SNP molecular marker linked to w gene for cucumber peel color identification and its application

Abstract

The present invention discloses a SNP molecular marker linked to a W gene for cucumber peel color identification and its application. The site of the SNP marker is at the 40,368,703 bp of the physical position of cucumber chromosome 3. The base of the site is A or T. The cucumber material with the base of the site A is a cucumber tender fruit white peel material, and the cucumber material with the base of the site T is a cucumber tender fruit green peel material. The SNP marker obtained by the present invention can be used to judge the cucumber peel color of the cucumber candidate material at any stage. The marker has the advantages of high efficiency, few restrictions and accuracy, improves the efficiency of breeding cucumber materials with white peels, and shortens the breeding cycle.

Description

Technical Field

[0001] The invention relates to the technical field of biotechnology-assisted breeding, and in particular to a SNP molecular marker linked to a W gene for cucumber peel color identification and an application thereof. Background Art

[0002] Cucumber (Cucumis sativus L.) belongs to the Cucurbitaceae family and is one of the main cultivated vegetable crops in my country. After a long period of natural evolution and artificial selection, cucumber varieties have become increasingly abundant, consumers have more choices in cucumber varieties and types, and their requirements for product quality are also getting higher and higher. Peel color is an important quality trait of cucumber and has an important impact on the commercial value of cucumber. It is mainly determined by the composition and content of chlorophyll, carotenoids and anthocyanins (Allan et al., 2008).

[0003] At present, molecular markers for dark green (DG), green (dg), yellow-green (yg) and white (w) peels of cucumber have been reported (Pierce et al., 1990; Wehner et al., 1997; Gu Xingfang et al., 2005b). Li Yali (2008) used green-skinned cucumbers and white-skinned cucumbers as materials and used BSA and SRAP methods to discover molecular markers ME8EM14-425 and ME9EM1-309 related to green peels. Sun Xiaodan et al. (2011) used molecular markers to study yellow-green peels and milky white peels of cucumbers and found molecular markers E34M59 and E43M61 related to milky white peels. Dong Shaoyun et al. (2012) used SSR molecular markers to locate the white peel gene w on chromosome 3 and obtained linkage markers SSR23141 and SSR23517.

[0004] The white fruit peel trait of cucumber is controlled by a recessive single gene (w), and dark green is dominant over white (Dong Shaoyun, Miao Han, et al., "Study on the Genetic Law and Positioning of the White Fruit Peel Gene of Cucumber", 2012). Hanqiang Liu et al. ("Map-based cloning, identification and characterization of the w gene controlling white immature fruit color in cucumber (Cucumis sativus L.)", 2016) finely located the white fruit peel gene (w) of cucumber tender fruit, and limited the gene w to a 8.2kb physical region on chromosome 3, with four newly developed markers on both sides: InDel markers (LH39253, LH39258) and SNP markers (ASPCR39259, ASPCR39250).

[0005] In the study of functional genes related to cucumber peel color, Zhou et al. (2015) showed that Csa7G051430 is the gene that causes cucumber light green. Through further analysis, it was found that the amino acids in the GTPase domain of the conserved region of this gene have changed. Lun et al. (2016) found that the Ycf54 protein encoded by Csa6G133820 is involved in chlorophyll synthesis, resulting in a decrease in chlorophyll content in cucumber peel. Dong Xu et al. (2015) located the gene related to white peel on chromosome 3 and found that Csa3G904080 caused peel color differences by encoding pyruvate kinase. Liu et al. (2015) located the cucumber white peel gene on chromosome 3 and found APRR2 as a candidate gene controlling the white peel color of cucumber through positional cloning (Liu et al., 2016). Yang Kang et al. (2018) used sequence alignment and gene expression methods to determine that the candidate gene for the white-green peel of cucumber is Csa3G904140. Through further analysis, it was found that this gene has a certain regulatory effect on chlorophyll biosynthesis and chloroplast development. This positioning result is the same as the positioning result of APRR2. Huang Hongyu et al. (2021) used genome-wide association analysis (GWAS) combined with QTL positioning and other methods to locate the gene Csa3G91292 that controls the light green peel color of cucumber, and developed a KASP molecular marker.

