Disclosure of Invention
In view of the above, the invention aims to provide a dCAPS molecular marker related to the size of watermelon seeds and application thereof, and the dCAPS molecular marker can accurately identify the size of watermelon seeds through verification of 330 parts of watermelon germplasm resources, is fast and effective, can identify in a seedling stage, greatly shortens the breeding period, and can be applied to mass production.
The invention provides application of a reagent for detecting SNP loci related to the size and shape of watermelon seeds in watermelon breeding, wherein the SNP loci are located at 5389022 th nucleotide of chromosome 6 of a watermelon genome, when genotypes of the SNP loci are GG homozygosity or T/G heterozygosity, the length of the watermelon seeds is less than 10.5mm, and when genotypes of the SNP loci are TT homozygosity, the length of the watermelon seeds is more than 10.5mm.
The invention provides a dCAPS molecular marker for detecting the size of watermelon seeds, which comprises an upstream primer N022-dCAPS_S6F and a downstream primer N022-dCAPS_S6R; the nucleotide sequence of the upstream primer N022-dCAPS_S6F is shown in SEQ ID NO. 1; the nucleotide sequence of the downstream primer N022-dCAPS_S6R is shown in SEQ ID NO. 2.
The invention provides a kit for detecting the size of watermelon seeds, which comprises the dCAPS molecular marker.
Preferably, the kit further comprises detection reagents including Easy Taq Buffer and Easy Taq DNA Polymerase.
The invention provides a method for detecting the size of watermelon seeds, which comprises the following steps:
1) Taking the total DNA of the watermelon plant as a template, and carrying out PCR amplification by taking the dCAPS molecular marker as a primer to obtain an amplification product;
2) Performing enzyme digestion on the amplification product, and then performing electrophoresis to determine the size of the watermelon seeds according to an electrophoresis result;
the enzyme for enzyme digestion is Hpa I or Hinc II restriction enzyme;
when the electrophoresis result shows that the single band of 279bp exists, the genotype of the SNP locus is TT homozygosity, and the length of the watermelon seeds is more than 10.5mm;
when the electrophoresis result shows that only one band of 251bp exists, the genotype of the SNP locus is GG homozygote, and the length of the watermelon seeds is less than 10.5mm;
when the electrophoresis result has two bands of 251bp and 279bp simultaneously, the genotype of the SNP locus is T/G heterozygous, and the length of the watermelon seeds is less than 10.5mm.
Preferably, the amplification system of the PCR amplification in step 1) comprises the following components in 20. Mu.L: 1 mu L, N of total DNA of watermelon plant, 1 mu L, N of 022-dCAPS 6F 1 mu L, N of 022-dCAPS 6R 1 mu L of total DNA of watermelon plant, 10 XEasy Taq Buffer 2 mu L, dNTPs 1.6.6 mu L of total DNA of Easy Taq DNA Polymerase, 0.2 mu L, ddH2 O 13.2μL。
Preferably, the concentration of the total DNA of the watermelon plant is 80-120 ng/. Mu.L, and the concentrations of the N022-dCAPS_S6F and the N022-dCAPS_S6R are independently 8-12 mu.M.
Preferably, the amplification procedure of the PCR amplification in step 1) is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 55℃for 1min, extension at 72℃for 30s,32 cycles; extending at 72℃for 8min.
Preferably, the system of cleavage in step 2) comprises 2. Mu.L of amplification product, 1. Mu.L of Hpa I or Hinc II restriction enzyme, 10 Xbuffer 2. Mu.L, ddH2 O15. Mu.L; the enzyme digestion procedure is carried out at 37 ℃ for 1-2 h.
The invention provides the dCAPS molecular marker, the kit and the application of the method in watermelon auxiliary breeding.
