A SNP molecular marker for identifying watermelon rind thickness and its application

Abstract

The present invention relates to a SNP molecular marker for identifying the thickness of watermelon rind and an application thereof, and relates to the field of biotechnology. The SNP molecular marker is located at the 32344170th base of chromosome 2 of the watermelon genome, and the base is C or T. If the genotype is CC, the corresponding watermelon rind thickness is less than or equal to 0.6 cm, and if the genotype is GG, the corresponding watermelon rind thickness is greater than 0.6 cm. It effectively accelerates the breeding process and improves the breeding efficiency, which is of great significance to watermelon breeding.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a SNP molecular marker for identifying watermelon rind thickness and an application thereof. Background Art

[0002] As an important cucurbit crop, watermelon is an important fruit for people to cool down and quench their thirst in summer, and it occupies a very important position in the world's horticultural crops. China is the world's largest producer and consumer of watermelon. Peel thickness is one of the important agronomic traits of watermelon, which directly affects the edible rate and commercial rate of watermelon and is an important breeding indicator for watermelon. At present, the research on watermelon peel traits mainly focuses on the gene positioning and marker development of peel color, texture, wax powder, hardness, etc., but there is no report on the research on watermelon peel thickness.

[0003] In recent years, multi-gene marker screening methods have developed rapidly, and Genome-Wide Association Study (GWAS) has become one of the mainstream methods, which can screen the relevant variation of a trait in the whole genome. The identification of genetic markers of low heritability traits through genome-wide association analysis has become a common method in modern breeding. SNP molecular markers (single nucleotide polymorphisms) refer to changes in DNA sequences caused by mutations of single nucleotides at the genome level. They are widely present in the genome and have the characteristics of high throughput, simplicity, stability, high sensitivity, etc. They are molecular markers with great development prospects in molecular marker-assisted breeding.

[0004] With the advent of the watermelon genome era, clarifying the gene functions of important agronomic traits of watermelon, finding the key genes controlling these traits, developing corresponding molecular markers, and applying molecular markers to breeding are important contents of current watermelon breeding. Therefore, the present invention provides a SNP molecular marker for identifying watermelon rind thickness and its application. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a SNP molecular marker for identifying watermelon rind thickness and its application. The purpose is to develop molecular markers related to watermelon rind thickness and apply them to watermelon breeding to accelerate the watermelon breeding process.

[0006] In order to solve the above technical problems, the first purpose of the present invention is to provide a SNP molecular marker for identifying the thickness of watermelon rind, wherein the SNP molecular marker is located at the 32344170th base of chromosome 2 of the watermelon genome, and the base is C or T.

[0007] The invention has the following beneficial effects: the invention uses genome-wide association analysis (GWAS) to identify 5 significant SNP sites related to watermelon peel, and then combines transcriptome sequencing to analyze candidate genes near 100kb upstream and downstream of the significant SNP sites, and determines the candidate genes related to watermelon peel thickness. Based on the candidate genes, it is finally determined that the 32344170th SNP site of chromosome 2 of the watermelon genome is closely linked to the watermelon peel thickness. Therefore, the SNP marker related to watermelon peel thickness of the invention can promote molecular marker-assisted selection of watermelon peel thickness, effectively accelerate the breeding process, improve breeding efficiency, and has important significance for watermelon breeding.

[0008] Based on the above technical solution, the present invention can also be improved as follows.

[0009] Furthermore, if the genotype is CC, the corresponding watermelon rind thickness is less than or equal to 0.6 cm, and if the genotype is GG, the corresponding watermelon rind thickness is greater than 0.6 cm.

[0010] Furthermore, the watermelon is watermelon 97103.

[0011] The second purpose is to provide an application of a SNP molecular marker for identifying the thickness of watermelon rind, the application of the above-mentioned SNP molecular marker in the identification of watermelon rind thickness; or the application of the above-mentioned SNP molecular marker in watermelon breeding.

[0012] In the above application, the polymorphic substance of the SNP molecular marker is a reagent, a kit or an instrument for detecting the SNP molecular marker using whole genome sequencing, and can be a primer pair, a probe or a chip for detecting the SNP marker.

[0013] The third object is to provide a watermelon breeding method, comprising the following steps:

[0014] The sample watermelon with the SNP molecular marker CC in the genome detected was used as a parent for watermelon breeding.

[0015] The present invention also protects a gene chip comprising the above-mentioned SNP molecular marker or the above-mentioned SNP molecular marker combination.

[0016] Primer pairs, probes or chips used to detect the above-mentioned SNP molecular markers and their corresponding gene chips or SNP molecular marker combinations and their corresponding gene chips are also within the protection scope of the present invention.

[0017] The use of the above-mentioned SNP molecular markers or SNP molecular marker combinations and primer pairs, probes or chips for detecting the above-mentioned SNP molecular markers or SNP molecular marker combinations in watermelon whole genome selection is also within the protection scope of the present invention.