[0006] Genome-wide association study (GWAS) is a method for overall association analysis of common genetic variations (single nucleotide polymorphisms and copy number) in the whole genome. This method takes natural populations as the research object and is based on linkage disequilibrium (LD) between genes (locus) retained after long-term recombination. It combines the diversity of target trait phenotypes with the polymorphism of genes (or marker loci) for analysis, and can directly identify gene loci or marker loci that are closely related to phenotypic variation and have specific functions.

[0007] The cleaved amplified polymorphic sequence (CAPS) marker is a DNA marker for detecting SNP sites that is generated by combining specific primer PCR with restriction enzyme digestion. The principle is that the SNP mutation site is located on the recognition sequence of a certain endonuclease. After PCR amplification with specific primers, the PCR product is digested with the corresponding endonuclease to produce polymorphisms in the digested fragments, which can be displayed by agarose or polyacrylamide gel electrophoresis and staining. However, it is relatively rare for SNP to be located at a certain restriction enzyme digestion site. Michaels and Amasino (1998) and Neff (1998) developed a derived cleaved amplified polymorphic sequence (dCAPS) marker with artificially introduced mismatched bases based on the CAPS marker, which can convert almost all SNP sites into markers that can be detected by electrophoresis. Both CAPS and dCAPS have the characteristics of co-dominance, site specificity, simple operation, rapid detection, low cost and independence from precision instruments and equipment. They can be used for plant genotyping, positioning, genetic diversity analysis, variety identification and other aspects.

[0008] So far, there has been no report on SNP markers associated with white peel of cucumber developed based on dCAPS markers. Summary of the invention

[0009] Based on the above-mentioned gap in the prior art, the purpose of the present invention is to provide a SNP molecular marker linked to the w gene for cucumber peel color identification and its application.

[0010] In order to achieve its purpose, the present invention adopts the following technical solution:

[0011] A SNP molecular marker linked to the w gene for cucumber peel color identification, wherein the site of the SNP marker is at the 40,368,703bp physical position of cucumber chromosome 3, the base of the site is A or T, the cucumber material with the base A at the site is a cucumber tender fruit white peel material, and the cucumber material with the base T at the site is a cucumber tender fruit green peel material.

[0012] The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3, wherein the 205th base is A or T.

[0013] A specific primer pair for detecting the above-mentioned SNP molecular marker, comprising primers w-SNP8703-F and w-SNP8703-R, wherein the nucleic acid sequences of the primers are as follows:

[0014] w-SNP8703-F: 5'-CCATGTGGTGGGGGTCTCGAGA-3',

[0015] w-SNP8703-R: 5'-GAGACCATAGCCTTTATCTACAACAATTCC-3'.

[0016] A kit for detecting the above-mentioned SNP molecular markers comprises the above-mentioned specific primer pair.

[0017] Preferably, the kit further comprises a cucumber genomic DNA extraction reagent, a PCR amplification reaction reagent, a PCR amplification product sequencing reagent or a SNP differential site recognition reagent; preferably, the SNP differential site recognition reagent comprises a restriction endonuclease capable of recognizing SNP differential sites, and preferably further comprises a polyacrylamide gel electrophoresis reagent; preferably, the restriction endonuclease is MfeI.

[0018] The above-mentioned SNP molecular marker is used in any of the following (1)-(5):

[0019] (1) Identify or assist in identifying white peel / green peel materials of cucumber tender fruits;

[0020] (2) Identify or assist in identifying the gene w, which is the white fruit skin gene of cucumber tender fruit;

[0021] (3) Screening or assisting in the screening of tender-fruited white-skinned cucumber varieties;

[0022] (4) Cultivate or assist in the cultivation of tender-fruit white-skinned cucumber varieties;

[0023] (5) Cucumber breeding.

