CN113186323A - KASP molecular marker related to strawberry petal shedding speed character and application thereof - Google Patents
KASP molecular marker related to strawberry petal shedding speed character and application thereof Download PDFInfo
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Abstract
The invention discloses a KASP molecular marker related to the fast and slow character of strawberry petal shedding and application thereof, wherein the molecular marker is positioned on chromosome 2-2 of strawberry, a base A at 776356bp is mutated into G, the strawberry with the genotype of GG shows that the petal shedding is fast, and the correlation between the genotype and the phenotype is verified in an F1 group and a resource group.
Description
Technical Field
The invention belongs to the technical field of molecular biology and genetic breeding, and particularly relates to a molecular marker related to the fast and slow shedding traits of strawberry petals and application thereof.
Background
Strawberry is an important horticultural economic crop, is widely planted all over the world, and has high nutritive value and short production period. China is the first big strawberry producing country and consuming country in the world, the strawberry planting area in China in 2018 is 11 ten thousand hectares, and the yield reaches 296 thousand tons (FAO, 2018). The gray mold is one of the most serious diseases causing the yield loss of the strawberries, can occur in the growth period and the storage period of the strawberries, and causes the annual yield loss of the strawberries to be at least 10-30 percent, and can reach more than 50 percent when the strawberries are serious. Botrytis cinerea can infect stem leaves, flowers, fruits and almost all overground parts of strawberries, wherein aged petals are an important way for infection of Botrytis cinerea. Earlier researches show that the incidence rate of gray mold of fruits can be reduced by removing aged petals on strawberry fruits. The field test shows that the 7 th day petal shedding rate after the strawberry budding is obviously negative relative to the incidence rate of the fruit gray mold, and the petals of different strawberry varieties fall at different speeds, so that the incidence rate of the strawberry gray mold can be effectively reduced by breeding the strawberry variety with the quick petals falling, the pesticide use amount and the production cost are reduced, the green and safe production of the strawberry is effectively realized, and the economic income is increased.
Molecular marker assisted selection is a method which can realize the rapid breeding of varieties with specific characters and depends on molecular markers closely linked with target genes. At present, researches related to the shedding speed of strawberry petals are few, so that the molecular assisted breeding of the shedding speed of the petals is to be realized, and the excavation of genetic sites, key genes and related molecular markers for regulating the shedding speed of the strawberry petals is urgent. According to the invention, through carrying out bulk Segregant RNA sequencing (BSR) positioning on a strawberry petal shedding segregation population, a key gene is screened out, a KASP primer is designed and developed according to a key gene sequence, and the primer can be used for carrying out auxiliary selection on the strawberry petal shedding speed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to locate the genetic locus with fast and slow strawberry petal shedding, identify candidate genes in the genetic locus, develop KASP molecular markers based on candidate gene SNP information, verify the correlation between genotypes and phenotypes in a population, assist in screening varieties with fast and slow strawberry petal shedding and accelerate the breeding process.
In order to achieve the purpose, the invention adopts the following technical scheme:
the KASP molecular marker is positioned on chromosome 2-2 on an octaploid genome of the strawberry, the base A is mutated into G at 776356bp, and the strawberry with the genotype of GG shows that the petals fall fast.
The sequence of the specific primer for amplifying the KASP molecular marker is shown as SEQ ID NO. 2-4.
The application of the KASP molecular marker in identification of strawberry petal shedding speed traits or molecular breeding comprises the following steps:
(1) extracting the genome DNA of the strawberry to be detected;
(2) taking the strawberry genome DNA to be detected as a template, and carrying out PCR amplification reaction by using the primer;
(3) after the PCR reaction is finished, collecting the fluorescence signal generated by each reaction hole, judging the genotype of the molecular marker according to the type of the fluorescence signal, if the genotype of the KASP molecular marker is GG, the strawberry is expressed as fast petal falling, and if the detected genotype is GA, the strawberry is expressed as slow petal falling.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention provides a method for developing KASP (Kaempferi protein) marks according to positioning of a strawberry petal-shedding genetic locus, screening of key genes in the genetic locus and utilization of SNP (single nucleotide polymorphism) information of the key genes, which can convert the genetic locus and the key genes into molecular mark sequences for molecular breeding, and lays an important foundation and a necessary premise for realizing molecular assisted breeding and selecting varieties with rapid and slow petal shedding.
