Method for transforming cucumis melo female line through high-throughout molecular marker and special primer thereof

A technology for female lines and melons, applied in the field of molecular biology, can solve problems such as cumbersome processes, and achieve the effects of high analytical throughput, accelerated breeding, and time and labor cost savings.

Active Publication Date: 2016-01-20
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] However, since the CAPS marker developed for the A / a gene needs to digest the PCR product, the SCAR designed for the G / g gene needs to amplify the specific fragments of the G and g genes respectively, and then use polyacrylamide gel to amplify the target fragments. The procedures such as separation and detection of fragments are relatively cumbersome, which limits the application of these markers in molecular marker-assisted breeding of melon female lines

Method used

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  • Method for transforming cucumis melo female line through high-throughout molecular marker and special primer thereof
  • Method for transforming cucumis melo female line through high-throughout molecular marker and special primer thereof
  • Method for transforming cucumis melo female line through high-throughout molecular marker and special primer thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. High-throughput KASP marker design of melon sex gene and development of special primer sequences

[0050] The melon sex genes involved in the present invention are CmACS7 gene (a gene) and CmWIP1 gene (g gene).

[0051] 1. Extraction of tested muskmelon materials and their genomic DNA

[0052] A. Selection of test materials

[0053] Test material: melon hermaphrodite strain M26 (aagg) and monoecious strain M29 (AAGG) with known genotypes of CmACS7 gene (a gene) and CmWIP1 gene (g gene). Among them, the genotypes of the two parents were marked by CAPS-AluI (for details, see "BoualemA, FerganyM, FernandezR, et al. Conserved MutationinanEthylene Biosynthesis Enzyme Leadsto AndromonoecyinMelons. Science, 2008Aug8; 321 (5890): 836-8") and SCAR marks (for details, see "MartinA, TroadecC, BoualemA, et al. A transposon-induced epigenetic change leads to sex determination in melon. Nature. 2009 Oct 22; 461(7267): 1135-8") detected and verified by sequencing.

[005...

Embodiment 2

[0112] Example 2. Establishment of a method for detecting melon sex genes using high-throughput KASP markers

[0113] 1. Genomic DNA extraction

[0114] See Step 1 of Example 1.

[0115] 2. PCR amplification

[0116] Using the genomic DNA extracted in step 1 as a template, PCR amplification was performed with the KASP-marked special primers developed in Example 1 for detecting two melon sex genes (CmACS7 gene and CmWIP1 gene), respectively, to obtain PCR amplification products.

[0117] KASP genotyping PCR reaction system:

[0118] 96-well plate: 10ng genomic DNA, 5μl KASPV4.02×MasterMix, 0.14μl KASP72×assaymix, add ddH 2 0 to 10 μl.

[0119] 384-well plate: 5ngDNA, 2.5μl KASPV4.02×MasterMix, 0.07μl KASP72×assaymix, add ddH 2 0 to 5 μl.

[0120] 1536-well plate: 5ngDNA, 2.5μl KASPV4.02×MasterMix, 0.07μl KASP72×assaymix, add ddH 2 0 to 5 μl.

[0121] Among them, KASPV4.02×MasterMix is ​​a product of LGC Company, and the catalog number of KASPV4.02×MasterMix for 96 / 384-w...

Embodiment 3

[0135] Example 3. Verification of the detection of melon sex genes by high-throughput KASP markers and its application in breeding

[0136] 1. Validation of high-throughput KASP molecular markers

[0137] 1. Selection of test materials

[0138] The materials for the test body include: the F 1 Generation, F 1 F 2 generation. The known genotypes of melon variety WMR29 (aa genotype), melon variety Védrantais (aa genotype) and melon variety PI124112 (AA genotype) were selected as controls.

[0139] Above F 2 The first-generation segregation population was cultivated and collected in Hainan in the autumn of 2012. It was germinated and broadcast live in a greenhouse in August 2013. It was planted in the Sijiqing Experimental Base of the Vegetable Research Center of the Beijing Academy of Agriculture and Forestry Sciences. The male parent, female parent, and F 1 , WMR29 (aa genotype), Védrantais (aa genotype) and PI124112 (AA genotype) each colonized 20 plants, F 2 120 plants ...

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Abstract

The invention discloses a method for transforming a cucumis melo female line through a high-throughout molecular marker and a special primer thereof. The special primer is a KASP primer set used for detecting cucumis-melo sex genes CmACS7 and CmWIP1, and the specific nucleotide sequence of the primer is the first sequence to the sixth sequence in a sequence table. The types of the cucumis-melo sex genes are detected through a high-throughout molecular mark system based on a PCR SNPLine platform provided by the method, the operation process is full automatic, and human errors are reduced; the analysis throughout is high, and the method and the special primer are quite suitable for detecting a large number of samples at the same time. According to the method and the special primer, the high-throughout detection molecular marker is designed based on the sequences of the cucumis-melo instaminate flower development A/a genes and the G/g genes and applied to transforming of the cucumis-melo female-line genes, time cost and manual cost can be greatly saved, the molecular-marker-assisted selection seed breeding efficiency can be improved, and transformation of cucumis-melo female characters to excellent key inbred lines is accelerated.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a method for using high-throughput molecular markers to transduce muskmelon female lines and special primers thereof. Background technique [0002] Melon (Cucumismelo L.) is an important economic crop of Cucurbitaceae Cucumber. The sexual types of melon plant flowers mainly include the following phenotypes: andromonoecious, dioecious, hermaphrodite, monoecious, trisexual, all female (female), all male . At present, the vast majority of commercially cultivated melon varieties are andromoneous. Since the fruiting flowers are hermaphroditic flowers, manual emasculation, bagging, pollination, marking and other operating procedures must be carried out before cross-pollination, which is labor-intensive and costly for seed production. High; and artificial pollination often hurts the pistil, resulting in low pollination rate, decreased seed setting rate, and reduced yield. If artificial...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11A01H1/02
CPCA01H1/02C12Q1/6895C12Q2600/156C12Q2600/172
Inventor 许勇张春秋史建廷宫国义张海英
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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