Molecular marker based on SNP site of melon leaf yellowing characteristics and application thereof

Abstract

The present application relates to a melon leaf yellowing characteristic SNP marker, a dCAPS molecular marker, primers and applications thereof. The SNP site of the melon leaf yellowing characteristic is nucleotide A to G at position 1609229 on melon chromosome 8 MELO3C007233. The primer set of the dCAPS marker includes a forward primer 5'-CATTTCCATTTCCCATCCT-3' and a reverse primer 5'-CTCCTCGACAATCTTCACTCT-3'. The SNP site or dCAPS molecular marker and its primers can be applied to the identification and assisted screening breeding of melon leaf yellowing characteristics. The SNP site and dCAPS marker identified in the present application have the characteristics of rapidness, directness, high specificity, etc., and can complete the screening and prediction work of a large number of melon leaf yellowing characteristic materials in a short time, and can effectively serve the breeding work of melon leaf yellowing characteristics.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of melon breeding, and particularly relates to a molecular marker based on a SNP site of a melon leaf yellowing characteristic and application thereof. BACKGROUND

[0002] Melon (Cucumis melo L.) belongs to the melon species of the Cucurbitaceae family and is an annual climbing herbaceous plant and one of the world's major fruits. A key point in the melon seed production industry is seed purity identification work, but most of the existing seed purity test techniques are generation planting, and seed purity is identified by phenotypic traits of the offspring, which has the disadvantages of time-consuming and labor-intensive. Therefore, there is an urgent need for a more convenient and fast seed purity identification technique. The melon leaf color mutant can well solve this problem. By using a melon leaf color yellow mutant as a female parent and a normal leaf color melon material as a male parent for hybrid breeding, the seed purity of the offspring can be identified at the seedling stage. Therefore, the yellow leaf melon material is improved and utilized to breed a yellow leaf melon line with excellent traits, which can shorten the seed production process and reduce the seed production cost.

[0003] However, there have been reports on the study of melon leaf color mutation genes in China. Zheng Jing et al. studied the natural yellowing mutant material '1521' of thin-skinned melon and found that the yellowing trait is controlled by a pair of recessive genes. Zhu Huayu et al. preliminarily located the ygl gene on the 11th chromosome of melon by studying the yellow-green leaf color material M68 of thick-skinned melon. Wang Xiaojuan preliminarily analyzed the genetic regulation network and molecular mechanism of the gene CmGLK that controls the yellow-green leaf color trait of the material M68 to regulate the development of melon chloroplast. However, due to different breeding needs, more leaf yellowing materials with good growth advantages and corresponding identification molecular markers are needed to be obtained. SUMMARY

[0004] The inventors found that the yellowing trait mutation of the yellowing mutant material C40 occurs on the 8th chromosome MELO3C007233 of melon, and an A→G mutation occurs at 1609229 bp, which causes the leaf color of melon to change from green to yellow. Based on this research, the dCAPS molecular marker C-43 for identifying the yellowing mutation of melon was developed. The primer designed according to the marker can quickly and accurately identify whether the melon variety or strain carries the yellowing mutation gene and its homologous gene, and can also quickly identify the seed purity of the melon new variety.

[0005] The first aspect of the present application provides a SNP marker for detecting the leaf yellowing characteristic of melon, wherein the SNP marker is a mutation of the 1609229th nucleotide from A to G on the 8th chromosome MELO3C007233 of the melon genome.

[0006] The second aspect of the present application provides a dCAPS marker for detecting the leaf yellowing trait of melon, the primer sequences of which are as follows:

[0007] Upstream primer: 5'-CATTTCCATTTCCCATCCT-3',

[0008] Downstream primer: 5'-CTCCTCGACAATCTTCACTCT-3'.

[0009] Further, the restriction enzyme of the dCAPS molecular marker of the SNP site is RsaI.

[0010] The third aspect of the present application provides a method for identifying the leaf yellowing trait of melon by using a dCAPS marker: extracting the whole genome DNA of melon leaves;

[0011] Using the primer pair of the dCAPS marker to perform PCR amplification on the genomic DNA of watermelon;

[0012] Enzymatically digesting the PCR amplification product;

[0013] Electrophoresis of the enzymatic digestion product, judging the phenotype of the leaf yellowing trait of melon according to the band type of the enzymatic digestion product, the leaf yellowing trait of melon showing a band type of 154 bp, and other leaf traits showing a band type of 134 bp or two bands of 154 bp and 134 bp.