(2) The KASP labeled primers developed by the invention are used for detecting petal shedding segregation population, wherein 6 samples with fast petal shedding, GG genotypes and 7 samples with slow petal shedding are totally contained in 13 samples, 6 samples are GA, 1 sample is GG, and the coincidence rate of the markers and the phenotype is 92.3%.
(3) The KASP labeled primer developed by the invention is used for detecting resource materials, 14 petals fall off fast and slowly and correspond to genotypes, and the percentage is 82.4%.
Drawings
Fig. 1 shows BSR positioning results in embodiment 1 of the present invention. The upper and lower graphs are the positioning results calculated by the calculation method of Euclidean distance and SNP-Index respectively, the threshold line is represented by a dotted line in the graph, the red arrow marks the genetic locus which is positioned on chromosome 2-2 and used for controlling the shedding speed of strawberry petals and is positioned in the two methods, and the key candidate gene maker-Fvb2-2-augustus-gene-7.55 is positioned at the locus.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments and with reference to the accompanying drawings. The following examples are merely illustrative of the present invention and should not be construed as limiting thereof. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
The embodiment provides a method for obtaining KASP molecular markers related to strawberry petal shedding genes, which specifically comprises the following steps:
(1) the construction of the petal shedding separation group takes red as a female parent, the petal shedding rate of the petals reaches more than 80% in 14 days, the male parent is sweet charles, the petal shedding rate of 28 days is lower than 80%, and the petal shedding speed is identified in the flowering period after each single plant of the group is planted;
(2) selecting single plants with fast and slow petal falling according to the identification result, and positioning by using a Bulked seggregant RNA sequencing (BSR) method to obtain a site for regulating the fast and slow petal falling of the strawberry;
(3) selecting candidate genes from the sites located in the step (2), designing a KASP primer sequence according to the candidate gene sequence and SNP information, and detecting the correlation between the sequence and the petal falling speed in F1 groups and other strawberry resources;
(4) selecting 15 petals which fall off fast and slowly according to the result in the step (1), forming a mixed pool, carrying out bulk Segregant RNA sequencing (BSR) analysis, and carrying out bioinformatics analysis on BSR data by using Euclidean distance and SNP-Index methods respectively to identify loci which can be positioned by the two methods, including genetic loci located on chromosome 2-2, as shown in figure 1;
(5) screening out candidate genes maker-Fvb2-2-augustus-gene-7.55 located in chromosome 2-2 from 365 genes located in the 0-2.01Mb interval of chromosome 2-2 according to the analysis result in (3) in combination with the literature; the gene has 24 SNPs, an SNP variation from A (sweet physical and fast shedding) to G (red and fast shedding mixed pool) is formed at 776356bp, a sequence (SEQ ID NO.1) of 100bp at the upstream and the downstream of the SNP site is extracted, and a KASP primer is designed according to the sequence; forward primer F1 of Fvb2-2_ 776356: 5'-ACTATACCTGGTGAATAATGCCATCT-3' (SEQ ID NO. 2); forward primer F2: 5'-CTATACCTGGTGAATAATGCCATCC-3' (SEQ ID NO. 3); reverse primer R1: 5'-AGCTTTGCAGAAAACGATAGCATTGCAA-3' (SEQ ID NO.4), wherein the 5 ' ends of the forward primers F1 and F2 are respectively connected with a fluorescent tag sequence FAM (GAAGGTGACCAAGTTCATGCT) and HEX (GAAGGTCGGAGTCAACGGATT);
(6) the KASP primers were used to detect genotypes of F1 isolate with rapid and slow petal shedding, and the PCR system was (1. mu.l for example): DNA template 10ng, 0.5. mu.L 2 XKASP master mix and 0.014. mu.L mixed primer. PCR program Touchdown PCR program was used: pre-denaturation at 94 ℃ for 15min, followed by 10 Touch down cycles (denaturation at 94 ℃ for 20s, annealing at 61 ℃ for the start annealing temperature, 0.6 ℃ reduction per cycle, and extension time for 60s), followed by 26 cycles of denaturation at 94 ℃ for 20s, annealing at 55 ℃ and extension for 60 s.