Compared with the prior art, the invention has the following beneficial effects:
the SNP marker and dCAPS molecular marker closely linked with the target trait can be used for auxiliary selective breeding of watermelon molecular markers, and can improve the selection of the size trait of watermelon seedsAccuracy, speed up the breeding process. Pair F using SNP and dCAPS molecular markers of the present invention2 The population totally carries out phenotype verification on 751 watermelon plants to be detected, and the result shows that the accuracy rate of identifying the size of the watermelon seeds by the SNP molecular marker reaches 100%. The SNP marker and the dCAPS molecular marker are utilized to verify 330 parts of watermelon germplasm resources with different seed sizes, and the result shows that the accuracy of the molecular marker in identifying the large seed and the small seed watermelons is 100 percent, and the identification of the watermelon seed size by utilizing the SNP marker and the dCAPS molecular marker has the advantages of simplicity, rapidness and high accuracy and can be applied to mass production.
The invention also provides application of the dCAPS molecular marker and the kit in auxiliary breeding of watermelons, and provides a new means for detecting the size of watermelon seeds, so that the improvement process of the size characters of the watermelon seeds is accelerated, and the breeding accuracy and the selection efficiency are improved.
Detailed Description
The invention provides application of a reagent for detecting SNP loci related to the size and shape of watermelon seeds in watermelon breeding, wherein the SNP loci are located at 5389022 th nucleotide of chromosome 6 of a watermelon genome, when genotypes of the SNP loci are GG homozygosity or T/G heterozygosity, the length of the watermelon seeds is less than 10.5mm, and when genotypes of the SNP loci are TT homozygosity, the length of the watermelon seeds is more than 10.5mm. In the following description, a seed having a length of less than 10.5mm is simply referred to as a small seed, and a seed having a length of more than 10.5mm is simply referred to as a large seed.
In the invention, the watermelon genome data is derived from a network, and the website is as follows: http:// www.cucurbitgenomics.org/organism/2.
The invention provides a dCAPS molecular marker for detecting the size of watermelon seeds, which comprises an upstream primer N022-dCAPS_S6F and a downstream primer N022-dCAPS_S6R; the nucleotide sequence of the upstream primer N022-dCAPS_S6F is shown in SEQ ID NO. 1; the nucleotide sequence of the downstream primer N022-dCAPS_S6R is shown in SEQ ID NO. 2.
In the present invention, the concentration of the upstream primer N022-dCAPS_S6F and the downstream primer N022-dCAPS_S6R is preferably 8 to 12. Mu.M, more preferably 10. Mu.M.
The invention also provides a kit for detecting the size of the watermelon seeds, which comprises the dCAPS molecular marker.
In the present invention, the kit preferably further comprises a detection reagent, preferably comprising Easy Taq Buffer and Easy Taq DNA Polymerase; in the present invention, the Easy Taq Buffer and Easy Taq DNA Polymerase are preferably derived from commercial products.
In the present invention, the kit preferably further comprises dNTPs, and the dNTPs are preferably used at a concentration of 2.4 to 2.6mM, more preferably 2.5mM.
The invention also provides a method for detecting the size of the watermelon seeds, which comprises the following steps: 1) Taking the total DNA of the watermelon plant as a template, and carrying out PCR amplification by taking the dCAPS molecular marker as a primer to obtain an amplification product; 2) Performing enzyme digestion on the amplification product, and then performing electrophoresis to determine the size of the watermelon seeds according to an electrophoresis result; the enzyme for enzyme digestion is Hpa I or Hinc II restriction enzyme; when the electrophoresis result shows that the single band of 279bp is available, the genotype of the SNP locus is TT homozygosity, and the watermelon seeds are large; when the electrophoresis result is that only one band of 251bp exists, the genotype of the SNP locus is GG homozygote, and the watermelon seeds are small; when the electrophoresis result has two bands of 251bp and 279bp at the same time, the genotype of the SNP locus is T/G heterozygous, and the watermelon seeds are small.
The extraction method of the total DNA of the watermelon plant is not particularly limited, and a plant genome DNA extraction method conventional in the art is adopted, and in the specific implementation process of the invention, a CTAB method is preferably adopted for extraction. In the present invention, the concentration of the total DNA of the watermelon plant is preferably 80-120 ng/. Mu.L, more preferably 90-110 ng/. Mu.L, and still more preferably 100 ng/. Mu.L.