[0014] The one or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0015] The dCAPS marker designed based on the SNP site discovered in the present application has the characteristics of rapidness, directness, high specificity, etc., and can complete the screening and prediction work of a large number of melon leaf yellowing trait materials in a short time, and can effectively serve the melon breeding and seed purity identification involving the leaf yellowing trait of melon. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 For the QTL-seq, Euclidean Distance (ED), and G' value statistical correlation analysis results in an embodiment of the present application.

[0017] Figure 2 For the enzyme digestion identification results in the genotyping of the dCAPS molecular marker in identifying the BC1 population in an embodiment of the present application. DETAILED DESCRIPTION

[0018] The following will be described in detail with reference to the drawings and embodiments. However, the embodiments are only used to illustrate the present application in detail, and do not limit the scope of the present application in any way.

[0019] In the following examples, the instruments and equipment are conventional unless otherwise specified; the reagents are commercially available unless otherwise specified; the test methods are conventional unless otherwise specified. In order to better understand the technical solutions of the present application, the above technical solutions will be described in detail below in combination with the drawings and specific embodiments of the specification.

[0020] Example 1: Breeding test of melon leaf color mutant

[0021] 1. Test materials:

[0022] Select the yellowing mutant material C40 in the melon germplasm resource of the Institute of Fruit Trees, Chinese Academy of Agricultural Sciences. It was found by Shang Jian in 2008 when he investigated the resources in the experimental base of the middle-term library in Liucun, Xiangfu, Bagang Town, Zhengzhou, Zhongmu County. After observing the stable traits after self-crossing for many generations, it was named yellowing mutant No. 1, and the material number is C40.

[0023] 2. Breeding method

[0024] (1) Select varieties with excellent traits such as early maturity, disease resistance, high yield, and high quality as the object of transformation, hoping to obtain a batch of excellent lines with melon leaf yellowing traits, other excellent traits, and similar to the original variety. First, cross the melon leaf yellowing line C40 with the above transformation materials to obtain the first hybrid. Then, select and leave single plants with melon leaf yellowing traits in the seedling stage of the first hybrid offspring or the backcross offspring of C40. Then, according to the purpose of transformation, according to the method of melon hybrid selection and utilization, select and leave single plants with the required traits for self-crossing selection, and finally obtain new self-crossing lines with melon leaf yellowing traits, fruit, flesh color, and seed characteristics consistent. Studies have shown that this trait is controlled by a pair of alleles, and the F2 and BC1 yellow leaf and green leaf separation ratios are 1:3 and 1:1, respectively. To ensure the screening population, 80-100 seeds of each material were sown for hybrid self-crossing offspring, and 40-60 seeds of each material were sown for hybrid backcross offspring. The breeding time is generally 6-7 generations.

[0025] (2) Then cross the above melon leaf yellowing line as the female parent and the normal green leaf line as the male parent to obtain the first hybrid.

[0026] (3) Finally, purity identification was carried out by sowing and observing after harvesting of the first generation hybrid, and the single plant with normal green cotyledon or seedling leaf was a successful hybrid plant, and the single plant with yellow leaf was a hybrid failure plant. If the number of plants with green leaf color in 100 hybrid offspring is n, then the purity of the offspring seed is n%.

[0027] Example Two, Acquisition of Melon Leaf Yellowing Characteristic SNP Site

[0028] 1. Test materials:

[0029] T27 (female parent) and C40 (male parent) selected from the medium-term library of the West Melon Germplasm Resource of the Zhengzhou Fruit Tree Research Institute of the Chinese Academy of Agricultural Sciences were used as parents, F1 was obtained by hybridization of the two parents, and BC1 population was obtained by hybridization of F1 and the female parent. T27 is green leaf, and C40 is yellow leaf.

[0030] 2. dCAPS marker development method:

[0031] (1) Through BSA association analysis technology, a sequencing library was established, and then after the Illumina NovaSeq sequencing data (Raw Data) was downloaded, the low-quality data was filtered to obtain high-quality data (Clean Data). The Clean Data was aligned to the reference genome sequence reference genome CM4.0 from the Cucurbit Genomics Information Network CucCAP of the United States Cornell University http: / / cucurbitgenomics.org / v2 / ftp / genome / melon / DHL92 / v4.0 / using BWA software, and the sequence position attribution (i.e. BAM file) was obtained. The BAM file was corrected using the Best Practices process of GATK software, and SNP markers were detected. The SNPEff software and the gene prediction information of the reference genome were used for variation function annotation, and further SNP and InDel were used for trait positioning analysis. Based on the variation detection results, the genetic markers with homozygous difference between the two parents (except F1 population) were screened, SNP markers and InDel markers were selected, and the SNP and INDEL sites with difference between the parents obtained based on marker filtering were subjected to QTL-seq, Euclidean Distance (ED), and G’value statistical methods for association analysis (results are shown in Figure 1 ).