The results of enzyme-labeled detection PCR show that: of the 13 samples, there were 6 samples in total with rapid petal shedding, all genotypes were GG, and 7 samples in total with slow petal shedding, 6 of which were GA and 1 of which was GG, and the coincidence rate of the marker and phenotype was 92.3%, and the specific results are shown in table 1.
TABLE 1F 1 group petal drop and KASP primer test results
Example 2
The KASP primer obtained in the embodiment 1 is used for the universality verification of strawberry petal shedding speed identification, and the specific steps are as follows:
(1) selecting 17 parts of single plant or strawberry resource materials of different groups, and counting the speed of petal falling in a flowering season;
(2) the method for extracting the genomic DNA of the strawberry material and utilizing the KASP labeled primer developed by the invention to carry out PCR amplification is the same as that in the example 1, the results are shown in the table 2, and 14 phenotype results with rapid and slow petal shedding in 17 parts of strawberry resource materials are consistent with the genotype results and account for 82.4 percent.
TABLE 2 strawberry resource material petal drop and KASP primer detection results
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Sequence listing
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Claims (5)
1. The KASP molecular marker related to the fast and slow strawberry petal shedding property is characterized in that the molecular marker is located on chromosome 2-2 on an octaploid genome of a strawberry, a basic group A is mutated into G at the 776356bp position, and the strawberry with the genotype of GG shows fast petal shedding.
2. Specific primers for amplifying the KASP molecular marker of claim 1, wherein the primer sequences are shown in SEQ ID No. 2-4.
3. The application of the molecular marker of claim 1 in identification of strawberry petal shedding speed and slow trait.
4. The use of the molecular marker of claim 1 in strawberry petal abscission molecular breeding.
5. Use according to claim 3, characterized in that it comprises the following steps:
(1) extracting the genome DNA of the strawberry to be detected;
(2) performing PCR amplification reaction by using the strawberry genomic DNA to be detected as a template and the primer of claim 2;
(3) and detecting the PCR product, wherein if the genotype of the KASP molecular marker is GG, the strawberry shows that the petal shedding is fast, and if the detected genotype is GA, the strawberry shows that the petal shedding is slow.
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| CN116875728A (en) * | 2023-07-20 | 2023-10-13 | 沈阳农业大学 | KASP molecular marker group for identifying five-leaf strawberries and application thereof |
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| US20100064392A1 (en) * | 2008-06-10 | 2010-03-11 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring improved agricultural and/or ornamental characteristics to plants by modulating abscission |
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| CN111944921A (en) * | 2020-08-26 | 2020-11-17 | 中国农业科学院油料作物研究所 | Application of brassica napus BnaA08.PDS3 gene in breeding of color traits of brassica napus petals |
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| US20100064392A1 (en) * | 2008-06-10 | 2010-03-11 | Ceres, Inc. | Nucleotide sequences and corresponding polypeptides conferring improved agricultural and/or ornamental characteristics to plants by modulating abscission |
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| CN108546773A (en) * | 2018-05-28 | 2018-09-18 | 沈阳农业大学 | The primer of SSR molecular marker for identifying safflower fraise character and application |
| CN111944921A (en) * | 2020-08-26 | 2020-11-17 | 中国农业科学院油料作物研究所 | Application of brassica napus BnaA08.PDS3 gene in breeding of color traits of brassica napus petals |
Non-Patent Citations (2)
| Title |
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| CN116875728A (en) * | 2023-07-20 | 2023-10-13 | 沈阳农业大学 | KASP molecular marker group for identifying five-leaf strawberries and application thereof |
| CN116875728B (en) * | 2023-07-20 | 2024-03-08 | 沈阳农业大学 | KASP molecular marker group for identifying five-leaf strawberries and application thereof |
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