In the present invention, the amplification system for PCR amplification preferably comprises the following components in 20. Mu.L: 1 mu L, N of total DNA of watermelon plant, 1 mu L, N of 022-dCAPS 6F 1 mu L, N of 022-dCAPS 6F 1 mu L of total DNA of watermelon plant, 10 XEasy Taq Buffer 2 mu L, dNTPs 1.6.6 mu L of total DNA of Easy Taq DNA Polymerase, 0.2 mu L, ddH2 O 13.2μL。
In the present invention, the amplification procedure for PCR amplification is as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, renaturation at 55℃for 1min, extension at 72℃for 30s,32 cycles; extending at 72℃for 8min.
After the amplification product is obtained, the amplification product is subjected to enzyme digestion; the system for cleavage preferably comprises, in 20. Mu.L: amplification product 2. Mu.L, hpa I or Hinc II restriction endonuclease 1. Mu.L, 10 Xbuffer 2. Mu.L, ddH2 O15. Mu.L; the procedure for the cleavage is preferably 37℃for 1 to 2 hours, more preferably 37℃for 1.5 hours.
The invention also provides the dCAPS molecular marker, the kit and the application of the method in watermelon auxiliary breeding. In the breeding process of watermelons, the dCAPS molecular markers provided by the invention are used for detecting the genotypes of SNP of each individual plant in a breeding offspring group, classifying different individual plants according to the genotypes, and screening the individual plants with target characters for subsequent breeding. For example, when fresh watermelons are screened, small-seed-trait individuals are selected for subsequent breeding, and when seed-reserved watermelons are screened, large-seed-trait individuals are selected for subsequent breeding.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Discovery of SNP (Single nucleotide polymorphism) sites closely linked with watermelon seed size and shape and development of dCAPS molecular markers
1. Genetic rule analysis of watermelon seed size gene
"BW85" (P) using large seed female parent of watermelon1 ) And a small seed male parent "F211" (P2 ) Hybridization, selfing and backcrossing to obtain six-generation population (P respectively1 、P2 、F1 、F2 、BC1 P1 、BC1 P2 ) The seed length, width and hundred grain mass were measured. Combining the parents, F1 ，BC1 P1 ，BC1 P2 And 221F2 And separating the size phenotype identification result of the colony seeds to obtain the dominant of the watermelon seed size gene controlled by a major gene from small seeds to large seeds.
2. Watermelon seed size-related gene targeting
Obtaining F by hybridization of large seed female parent BW85 and small seed male parent F211 of watermelon2 Segregating populations from F2 And selecting 30 watermelon plants with large seeds and small seeds from the generation separation population, respectively extracting leaf genome DNA, mixing together genome DNA of 30 large-seed watermelon plants and genome DNA of 30 small-seed watermelon plants. Parent and F using an Illumina HiSeq 2000 sequencer2 The generation mix samples were sequenced to obtain 47.68G effective data, which was compared to watermelon reference genome 97103 (http:// www.cucurbitgenomics.org/organoprism/2). 214582 SNP loci are identified, and the SNP-index value is calculated by utilizing the SNPs to carry out bioinformatics analysis, so that the candidate interval of the gene related to the seed size trait is positioned between 4847169 th nucleotide and 5419136 th nucleotide of a No. 6 chromosome of a watermelon genome.
3. Obtaining candidate SNP loci
SNP site between 4847169 ～ 5419136 nucleotide of watermelon genome chromosome 6 obtained by BSA-seq for F2 And (3) carrying out genotype analysis on the group single plants, combining single plant phenotype data (the seeds are large seeds or small seeds), screening the recombined single plants, continuously identifying the genotype of the recombined single plants by utilizing SNP in the interval to obtain new recombined single plants, gradually shortening candidate intervals from two ends, and finally finely positioning target genes between 5363185 ～ 5419136 nucleotides. Genotype and phenotype of SNP detection at 5389022 th position in the intervalFull anastomosis, no exchange event occurred, and the SNP locus was determined to be closely linked with the seed size trait. The SNP locus is GG homozygous or T/G heterozygous in small seed plants, and the SNP locus is TT homozygous in large seed plants.
dCAPS molecular marker primer design
Designing a dCAPS molecular marker primer according to the SNP locus, wherein the dCAPS molecular marker primer consists of a primer N022-dCAPS_S6F and a primer N022-dCAPS_S6R.