[0018] The use of the above-mentioned SNP molecular marker or SNP molecular marker combination and the primer pair, probe or chip for detecting the above-mentioned SNP molecular marker or SNP molecular marker combination in watermelon breeding is also within the protection scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Manhattan plot of watermelon rind thickness based on GWAS. DETAILED DESCRIPTION

[0020] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] Example 1: Mining SNP markers associated with watermelon rind thickness based on genome-wide association analysis (GWAS)

[0022] 1. Materials and Methods

[0023] 1.1. Acquisition, quality control and typing of watermelon whole genome resequencing data

[0024] 112 natural watermelon population materials were selected from the watermelon mid-term library (see Table 1 for details) and planted in a greenhouse. Young leaves at the top of the plants were collected during the plant extension period, and complete genomic DNA was extracted using the traditional CTAB method. The quality of the extracted genomic DNA was tested using a Nanodrop ND1000 spectrophotometer, and the quality standard was met when the A260 / 280 ratio was between 1.8 and 2.0 and the A260 / 230 ratio was around 1.7 and 1.9.

[0025] The concentration of DNA samples that meet the standards was diluted to 50 ng / μl, and each DNA sample was resequenced using the sequencing platform of Illumina (average sequencing depth 9.24X). The sequencing reads of each variety were aligned to the watermelon reference genome (http: / / cucurbitgenomics.org / organism / V1) using SOAP2. The parameters were set as follows: -m100, -x888, -s35, -132, -v3, and duplicate removal. All reads on the double-end and single-end alignments were used for SNP calling using SOAPsnp software with the parameters: -L100-u-F156. SNPs were filtered and data with SNP quality ≥ 40 and base quality ≥ 40 were retained.

[0026] 1.2 Determination of peel thickness of 112 watermelon natural population materials

[0027] 112 natural populations were planted for 3 consecutive years. At the fruit maturity stage, 3 melons with the same node position and fruit shape were selected. The watermelon peel thickness was measured with a ruler. The average value was the final peel thickness of each material using the data of different field phenotypes for 3 years. The peel thickness of the 112 natural population materials of watermelon shown in Table 1 was measured. With 0.6 cm as the boundary, peel thickness less than or equal to 0.6 cm was considered thin peel, and peel thickness greater than 0.6 cm was considered not thin peel.

[0028] 1.3. Genome-wide association analysis

[0029] The mixed linear model in GEMMA 0.98.1 software was used, with Q matrix and kinship matrix as covariates, and Tassel V5.2.43 software was used to conduct genome-wide association analysis on 112 natural watermelon population materials. The Manhattan plot was drawn by R software, and the significance threshold was calculated by Bonferroni correction. -log10 (P) 0.1 / Ne (Ne = number of effective SNPs) and -log10 (P) 0.01 / Ne were set as the two threshold lines for screening significant SNPs. The results are shown in Figure 1 The watermelon rind thickness gene was located at the end of chromosome 2. There are 5 significant SNPs in this region, namely S2:32344170, S2:32321308, S2:32304738, S2:32328501, and S2:32311192.

[0030] 2. Results

[0031] Obtaining the target SNP: The expression and gene function analysis of 21 candidate genes near 100kb upstream and downstream of the significant SNP site found that the MADS family gene Cla97C02G044160 in watermelon may be a key candidate gene for regulating the thickness of watermelon fruit skin, and the significant SNP S2:32344170 upstream of this gene may be closely linked to the thickness of watermelon skin. Therefore, the SNP marker related to watermelon skin thickness is located at the 32344170th nucleotide on chromosome 2 of the watermelon genome (http: / / cucurbitgenomics.org / , V2), which is T or C. The homozygous genotypes of this SNP marker are TT genotype and CC genotype, respectively. The CC genotype is a thin-skinned watermelon in the watermelon variety, and the TT genotype is a non-thin-skinned watermelon in the watermelon variety. The detection efficiency is 91% (see Tables 1 and 2 for details).

[0032] Table 1 Peel thickness and genotypes of 112 natural watermelon populations

[0033]

[0034]

[0035]

[0036]

[0037] Table 2 Statistics of peel thickness and genotypes of 112 natural watermelon populations

[0038]

[0039] Example 2: Application Evaluation

[0040] 1.1. Validation of the identification method for watermelon rind thickness

[0041] The genotypes of 30 natural population materials at position 32344170 on chromosome 2 were detected by genome resequencing, and the accuracy of the SNP markers of the present invention in detecting thin-skinned watermelon materials was 97%, as shown in Tables 3 and 4.

[0042] Table 3 Peel thickness and genotypes of 30 natural watermelon populations

[0043]

[0044] Table 4 Statistical values ​​of peel thickness and genotypes of 30 natural watermelon populations

[0045]

[0046]

[0047] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An application of a SNP molecular marker in the identification of watermelon rind thickness, characterized in that: The SNP molecular marker is located at base 32344170 of chromosome 2 of the watermelon genome 97103V2, and the base is C or T.

2. The use of a SNP molecular marker in watermelon rind thickness identification according to claim 1, characterized in that: If the genotype is CC, the corresponding watermelon rind thickness is less than or equal to 0.6 cm. If the genotype is TT, the corresponding watermelon rind thickness is greater than 0.6 cm.

3. The use of a SNP molecular marker in watermelon rind thickness identification according to claim 1, characterized in that: The watermelon is watermelon 97103.

4. A watermelon breeding method, characterized in that: The steps include: The SNP molecular marker is located at the 32344170th base of chromosome 2 of the watermelon genome 97103V2, and the base is C or T; the sample watermelon with the SNP molecular marker CC in the genome is detected and used as a parent for watermelon breeding.

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