[0032] (3) According to the results of BSA analysis, the candidate interval is located between 1070000bp-1240000bp on chr12 and 52191bp-2683507bp on chr08, with a total length of about 2.67M, and InDel / SNP primers are designed in the candidate interval.

[0033] (4) The polymorphism is screened in T27 (the mother), C40 (the father) and F1 using the designed InDel / SNP primers, and then the genotypes are identified and the primers are screened in the BC1 population, so as to gradually shorten the candidate interval of the melon leaf color mutation gene and select the most closely linked molecular marker. After analysis, the closely linked molecular marker C-43 of the melon leaf yellowing trait is screened, which is located at 1609229bp on chromosome 8 of melon. A A→G mutation occurs at this site, and whether the mutation of the site in the melon genome is closely related to the yellowing of the melon leaf.

[0034] Example Three: Design and identification of dCAPS marker primer of melon leaf yellowing characteristic

[0035] After analysis, the closely linked molecular marker C-43 of the melon leaf yellowing trait is screened, which is located at 1609229bp on chromosome 8 of melon. A A→G mutation occurs at this site, and whether the mutation of the site in the melon genome is closely related to the yellowing of the melon leaf. According to the upstream and downstream sequences, the PCR primer sequences of the dCAPS molecular marker C-43 are as follows:

[0036] Upstream primer F1: 5'-CATTTCCATTTCCCATCCT-3'

[0037] Downstream primer R1: 5'-CTCCTCGACAATCTTCACTCT-3'

[0038] The method for identifying the melon leaf yellowing characteristic by using the above molecular marker mainly comprises the following steps:

[0039] (1) Extracting total DNA from leaves by CTAB method

[0040] ① Put 1g of fresh leaves into a mortar and add liquid nitrogen to grind them into powder uniformly;

[0041] ② After grinding, immediately transfer the leaf powder into a centrifuge tube containing 1mL of CTAB extraction solution, mix thoroughly, and then place in a 65℃ constant temperature water bath for 60 min, and mix 2-3 times during the process;

[0042] ③ After taking out from the water bath, centrifuge for 1 min at 8000 rpm;

[0043] (4) Take the supernatant to a new centrifugal tube, add an equal volume of chloroform: isopropyl alcohol (25:1, V / V), mix well;

[0044] (5) 10,000 rpm, centrifugal 5 min, at 10,000 rpm;

[0045] (6) Take the supernatant to a new centrifugal tube, add 0.7 times volume of isopropyl alcohol (pre-cooled on ice for 30 min), mix well, and freeze at -20℃ (not more than 30 min) to precipitate the DNA;

[0046] (7) Centrifugal 5 min, at 10,000 rpm;

[0047] (8) Discard the supernatant, wash the precipitate with anhydrous ethanol several times, pour off the soaking liquid, and dry on the clean bench (about 10 min);

[0048] (9) Add 200 μL of distilled water to dissolve the DNA;

[0049] (10) Use a UV spectrophotometer to determine the concentration of the DNA, and store in a refrigerator at -20℃ for standby use.

[0050] (2) Primer design:

[0051] (1) According to the BSA primary positioning region in Example 1 and the parent resequencing results, design primers uniformly in the candidate region;

[0052] (2) Use a PERL self-programming program to extract the sequences of 500 bp before and after the corresponding position of the insertion and deletion, and use Primer 5 software to design primers.

[0053] (3) The PCR reaction system is as follows: melon leaf total DNA (100 ng / μL) 1 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, 2×Power Taq PCR MasterMix 12.5 μL, ddH2O 9.5 μL.

[0054] (4) The PCR reaction program is as follows: 94℃, 5 min; 94℃, 20 s, 55℃, 1 min, 72℃, 30 s, for a total of 35 cycles; 72℃, 5 min.

[0055] (5) Enzymatic digestion: use the following enzyme digestion system for the PCR product: PCR product 2 μL, RsaI enzyme 0.2 μL, Buffer 1 μL, ddH2O 6.8 μL, and the enzyme digestion program is 37℃, 45 min.

[0056] (6) Preparation of electrophoresis buffer 5xTBE electrophoresis buffer: Tris 26.95 g, EDTA 1.86 g, boric acid 13.75 g, deionized water to 500 mL.

[0057] (7) Preparation of gel: 40% polyacrylamide solution: polyacrylamide 77.34 g, methylene bisacrylamide 2.66 g, deionized water to 200 mL.

