Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof

A molecular marker and gene restoration technology, applied in the field of plant biotechnology and molecular breeding, can solve the problems of chain drag and loss of target genes, and achieve the effect of overcoming the chain drag.

Active Publication Date: 2015-07-22
CHINA NAT SEED GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These two types of markers have certain defects in large-scale selection. The former will lose the target gene, and the latter can only identify the genotype of one of the gene loci in breeding practice, and both markers will be accompanied by unexpected traits Bringing in, that is, there is a problem of chain burden

Method used

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  • Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof
  • Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof
  • Molecular markers for haplotype identification of paddy rice CMS restoring gene Rf-1 segment and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Design of Molecular Marker of Rice CMS Restoration Gene Rf-1 Segment

[0058] Using next-generation sequencing technology (Illumina MiSeq sequencer), the parents of the rice wild-type and Baotai-type CMS restorer lines Minghui 63, the parent of the Honglian type CMS restorer line 9311, the self-developed excellent restorer line G527 and the rice wild-type restorer line The maintainer parent Zhenshan 97 was resequenced with an average 20-fold coverage of the whole genome, and the Rf-1 gene and its upstream and downstream regions of 200kb (genetic distance of about 1cM interval) were selected to discover the variation sites. The specific steps of marking design are as follows:

[0059] The first step is to compare the reference genome, compare the sequencing reads of each material to the Nipponbare genome, and use the software BWA for comparison. The reference genome sequence is the genome sequence of MSU version 6.1 Nipponbare.

[0060] The second step is to i...

Embodiment 2

[0063] Example 2 Preparation of Rice CMS Restoration Gene Rf-1 Segment OA Chip

[0064] Submit 128 sequences SEQ ID No.1-SEQ ID No.128 of 32 molecular markers obtained in Example 1 to Life Technologies to synthesize corresponding primers and TaqMan-MGB probes, and make an OpenArray chip, which can detect 96 simultaneously sample.

Embodiment 3

[0065] Example 3 Validation of the application effect of molecular markers for identifying the haplotype of the rice CMS restorer gene Rf-1 segment in the detection of rice DNA samples

[0066] In order to verify the haplotype identification method of the rice CMS recovery gene Rf-1 segment, it is necessary to test the primer probe combination, the actual detection and typing effect of the OA chip, and compare whether the design reference sample genotype is consistent with the actual detection genotype , so as to determine whether the primer probe set and OA chip have achieved the expected effect of design. The concrete steps of this embodiment are as follows:

[0067] 1. Rice genomic DNA extraction:

[0068] Genomic DNA was extracted from rice seeds, leaves and other tissues according to the detection requirements, and the DNA of young rice leaves was extracted using the standard procedure of the Promega Plant Genome Extraction Kit.

[0069] 2. DNA sample quality testing:

...

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Abstract

The invention provides molecular markers for haplotype identification of a paddy rice CMS restoring gene Rf-1 segment and applications thereof, and belongs to the fields of plant biology and molecular breeding. By comparing various types of restorer parent sequencing data of paddy rice, 32 sites of restoring gene Rf-1 segment polymorphism are screened, and a plurality of primer pairs and TaqMan-MGB probe compositions are designed. Sequences of the TaqMan-MGB probe compositions are respectively shown as SEQID NO.1-128. The 32 primer pairs and the TaqMan-MGB probe compositions are further prepared into an OpenArray chip. Haplotype identification of the paddy rice CMS restoring gene Rf-1 segment can be achieved by utilization of the molecular markers or the chip. The molecular markers can be applied for haplotype identification of the CMS restoring gene Rf-1 segment in the paddy rice resource and for breeding of three-line hybrid rice.

Description

technical field [0001] The invention relates to the field of plant biotechnology and molecular breeding, more specifically, to a molecular marker for identifying the haplotype of the rice CMS restorer gene Rf-1 segment and its application. Background technique [0002] Three-line hybrid rice has always been in the leading position in the planting area due to its advantages of stable and high yield, and has made great contributions to my country's food security. CMS (Cytoplasmic Male Sterility), that is, cytoplasmic male sterility, is the material basis for the development and utilization of three-line hybrid rice. The three-line matching developed on this basis specifically refers to the CMS restorer line, CMS maintainer line and CMS sterile line. The breeding process needs to complete the preservation of the three lines and the preparation of hybrids. The specific operations are self-propagation of the CMS maintainer line and the CMS restorer line; hybridization of the CMS ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C40B40/06
CPCC12Q1/6895C12Q2600/156C40B40/06
Inventor 周发松喻辉辉刘刚张小波陆青张学堂宋丁丁王乃元姚玥韦懿周莹陈瑶余千
Owner CHINA NAT SEED GRP
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