SNP molecular marker closely linked to tomato male sterility gene and its obtaining method and application

Abstract

The invention belongs to the technical fields of bio-technology engineering and tomato male sterile genetics and breeding, and in particular discloses a single nucleotide polymorphism (SNP) molecularmarker closely linked to tomato male sterile gene as well as an obtaining method and application of the SNP molecular marker. The SNP loci are obtained by means of associative analysis on the basis ofsimplified genome sequencing; the SNP223 marker is obtained by using polymorphic DNA fragments which contain the SNP loci and designing specific primers. Experiments show that the SNP marker closelylinked to the tomato male sterile gene can be used for assisted selection breeding of tomato male sterile lines. Compared with the second generation molecular markers, the SNP molecular marker is moreaccurate in detection way and higher in detection efficiency.

Description

technical field

[0001] The invention belongs to the technical field of biological genetics and breeding, and specifically discloses a SNP molecular marker closely linked with a tomato male sterility gene, an obtaining method and application thereof. Background technique

[0002] Heterosis is a common phenomenon in the biological world, and it is an important way to improve crops and greatly increase yield. Since the beginning of the 20th century, male sterility has been found in various crops, and male sterility has been used in production to produce hybrids. In some crops, such as corn, sorghum, rice and other crops, great achievements have been made. Tomato is a self-pollination crop, and the heterosis is very obvious. The yield of the hybrid is 20%-30% higher than that of the common variety, and it has high uniformity and strong stress resistance. With the increasing difficulty of tomato multi-objective breeding such as high yield, high quality, and stress resistance, i...

Examples

Embodiment 1

[0022] Embodiment 1, the method for the SNP locus acquisition and marker development that are closely linked with the tomato male sterility gene, comprises the following steps:

[0023] (1) Using the tomato male sterile line BY-17 as the female parent (the original number was 2-031, introduced from the Tomato Genetics Center, which is an open institution, ordinary researchers can apply for the introduction of materials), and the male sterile line Line KY-113 was used as the male parent (the original number was LA0012, which was also introduced from the Tomato Genetics Center), and the F 2 genetic population;

[0024] (2) Extract 30 strains of F 2 generation male sterile single plant and 30 F 2 The genomic DNA of the male fertile single plant of the first generation was used to construct the male sterile gene pool and the male fertile gene pool, and the genomic DNA of the parents was extracted at the same time to establish the parent gene pool;

[0025] (3) Use restriction s...

Embodiment 2

[0029] Example 2, Detection and Identification of SNP Sites and Markers Closely Linked to Tomato Male Sterility Gene

[0030] The tomato male sterile line BY-17 was used as the female parent, and the male sterile line KY-113 was used as the male parent for crossing, and then the F 1 F 2 Substitute, with F 2 Using the specific primers of the molecular marker SNP223 as the object, the genomic DNA of each individual plant is amplified, and then the amplified product is sequenced to detect the genotype of the SNP223 locus, so that it can be judged at the seedling stage whether the individual plant is male infertility. Breeding plants.

[0031] The method for identifying tomato male sterility comprises the steps of using specific primers: upstream primer: 5'-GGTGATGGCGTCAGTGATTA-3'; downstream primer 5'-AATGGAATTCCCCAGTACCC-3' for amplification. The amplified product was sequenced to detect the genotype of the SNP223 site.

Embodiment 3

[0032] Embodiment 3, develop SNP molecular marker according to polymorphic SNP site, and calculate the linkage distance of SNP marker and male sterility gene, its method is:

[0033] (1) Using simplified genome sequencing to perform polymorphism analysis of SNP sites on specific DNA fragments in male sterile parents, male fertile parents, male sterile gene pools, and male fertile gene pools;

[0034] (2) Obtain the SNP site SNP223 significantly associated with the male sterility gene through association analysis, and the genotype of the SNP223 site is A / A homozygous, T / T homozygous or A / T heterozygous;

[0035] (3) Using the DNA fragment sequence where the SNP223 site is located, design specific primers to obtain the SNP223 molecular marker;

[0036] (3) Linkage analysis of SNP223 molecular markers: F obtained from sterile parents and fertile parents 2 Individual plants were identified in the field at the flowering stage, and the investigation of F 2 For the fertility of a s...

technical field

[0001] The invention belongs to the technical field of biological genetics and breeding, and specifically discloses a SNP molecular marker closely linked with a tomato male sterility gene, an obtaining method and application thereof. Background technique

[0002] Heterosis is a common phenomenon in the biological world, and it is an important way to improve crops and greatly increase yield. Since the beginning of the 20th century, male sterility has been found in various crops, and male sterility has been used in production to produce hybrids. In some crops, such as corn, sorghum, rice and other crops, great achievements have been made. Tomato is a self-pollination crop, and the heterosis is very obvious. The yield of the hybrid is 20%-30% higher than that of the common variety, and it has high uniformity and strong stress resistance. With the increasing difficulty of tomato multi-objective breeding such as high yield, high quality, and stress resistance, i...

Examples

Embodiment 1

[0022] Embodiment 1, the method for the SNP locus acquisition and marker development that are closely linked with the tomato male sterility gene, comprises the following steps:

[0023] (1) Using the tomato male sterile line BY-17 as the female parent (the original number was 2-031, introduced from the Tomato Genetics Center, which is an open institution, ordinary researchers can apply for the introduction of materials), and the male sterile line Line KY-113 was used as the male parent (the original number was LA0012, which was also introduced from the Tomato Genetics Center), and the F 2 genetic population;

[0024] (2) Extract 30 strains of F 2 generation male sterile single plant and 30 F 2 The genomic DNA of the male fertile single plant of the first generation was used to construct the male sterile gene pool and the male fertile gene pool, and the genomic DNA of the parents was extracted at the same time to establish the parent gene pool;

[0025] (3) Use restriction s...

Embodiment 2

[0029] Example 2, Detection and Identification of SNP Sites and Markers Closely Linked to Tomato Male Sterility Gene

[0030] The tomato male sterile line BY-17 was used as the female parent, and the male sterile line KY-113 was used as the male parent for crossing, and then the F 1 F 2 Substitute, with F 2 Using the specific primers of the molecular marker SNP223 as the object, the genomic DNA of each individual plant is amplified, and then the amplified product is sequenced to detect the genotype of the SNP223 locus, so that it can be judged at the seedling stage whether the individual plant is male infertility. Breeding plants.

[0031] The method for identifying tomato male sterility comprises the steps of using specific primers: upstream primer: 5'-GGTGATGGCGTCAGTGATTA-3'; downstream primer 5'-AATGGAATTCCCCAGTACCC-3' for amplification. The amplified product was sequenced to detect the genotype of the SNP223 site.

Embodiment 3

[0032] Embodiment 3, develop SNP molecular marker according to polymorphic SNP site, and calculate the linkage distance of SNP marker and male sterility gene, its method is:

[0033] (1) Using simplified genome sequencing to perform polymorphism analysis of SNP sites on specific DNA fragments in male sterile parents, male fertile parents, male sterile gene pools, and male fertile gene pools;

[0034] (2) Obtain the SNP site SNP223 significantly associated with the male sterility gene through association analysis, and the genotype of the SNP223 site is A / A homozygous, T / T homozygous or A / T heterozygous;

[0035] (3) Using the DNA fragment sequence where the SNP223 site is located, design specific primers to obtain the SNP223 molecular marker;

[0036] (3) Linkage analysis of SNP223 molecular markers: F obtained from sterile parents and fertile parents 2 Individual plants were identified in the field at the flowering stage, and the investigation of F 2 For the fertility of a s...

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