G665390 gene in sf1.1.

[0018] Figure 5 This is the result of agarose gel electrophoresis detection of Csa1G665390 in the example.

[0019] Figure 6 It is the PCR amplification effect of the molecular marker dCAPSCsSf1.1 in the embodiment, wherein WD1 and 9930 are parents of long-fruited cucumbers, sf1.1 is a parent of short-fruited cucumbers, and the F2 population consists of long-fruited plants and short-fruited plants. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiment of the present invention clearer, the technical solution of the embodiment of the present invention will be clearly and completely described in conjunction with the drawings of the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0021] The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions, such as those described in Sambrook et al. Molecular Cloning, Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or according to the conditions recommended by the manufacturer.

[0022] Example 1

[0023] This example provides the construction of a genetically isolated population and the identification of cucumber fruit length genes, as follows:

[0024] (1) Materials

[0025] Cucumber parents WD1 and 9930 are both cultivated cucumber inbred lines in North China. WD1 is used to construct a cucumber EMS-induced mutant library. This line is a high-generation self-pollination homozygous material, and its commercial melon length is about 35 cm. After sowing the seeds treated with EMS mutagenesis, the single plant is harvested to obtain M1. The M1 generation is self-pollinated to obtain the M2 generation. In the M2 generation, mutants with fruit length significantly shorter than the wild type are identified. Untreated WD1 is used as a control to observe whether the phenotypes of offspring plants of different strains in the M1 population have changed. A short fruit length mutant was found in one strain, numbered 2-91-1, which was self-pollinated to obtain the M2 population. The short fruit trait of the offspring of the M2 population is all homozygous. Among them, the short fruit length mutant 2-91-1 was used as the male parent and 9930 as the female parent, and the F1 was hybridized to obtain the F2 population with separated traits. At the same time, the BC1 population was backcrossed with the recessive parent 2-91-1 for genetic analysis. The phenotypic characteristics are as follows Figure 1-3 shown.

[0026] (2) Methods

[0027] The measurement of fruit length is chosen 40 days after flowering, when the fruit is fully ripe, so that the length of the fruit can be judged clearly and accurately, and 1 standard fruit is surveyed for each plant.

[0028] Take the young cotyledons of cucumber, put them into 2 mL centrifuge tubes, quickly freeze them with liquid nitrogen, and store them in a -80°C refrigerator for genomic DNA extraction.

[0029] The BSA analysis method was used to preliminarily locate the CsSf1.1 locus. In the F2 segregation population composed of the wild type 9930 and the mutant sf1.1 (9930×sf1.1), 10 short-fruited plants and 10 long-fruited plants were randomly selected from the F2 population to construct the cucumber short-fruit gene pool and long-fruit gene pool.

[0030] A large population of 1625 9930×sf1.1 F2 and 90 BC1F1 were planted, and fresh cotyledon DNA was extracted at the seedling stage. The markers on both sides of the preliminary positioning interval were used to genotype these individuals, and the lines with exchange between the two markers were selected.

[0031] Based on the cucumber reference genome sequence (cucumber (Chinese Long) genome V2), candidate genes within the fine-mapping interval of CsSf1.1 were identified; the candidate gene sequences were amplified using high-fidelity enzymes for first-generation sequencing, and the mutation sites of the CsSf1.1 gene were confirmed in combination with the retest data of sf1.1; the function and structure of the CsSf1.1 gene were analyzed using the cucumber genome website (http: / / cucurbitgenomics.org / organism / 2) and the NCBI website.

[0032] (3) Results

[0033] According to the gene sequence in the cucumber genome database, the gene structure and coding sequence were predicted using Geneious software. In 9930 and WD1, the genomic DNA length of CsSf1.1 (Csa1G665390) was 14084bp, including 28 exons and 27 introns. Analysis found that the single base mutation of the mutant sf1.1 occurred in the last base of the 22nd exon of the 21st intron of the Csa1G665390 gene (from the 'G' of the wild-type WD1 to the 'A' of sf1.1). No other base differences were found between WD1 and sf1.1 in exons and other non-coding regions ( Figure 4 a, 4b), this base mutation caused the splicing site of the 21st intron of the Csa1G665390 gene to change from GT-AG of WD1 to GT-AA of sf1.1 ( Figure 4 a, 4b); Sequencing and comparison of the cDNA of Csa1G665390 gene in WD1 and sf1.1 revealed a 42 bp deletion at 2191-2332 bp in the cDNA coding region of Csa1G665390 gene in sf1.1 ( Figure 4 c, 4d), the deletion is due to the mutation of the splicing site of the 21st intron of Csa1G665390 in sf1.1, which results in the excision of 42 bases of the 22nd exon during the splicing of the intron. Based on the above results, Csa1G665390 is the candidate gene controlling the length of cucumber fruit, named CsSf1.1.