[0024] The application comprises the following steps: extracting genomic DNA of the sample to be tested as a template, performing PCR amplification using the amplification primers of the above-mentioned SNP molecular markers, and sequencing or enzyme-cutting electrophoresis detection on the PCR amplification products. The enzyme-cutting electrophoresis detection refers to using a restriction endonuclease that can recognize the difference site of the SNP molecular marker to cut the PCR amplification products, and then performing electrophoresis on the amplification products. Preferably, the electrophoresis uses non-denaturing polyacrylamide gel electrophoresis.

[0025] In the above-mentioned application technology solution, the amplification primers of the SNP molecular marker are as follows:

[0026] w-SNP8703-F: 5'-CCATGTGGTGGGGGTCTCGAGA-3',

[0027] w-SNP8703-R: 5'-GAGACCATAGCCTTTATCTACAACAATTCC-3';

[0028] A 235 bp fragment was obtained by PCR amplification. When sequencing was used for detection, the cucumber material whose 205th base of the PCR amplification fragment was A was a young fruit white peel cucumber material, and the cucumber material whose 205th base of the PCR amplification fragment was T was a young fruit green peel cucumber material;

[0029] When enzyme digestion and electrophoresis are used for detection, the restriction endonuclease is MfeI. If a 235bp band is obtained by electrophoresis after enzyme digestion, the detection object is a young fruit cucumber material with white peel; if a 201bp band is obtained by electrophoresis after enzyme digestion, the detection object is a young fruit cucumber material with green peel.

[0030] The reaction system for PCR amplification was as follows: 7.5 ng DNA template, 50 ng each of the forward primer and the reverse primer of the primer, 5 μl 2×3G Taq Master Mix for PAGE (Red Dye), and double distilled water to 10 ul;

[0031] The total reaction system was 10 μL, 10.0 ng DNA template, 25 ng each of forward and reverse primers, 5 μL 2×3G TaqMaster Mix for PAGE (Red Dye), and the balance was double distilled water;

[0032] 10 μL of the enzyme digestion reaction system contains: 1 μL 10×Cutsmart Buffer, 0.2 μL MfeI restriction endonuclease, 3 μL PCR product, and 5.8 μL double distilled water.

[0033] The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 51°C for 15 seconds, extension at 72°C for 30 seconds, 35 cycles; incubation at 72°C for 5 minutes, and storage at 10°C;

[0034] The enzyme digestion temperature was 37°C and the enzyme digestion time was 3h.

[0035] The beneficial effects of the present invention are:

[0036] The SNP marker obtained by the present invention can be used to determine the cucumber peel color at any stage of the cucumber candidate material. The marker has the advantages of high efficiency, few restrictions and accuracy, which improves the efficiency of breeding cucumber materials with white peel and shortens the breeding cycle.

[0037] The present invention lays a foundation for studying the molecular mechanism of peel regulation and for the precise positioning and molecular cloning of the white peel of cucumber by developing molecular markers linked to the white peel of cucumber. It also provides an efficient way for the use of molecular markers to assist in the selection of new varieties with white peel. The white regulatory genes can be quickly introduced into excellent varieties by combining hybridization and backcrossing techniques, providing support for the selection of high-quality new varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the detection result of SNP marker SNPw-8703 on cucumber parent materials CSIVF0254 (P1), CSIVF0021 (P2), and F1 generation individual plants.

[0039] Figure 2 It is the detection result of SNP marker SNPw-8703 on cucumber F2 population; 'a' represents the banding type: a band of 201bp is obtained, 'b' represents the banding type: a band of 235bp is obtained, 'h' represents the banding type: two bands of 201bp and 235bp are obtained, and 'u' represents no band; the lanes corresponding to the underlined fonts in the figure are individual plants whose field phenotypes are inconsistent with the molecular marker band verification. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0041] The experimental methods in the following embodiments are all conventional methods unless otherwise specified; the experimental reagents not otherwise specified are all conventional reagents in the art, which can be purchased commercially or prepared by conventional methods in the art, and the specifications are laboratory pure grade.

[0042] Materials and Methods

[0043] The experimental materials used in this study were 130 inbred lines and hybrids, including North China types, European greenhouse types, and hybrids of North China types and European greenhouse types.