N022-dCAPS_S6F (upstream primer):
5′-AACTCGTGGAATGAACTC-3′(SEQ ID NO.1)
N022-dCAPS_S6R (downstream primer):
5′-GCCTTTTAGTAACTGGTAAATGTGGGTTAA-3′(SEQ ID NO.2)。
the PCR sequence of the 279bp PCR product amplified by the primer and the small seed sample is shown as SEQ ID NO. 3:
AACTCGTGGAATGAACTCCAAAACTCTTAATTGCAACTCCTTATTAATCTTCATGATCATAGTCTTGAATAATGTTAGAATTCACATCACTCTAATTGATTCGTGAATTTGAATATATTTGAAATCACTATATATCCATAAATTCTTAGGACAATAATTGTGCTATAAAATTATTGAATAAACCACCTTAGATAACTCTTTGATAATCAAATCCTAAATCCCTTAGTTATTAGGGATCATACAAATTTGTTAACCCACATTTACCAGTTACTAAAAGGC。
the PCR sequence of the large seed sample is shown as SEQ ID NO. 4;
AACTCGTGGAATGAACTCCAAAACTCTTAATTGCAACTCCTTATTAATCTTCATGATCATAGTCTTGAATAATGTTAGAATTCACATCACTCTAATTGATTCGTGAATTTGAATATATTTGAAATCACTATATATCCATAAATTCTTAGGACAATAATTGTGCTATAAAATTATTGAATAAACCACCTTAGATAACTCTTTGATAATCAAATCCTAAATCCCTTAGTTATTAGGGATCATACAAATTTTTTAACCCACATTTACCAGTTACTAAAAGGC。
after cleavage by the DNA restriction enzyme Hpa I or Hinc II (Hpa I can recognize GTT/AAC, hinc II can recognize GTC/GAC in addition to GTT/AAC), the results after the cleavage in the test are consistent, and Hinc II has lower cost than Hpa I), the following situation occurs: cutting into two sections of fragments with the sizes of 251bp and 28bp (gel is run out in the electrophoresis process due to small molecular weight, and a 28bp band is not presented), wherein the SNP locus in the corresponding PCR product is GG; only the 279bp fragment was found, and the SNP site in the corresponding PCR product was TT.
Identification of watermelon seed size by dCAPS molecular markers
5.1 extracting genomic DNA of watermelon leaves by adopting a conventional CTAB method, removing RNA, measuring the OD value of the genomic DNA sample at 260nm and 280nm by using an ultraviolet spectrophotometer, calculating the DNA content and the ratio of OD260/280, and selecting the genomic DNA sample with the OD260/280 value between 1.8 and 2.0, wherein the concentration is diluted to 100 ng/mu L.
PCR reaction System and procedure
The reaction system: 1. Mu.l of total DNA of watermelon leaf (100 ng/. Mu.l), 1. Mu.l of N022-dCAPS_S6F1mu.l, 2.0. Mu.l of 10 Xeasy Taq Buffer, 1.6. Mu.l of dNTPs (2.5 mM), easy Taq DNA Polymerase 0.2.2. Mu. L, ddH2 O 13.2μL。
PCR reaction procedure: 94℃for 5min,32 cycles of 94℃for 30s,55℃for 1min,72℃for 30s, 72℃for 8min.
5.3. Enzyme cleavage reaction System and procedure
The reaction system: PCR products 2. Mu.L, hpa I or Hinc II restriction endonuclease 1. Mu.L, 10 Xbuffer 2. Mu.L, ddH2 O 15μL。
The reaction procedure: the temperature is kept at 37 ℃ for 1.5 hours.
5.4. And (3) performing 8% polyacrylamide gel electrophoresis, development, dyeing and band interpretation on the PCR product after enzyme digestion.