[0034] Using the cDNA of wild-type WD1 and short-fruit mutant sf1.1 as templates, primers were designed according to the Csa1G665390 sequence, amplified with high-fidelity enzymes, and PCR products were separated by 2% agarose gel electrophoresis and observed under ultraviolet light. The results showed that the length of the cDNA amplification product of the Csa1G665390 gene was about 2.9 kb, and the length was different between the wild-type strain WD1 and the short-fruit mutant sf1.1, but not significantly ( Figure 5 a); Primers designed in the coding region of exons 21 and 22 of the Csa1G665390 gene were used to perform electrophoresis analysis of PCR amplification products using cDNAs of WD1 and sf1.1 as templates. It was found that the length of the PCR amplification product in the wild-type WD1 was about 300 bp, and the length of the product in the short melon mutant sf1.1 was about 250 bp ( Figure 5 b), PCR amplification and electrophoresis analysis showed that the cDNA of Csa1G665390 gene in the short-fruit mutant sf1.1 had a sequence deletion.

[0035] Based on the SNP of cucumber fruit length gene CsSf1.1, a dCAPS (derived cleaved amplified polymorphic sequence) marker was designed using the dCAPS FINDER 2.0 online website. The wild type and mutant were amplified by PCR using the dCAPS marker. The PCR products were digested with restriction endonuclease PstI at 37°C for 5h, and then the enzyme digestion products were separated by 8% polyacrylamide gel electrophoresis to count the band patterns. Based on the difference in the gene sequence of CsSf1.1 gene in the short-fruit parent sf1.1 and the long-fruit parent WD1, as well as the sequence information on both sides of the differential site, a pair of specific dCAPS markers were obtained, as follows:

[0036] Forward primer (5′-TACTGAACTTATTTTTCTGCCTGCA-3′) (SEQ ID NO: 1);

[0037] Reverse primer (5'-GGTAACCCATAATCTGAACGTTTAT-3') (SEQ ID NO: 2).

[0038] The marker can be stably amplified by PCR and can clearly distinguish the genotypes of CsSf1.1 and CsSf1.1 by 8% polyacrylamide gel electrophoresis and is named dCAPSCsSf1.1.

[0039] Linkage analysis was performed on 320 F2 segregating populations (exchange plants and some population plants were selected from 1625 plants) using the dCAPSCsSf1.1 marker. The results showed that this marker was co-segregated with the cucumber short-fruit gene CsSf1.1, that is, the phenotypes of long-fruited and short-fruited plants in the population were consistent with the long-fruited and short-fruited parental bands amplified by the dCAPSCsSf1.1 marker, respectively. Since the short-fruit trait is controlled by a single recessive gene, the heterozygous band amplified by this marker corresponds to long-fruited plants, such as Figure 6 As shown. In the identification of cucumber seedlings, DNA was extracted from cotyledons at the cotyledon stage, and the dCAPSCsSf1.1 marker was used to perform genotyping of different individual plants, and the fruit length of the plant was predicted based on the genotyping results. The fruit length was then measured 40 days after flowering. Combining genotyping and fruit length phenotype data analysis, it was found that the dCAPSCsSf1.1 marker was co-segregated with the fruit length phenotype in the F2 population, indicating that the dCAPSCsSf1.1 marker can be used to perform genotyping of plants at the seedling stage, and the fruit length of each individual plant can be predicted based on the typing results, that is, molecular markers can be used to assist in breeding at the seedling stage to improve breeding efficiency.

[0040] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Application of a dCAPS molecular marker primer pair co-segregating with a cucumber fruit length gene in cucumber molecular marker-assisted breeding, the primer pair comprising a forward primer having a sequence as shown in SEQ ID NO: 1 and a reverse primer as shown in SEQ ID NO:

2.

2. The use according to claim 1, characterized in that: The dCAPS molecular marker primer pair co-segregating with the cucumber fruit length gene is used for co-segregation of the cucumber fruit length gene, that is, the long-fruit and short-fruit individual plant phenotypes of the population are respectively consistent with the long-fruit and short-fruit parent band types amplified by the dCAPS molecular marker primer pair, and the heterozygous band type amplified by the dCAPS molecular marker primer pair corresponds to the long-fruit type plant.

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