[0044] The validation population used in this study was the hybrid F1 obtained from the cucumber tender fruit white peel material CSIVF0021 and the cucumber tender fruit green peel material CSIVF0254 as parents, as well as the constructed F2 population containing 74 strains.

[0045] CSIVF0254(P1): A hybrid of the North China European type, with green fruit skin. This is a known variety.

[0046] CSIVF0021(P2): Indian cucumber with white skin. This is a known variety.

[0047] CSIVF0254 and CSIVF0021 are cucumber materials bred by the Cucumber Research Group of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences. They are preserved in this laboratory and guaranteed to be released to the public for verification experiments within twenty years from the date of application.

[0048] The dCAPS-MfeI labeled primers were designed by our laboratory based on the resequencing genome information using the CAPS Finder2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html) website and DNAMAN software, and synthesized at Beijing Bioengineering Co., Ltd.

[0049] Main reagents

[0050] PCR experiments used 2×3G Taq Master Mix for PAGE (RedDye) from Vazyme; enzyme digestion used NEBuffer cutsmart restriction endonuclease MfeI; gel electrophoresis used 40% non-denaturing polyacrylamide from Beijing Coolbo Technology Co., Ltd., which was diluted to 6% before use. Sequencing was performed at Beijing Bioengineering Co., Ltd.

[0051] Example 1. Obtaining SNP marker SNPw-8703 for detecting cucumber peel color

[0052] 1. Identification of cucumber peel color

[0053] The 130 inbred lines and hybrids selected by the cucumber genetics and breeding group of the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences were used as test materials. The phenotypes of the tender fruits were identified and divided into five grades: white, light green, flowery green, green and dark green. The phenotypes were determined by visual determination, and three replicates were measured for each material.

[0054] 2. Preliminary positioning - genome-wide association study (GWAS)

[0055] 130 test materials from our laboratory were used as genotype data for this experiment (genome information based on resequencing by the inventor's laboratory). The genotype data were filtered by removing SNP sites with minor allele frequency (MAF<0.05) and missing rate exceeding 20%, and finally 1,642,709 high-quality SNP sites were obtained. Association analysis was performed using a mixed linear model (MLM) with -log 10 (Pvalue) = 7.2 was the significant screening threshold, and the SNP (single nucleotide polymorphism) sites significantly associated with the white peel of young cucumber fruits were determined. Manhattan and QQ-plot graphs were drawn using the R language package CMplot.

[0056] Through genome-wide association analysis, the signal locus gcsc3.2 was repeatedly detected on chromosome 3 (Table 1).

[0057] Table 1 SNP loci significantly associated with white peel of young cucumber fruit

[0058]

[0059] 3. Development and application of SNP markers

[0060] Analysis-log 10 The site with a Pvalue above 7.2 was found at the physical position 40,368,703bp of chromosome 3. The base of this site in the genome of cucumber material CSIVF0254 (P1) was T, and the base of this site in the genome of material CSIVF0021 (P2) was A. Thus, the SNP molecular marker was found and recorded as SNPw-8703. The marker SNPw-8703 was significantly correlated with white peel.

[0061] Based on the obtained marker SNPw-8703 associated with the white peel of cucumber tender fruit, a dCAPS marker linked to the cucumber white peel gene w was developed. The DNA sequence of the above segment of the reference genome of cucumber chromosome 3 (the reference genome sequence of the North China cucumber variety "9930", version number Cucumber (Chinese Long) v3 Genome) was downloaded from the cucumber genome database website (http: / / cucurbitgenomics.org / ), and a pair of primers were designed using the dCAPS Finder2.0 (http: / / helix.wustl.edu / dcaps / dcaps.html) website and DNAMAN software according to the position of the SNP. The nucleotide sequence is as follows:

[0062] w-SNP8703-F (SEQ ID NO.1): 5'-CCATGTGGTGGGGGTCTCGAGA-3',

[0063] w-SNP8703-R (SEQ ID NO. 2): 5'-GAGACCATAGCCTTTATCTACAACAATTCC-3'.