[0058] ① Measure 10 mL of 40% polyacrylamide solution, add 5xTBE 5 mL, 10% TBE 200 μL to prepare 8% gel, shake well.

[0059] ② Pour into the gel preparation plate and install the comb, and pull out the comb after fully solidifying.

[0060] (8) Loading: 1 μL of PCR product is spotted into the loading well of the prepared 8% polyacrylamide gel, and appropriate markers are added into the other loading wells.

[0061] (9) Electrophoresis: 240 V, 200 mA, 35 min. The electrophoresis buffer is 1xTBE.

[0062] (10) Silver staining and development Silver staining solution: nitric acid 1 g, glacial acetic acid 5 mL, anhydrous ethanol 50 mL, deionized water to 500 mL. Developing solution: sodium hydroxide 15 g, formaldehyde (37%) 2.5 mL, deionized water to 500 mL.

[0063] (11) Band type interpretation: Place the naturally dried glass plate after development on the reading table and observe the position difference of the two parent bands with the naked eye.

[0064] Example Three: Identification and verification of dCAPS marker in melon leaves

[0065] Application of dCAPS marker in identification of melon leaf yellowing characteristics, the application steps are as follows:

[0066] (1) Use the dCAPs molecular marker and method steps described in Example 2 to genotype 96 known leaf yellowing characteristic plant individuals.

[0067] (2) According to the electrophoresis results of the enzyme digestion products, if the amplified product contains a 134 bp homozygous band or a 154 bp and 134 bp heterozygous band or a fragment as shown in C43.1 ( Figure 2 ), it is determined that the melon to be tested is green leaf. If the amplified product contains a 154 bp homozygous band or a fragment as shown in C43.2 ( Figure 2) the fragment shown in Fig. 2, it is determined that the melon to be tested is a yellow leaf. (3) The detection results of 92 melon germplasms show the genotype distribution of the dCAPS molecular marker c-43 (A is the maternal genotype, B is the paternal genotype, and H is the heterozygous genotype) in 96 BC1 populations (Table 1). Among the 96 BC1s, 48 are A, 0 are B, and 48 are H, and the accuracy rate of genotype identification reaches 100%.

[0068] .

[0069] .

Claims

1. A dCAPS marker for detecting yellowing characteristics of melon leaves, characterized in that, The following primers were designed based on the corresponding SNP sites: Upstream primer: 5'-CATTTTCCATTTCCCATCCT-3', Downstream primer: 5'-CTCCTCGACAATCTTCACTCT-3'; The SNP site is a mutation of A→G at nucleotide position 1609229 on chromosome 8 MELO3C007233 of the melon genome.

2. The dCAPS mark according to claim 1, characterized in that, The restriction endonuclease used in conjunction with the primers is RsaI.

3. A method for identifying the yellowing characteristics of melon leaves, characterized in that, Includes the following steps: a. DNA extraction: Total DNA was extracted from melon leaves using the CTAB method; b. PCR amplification: The reaction system consisted of 1 μL of total DNA from melon leaves, 1 μL of the upstream primer described in claim 1, 1 μL of the downstream primer described in claim 1, 12.5 μL of 2×Power Taq PCR MasterMix, and 9.5 μL of ddH2O. The reaction program was as follows: 94 °C for 5 min, followed by 35 cycles of 94 °C, 20 s, 55 °C, 1 min, 72 °C, 30 s, 72 °C, 5 min. c. Enzyme digestion reaction system and procedure: The reaction system consisted of 2 μL of PCR product, 0.2 μL of RsaI enzyme, 1 μL of buffer, and 6.8 μL of ddH2O; the reaction procedure was to treat at a constant temperature of 37℃ for 45 minutes. d. Electrophoretic pattern analysis: The enzyme digestion products were subjected to polyacrylamide gel electrophoresis, development, staining, and band pattern interpretation to find the target band. The genotype was determined according to the band size and positional relationship of the amplified products. The yellowing characteristics of melon leaves showed a 154 bp band, while other leaf characteristics showed a 134 bp band or two bands of 154 bp and 134 bp.

4. A kit comprising the primers of claim 1 and the restriction endonuclease RsaI.

5. The application of the SNP marker for yellowing leaf characteristics of melon, the dCAPS molecular marker of claim 1, or the kit of claim 4 in the identification and / or assisted screening of yellowing leaf characteristics in melon breeding, wherein the SNP marker for yellowing leaf characteristics of melon is the mutation of nucleotide 1609229 on chromosome 8 MELO3C007233 of the melon genome from A, which is a non-yellowing leaf characteristic, to G, which is a yellowing leaf characteristic.

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