After dCAPS amplification/enzyme digestion reaction is completed, 1 mu L of Loading Buffer (6×loading Buffer) is added into 5 mu L of amplification/enzyme digestion PCR product, and the mixture is uniformly mixed by a pipette, and 5 mu L of vertical spots are absorbed and added into 8% polyacrylamide gel for electrophoresis detection; electrophoresis at 260V for 1.5 h; and (3) silver staining, taking out the gel after the strips are clear, and photographing and storing. FIG. 1 is a pair F2 Separating the plant electrophoretogram of the population part, and obtaining accurate genotype from the size of the target band detected in the figure 1, thereby verifying the genotype and phenotype. Wherein A represents a small seed banding pattern, B represents a large seed banding pattern, and H represents a heterozygous genotype.
Example 2
Seed size identification of hybrid populations using SNP molecular markers
1. Selection of experimental materials
F was obtained as a hybrid combination BW85 (large) x F211 (small)2 751 individual plants of (2) are experimental materials, and the phenotype detection result shows that F2 In the generation group, 567 single plants are used for small seeds, 184 single plants are used for large seeds (the length of the seeds is more than 10.5 mm).
2. Pair F using SNP molecular markers2 Population identification
751F pairs of dCAPS molecular markers on chromosome 6 of watermelon obtained in example 1 and related to seed size trait2 The individual plants were identified by the specific identification method described in example 1. The identification result shows that the identification result of the dCAPS molecular marker and F2 The phenotypes of the groups are consistent, and the accuracy reaches 100%. Table 1 shows that the primer pair N022-dCAPS_S6 is in part F2 And (3) identifying and verifying the results in the population, wherein A represents a small seed genotype, B represents a large seed genotype, and H represents a heterozygous genotype (small seed).
TABLE 1N022-dCAPS_S6 primer pair at F2 Identification and verification in populations
The detection results of the other two molecular markers and the dCAPS molecular marker are compared with the detection results of the dCAPS molecular marker provided by the invention.
indel13_S6F:TGGTAGCAACATGGAAGAAG(SEQ ID NO.5)
indel13_S6R:AACTGCCAAGGTGAGAAAAC(SEQ ID NO.6)
This Indel marker is located at chromosome 5300529 of the Watermelon Genome (Watermelon (97103) v1 Genome), small seed base GGAGAGAGAGAGAGAGAGA, large seed base G; the amplified product of the large seed is 158bp, and the amplified product of the small seed is 176bp.
dcaps9_S6F:CTAAAAATACAGGATTAAAATTGTACATTC(SEQ ID NO.7)
dcaps9_S6R:TGTAAAACACATATATAACG(SEQ ID NO.8)。
The SNP marker is located at 5418365bp of a 6 th chromosome of a Watermelon Genome (Waterelon (97103) v1 Genome), small seed genotype GG and large seed genotype AA; restriction enzyme TaqI, the size of the target fragment of the large seed is 142bp, and the size of the target fragment of the small seed is 113bp.
As can be seen from the results in Table 2, the identification results of the two molecular markers of indel13_S6 and dcas9_S6 were inaccurate, and the identification results were inconsistent with the actual seed length.
TABLE 2 labeling of different dCAPS molecules at F2 Identification results in populations
Example 3
Identification of 330 parts of watermelon germplasm resources by using dCAPS molecular markers
1. Selection of experimental materials
330 parts of watermelon germplasm resources selected from the existing germplasm resources are experimental materials, wherein 41 parts of large-seed watermelons and 289 parts of small-seed watermelons are shown in table 3 as specific names and phenotypes.
2. Identification of 330 parts of watermelon germplasm resources by using dCAPS molecular markers
330 parts of watermelon germplasm resources were identified by using dCAPS molecular markers (N022-dCAPS_S6 primer pairs) on chromosome 6 of watermelon obtained in example 1 and the specific identification method is referred to in example 1. The identification result shows that the accuracy of the molecular marker for identifying the large seed and the small seed watermelons is 100%.
Table 3022-dCAPS_S6 primer pair identification and verification in 330 watermelon germplasm resources
According to the embodiment, the dCAPS molecular marker designed based on the SNP locus can accurately identify the size of the watermelon seeds, is quick and effective, can be identified in a seedling stage, greatly shortens the breeding period, and can be applied to mass production.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.