[0064] Due to the relationship of the SNPs obtained above (SNP=A / T), when base A exists, the recognition sequence of the endonuclease MfeI cannot be formed (the recognition sequence is C↓AATTG, ↓ is the enzyme cleavage site), and the amplified fragment cannot be cut by the endonuclease MfeI; when base T exists, the endonuclease recognition sequence is formed, and the amplified fragment can be cut by the endonuclease MfeI.

[0065] The above primers (w-SNP8703-F / w-SNP8703-R) were used to perform PCR amplification on the parental materials CSIVF0254 (P1) and CSIVF0021 (P2). In the material CSIVF0021 (P2) (white peel), a 235 bp band (nucleotide sequence as shown in SEQ ID NO.3) was obtained, and in the material CSIVF0254 (P1) (green peel), a 235 bp band (nucleotide sequence as shown in SEQ ID NO.4) was also obtained. The amplified fragment was digested with endonuclease MfeI to obtain a specific band ( Figure 1 ), the PCR amplification band of the white peel material CSIVF0021 was still 235 bp after the enzyme digestion step, and the band type was recorded as b; the PCR amplification band of the green peel material CSIVF0254 was digested to obtain a 201 bp band (the nucleotide sequence is shown in SEQ ID NO.5), and the band type was recorded as a; in the hybrid F1, two bands of 201 bp and 235 bp were detected at the same time, which was an intermediate type, and the band type was recorded as h.

[0066] The specific operation method of the detection is as follows:

[0067] Step 1. DNA extraction and PCR amplification

[0068] Young leaves of cucumber plants were taken and the genomic DNA of parents and F1 were extracted using the improved CTAB (cetyltrimethylammonium bromide) method.

[0069] The dCAPS-labeled PCR reaction system is as follows: total reaction system 10 μL, 1 μL DNA (10.0 ng·μL -1 ), forward and reverse primers (50 ng·μL -1 ), 0.5 μL each of 2×3G Taq Master Mix for PAGE (Red Dye) (product of Vazyme), and 3 μL of double distilled water.

[0070] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 51°C for 15 seconds, extension at 72°C for 30 seconds, 35 cycles; incubation at 72°C for 5 minutes, and storage at 10°C.

[0071] Step 2. Complete digestion of PCR product with MfeI

[0072] Enzyme digestion reaction system 10 μL: 1 μL 10×Cutsmart Buffer, 0.2 μL MfeI restriction endonuclease, 3 μL PCR product, 5.8 μL double distilled water.

[0073] Enzyme digestion reaction procedure: digest at 37℃ for 3h and store at -20℃.

[0074] The digested products were separated by 6% non-denaturing polyacrylamide gel, the electrophoresis buffer was 0.5×TBE, and the electrophoresis was performed at a constant power of 150V for 1h 30min. After electrophoresis, the products were silver stained and the band types were counted.

[0075] Step 3. Result determination

[0076] Method 1: Skip step 2, sequence the PCR product directly without enzyme digestion. The sequence obtained from the white peel material CSIVF0021 has an A at the 205th base; the sequence obtained from the green peel material CSIVF0254 has a T at the 205th base; the sequence obtained from F1 has two bases at this position, A and T coexisting.

[0077] Method 2: After complete digestion with endonuclease MfeI, the digestion products were separated by 6% non-denaturing polyacrylamide gel, the electrophoresis buffer was 0.5×TBE, and the electrophoresis was separated at a constant power of 150V for 1h 30min. After electrophoresis, silver staining was performed and the band types were counted.

[0078] A 235 bp fragment was obtained from the white peel material CSIVF0021, and the band pattern was recorded as b; a 201 bp fragment was obtained from the green peel material CSIVF0254, and the band pattern was recorded as a; two bands were detected simultaneously in F1, and the band pattern was recorded as h.

[0079] Example 2: F2 population verification

[0080] The F2 population containing 74 strains constructed using the cucumber white peel material CSIVF0021 and the cucumber green peel material CSIVF0254 as parents was tested and verified to determine the accuracy of the marker for molecular marker-assisted selection.

[0081] Young leaves of cucumber plants were taken, and the genomic DNA of each individual plant in the F2 population was extracted using the improved CTAB (cetyltrimethylammonium bromide) method, and then detected using the PCR amplification, enzyme digestion, and electrophoresis methods in Example 1.

