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SNP molecular marker co-segregated with cucumber thorn hardness gene hard and its application

A molecular marker, cucumber fruit technology, applied in the field of plant molecular genetics and breeding, can solve the problems of reduced breeding cost, shortened identification period, long identification period, etc., and achieves the effects of low cost, short time-consuming, and accelerated process.

Active Publication Date: 2020-09-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cost of traditional identification of varieties or evaluation of seed quality is high, and the identification cycle is long, while KASP technology can effectively avoid these two shortcomings, greatly reducing the cost of breeding, and the identification cycle is also significantly shortened. A preferred technique for seed quality control

Method used

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  • SNP molecular marker co-segregated with cucumber thorn hardness gene hard and its application
  • SNP molecular marker co-segregated with cucumber thorn hardness gene hard and its application
  • SNP molecular marker co-segregated with cucumber thorn hardness gene hard and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Acquisition of SNP molecular markers co-segregated with cucumber thorn hardness gene Hard

[0038] 1. Materials and reagents

[0039] Cucumber recombinant inbred lines 3541 (soft thorn) and 3542 (hard thorn) are a pair of near isogenic materials, except for the difference in thorn hardness, there is no other phenotypic difference.

[0040] The primers were synthesized by Shanghai Jierui Biotechnology Co., Ltd., and all were PAGE grade purified.

[0041] 2. Statistics on the genetic law of soft and hard thorns

[0042] Using 3542 (hard thorn) as the female parent and 3541 (soft thorn) as the male parent, the F1 generation was obtained, and then F1 was self-crossed to obtain the F2 generation population.

[0043] The identification method of the hardness of cucumber fruit thorns: the phenotype of hard thorns is: the base is spherical, with a large contact area with the epidermis; the phenotype of soft thorns is: the base is conical, with a small contact area w...

Embodiment 2

[0054] Example 2 KASP technology used for genotyping of cucumber thorn hardness

[0055] Preparation: PrimerMix consisting of two allele forward primers with different terminal bases and one reverse primer, the 5' ends of the two forward primers are respectively connected with different detection linker sequences; mixed with 2 fluorescent groups (FAM , HEX) Master Mix; DNA template.

[0056] The KSAP reaction system is specifically divided into: ①Dry DNA method: DNA (N / A), 2×Master mix (5μl), primer mix (0.14μl), H2O (5μl), Total reaction volume (10μl); ②Wet DNA method: DNA (5μl), 2×Master mix (5μl), primer mix (0.14μl), H2O (N / A), Total reaction volume (10μl).

[0057] KSAP reaction program: 94°C hot start activation for 15min; 94°C (20sec), 51-55°C (60sec), 0.6°C drop for each cycle, 10 cycles; 94°C (20sec), 55°C (60sec), 26 cycles cycle.

[0058] PCR reaction I: The denatured template is combined with the matching primer in Primer Mix and annealed, and the extended seque...

Embodiment 3

[0067] Example 3 Verification of co-segregated SNP markers in natural populations

[0068] Select 65 cucumber materials with different fruit thorn hardness and far away genetic background in the field. Hard thorns are common in natural populations, and soft thorn mutations are rare. Among these 65 materials, there is a soft thorn material, and the rest All are hard spine materials, and the accuracy of the SNP markers in Example 1 for molecular marker-assisted selection is determined through population verification. The thorns of these materials are different in hardness (discriminant standard: morphological difference, the phenotype of hard thorns is: the base is spherical, with a large contact area with the epidermis; the phenotype of soft thorns is: the base is conical, with a small contact area with the epidermis , the difference can be clearly seen in appearance). Cosegregated SNP-H was verified, and the results of natural population typing showed that individuals with hard...

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Abstract

The invention provides an SNP molecular marker co-segregated with the hardness gene Hard of fruit thorn of Cucumis sativus L. and application thereof. The SNP molecular marker is tested through threeprimers by means of the KASP technology, wherein the three primers respectively refer to two forward primers (SEQ ID NO.4+NO.1, SEQ ID NO.5+NO.2) of allele with different end basic groups and one reverse primer (SEQ ID NO.3), and SEQ ID NO.4 and SEQ ID NO.5 respectively refer to linker sequences of the two forward primers. The SNP molecular marker facilitates cloning of genes related to epidermalhair hardness of Cucumis sativus L. and building of molecular marker assistant selection breeding system of Cucumis sativus L.; besides, as the SNP molecular marker is co-segregated with hardness trait of fruit thorn, the molecular marker can be applied to screening of fruit thorn hardness difference type of Cucumis sativus L. with high throughput conveniently and rapidly and has high applicationvalue in improving breeding selection efficiency of Cucumis sativus L..

Description

technical field [0001] The invention belongs to the field of plant molecular genetic breeding, and in particular relates to a SNP molecular marker co-separated with cucumber thorn hardness gene Hard and its application. Background technique [0002] Cucumber (Cucumis sativus L.) is a perennial herbaceous plant belonging to Cucurbitaceae (Cucurbitaceae), and it is one of the main vegetable crops in my country. Cucumber is an important fruit vegetable, and fruit quality has always been the focus of cucumber breeding research. Fruit quality is divided into sensory quality, flavor quality and nutritional quality. The quality genes that have been identified so far are mostly sensory quality genes, including melon color, melon length, number of thorns, thorn color, fruit tumor size, and uniform fruit color. degree, gloss, fruit ribs, yellow stripes on the surface of the fruit, etc. Among them, the appearance quality has an important influence on the commerciality of cucumber. T...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 任华中薛舒丹刘兴旺蔡艳玲董明明张亚琦刘慧玲赵彬煜王亭蹇晨徐硕祁文竹
Owner CHINA AGRI UNIV
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