[0082] The phenotype of tender fruits of F2 plants was identified in the field and divided into five levels: white, light green, flowery green, green and dark green. Data analysis showed that the color of the peel of tender cucumber fruits was close to normal distribution, indicating that this trait was a quantitative trait in the F2 population. Green was incompletely dominant over white.

[0083] Results (such as Figure 2 (shown in Figure 2) found that among the 74 cucumber materials, the field phenotype identification was 16 tender fruit white peel materials and 58 tender fruit green peel materials (including light green, flower green, green and dark green). Through molecular detection, the field phenotype of white peel materials was detected, of which 12 materials had electrophoresis bands of type b; the field phenotype of green peel materials was detected, of which 55 materials had electrophoresis bands of type a or h. A total of 67 bands reflected phenotypic data consistent with the field survey results, with a calculated accuracy of 90.5%. The method of the present invention can be used to identify cucumber peel color for molecular-assisted breeding.

Claims

1. Application of a SNP molecular marker linked to the w gene for cucumber peel color identification in any of the following (1)-(4): (1) Identify or assist in identifying white peel / green peel materials of cucumber tender fruits; (2) Identify or assist in identifying the gene w, which is the white fruit skin gene of cucumber tender fruit; (3) Screening or assisting in the screening of tender-fruited white-skinned cucumber varieties; (4) Cultivate or assist in the cultivation of tender-fruit white-skinned cucumber varieties; The nucleotide sequence of the SNP molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.4, wherein the 205th base is A or T, and the cucumber material with the base A at this position is the cucumber young fruit with white peel material, and the cucumber material with the base T at this position is the cucumber young fruit with green peel material.

2. The use according to claim 1, characterized in that: The application comprises the following steps: extracting genomic DNA of the sample to be tested as a template, performing PCR amplification using the amplification primers of the SNP molecular marker, and sequencing or enzyme-cutting electrophoresis detection on the PCR amplification product. The enzyme-cutting electrophoresis detection refers to using a restriction endonuclease that can recognize the differential site of the SNP molecular marker to cut the PCR amplification product, and then performing electrophoresis on the amplification product.

3. The use according to claim 2, characterized in that: The electrophoresis adopts non-denaturing polyacrylamide gel electrophoresis.

4. The use according to claim 2, characterized in that: The amplification primers of the SNP molecular marker are as follows: w-SNP8703-F: 5'-CCATGTGGTGGGGTCTCGAGA-3', w-SNP8703-R: 5'-GAGACCATAGCCTTTATCTACAACAATTCC-3'; A 235 bp fragment was obtained by PCR amplification. When sequencing was used for detection, the cucumber material whose 205th base of the PCR amplification fragment was A was a young fruit white peel cucumber material, and the cucumber material whose 205th base of the PCR amplification fragment was T was a young fruit green peel cucumber material; When enzyme digestion and electrophoresis are used for detection, the restriction endonuclease is MfeI. If a 235bp band is obtained by electrophoresis after enzyme digestion, the detection object is a young fruit cucumber material with white peel; if a 201bp band is obtained by electrophoresis after enzyme digestion, the detection object is a young fruit cucumber material with green peel.

5. The use according to claim 4, characterized in that: The reaction system for PCR amplification was as follows: 10 μL total reaction system, 10.0 ng DNA template, 25 ng each of forward and reverse primers, 5 μL 2×3G Taq Master Mix for PAGE, and the balance was double distilled water; 10 μL of the enzyme digestion reaction system contains: 1 μL 10× Cutsmart Buffer, 0.2 μL MfeI restriction endonuclease, 3 μL PCR product, and 5.8 μL double distilled water.

6. The use according to claim 5, characterized in that: The reaction procedure of the PCR amplification is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 51°C for 15 seconds, extension at 72°C for 30 seconds, 35 cycles; incubation at 72°C for 5 minutes, and storage at 10°C; The enzyme digestion temperature was 37°C and the enzyme digestion time was 3h.

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