Efficient technical system for creating wide compatible rice recessive nucleic sterile line

A technique of recessive sterility and broad parenting, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of less markers, high cost, non-co-separation of markers and genes, etc., and speed up the breeding process , reduce the number of backcrossing, improve the effect of selection efficiency

Active Publication Date: 2011-07-13
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED +1
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  • Claims
  • Application Information

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Problems solved by technology

However, the current research on rice molecular-assisted selection breeding technology still has the following problems: 1) markers and genes do not co-segregate, resulting in markers that cannot replace genes, that is, markers cannot replace phenotypes; 2) fewer markers are used, and backgrou

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  • Efficient technical system for creating wide compatible rice recessive nucleic sterile line
  • Efficient technical system for creating wide compatible rice recessive nucleic sterile line
  • Efficient technical system for creating wide compatible rice recessive nucleic sterile line

Examples

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Effect test

Embodiment 1

[0033] Example 1: Construction of a rice mutant library and screening of rice nuclear recessive sterile mutants

[0034] Create a rice mutant library and screen out rice nuclear recessive male sterile mutants ms26 , the mutant is homozygous at the mutation site. attached Figure 1A nuclear recessive sterile mutant in rice ms26 Morphological manifestations of rice spikes and flower organs of plants. attached Figure 1B shows the rice ms26 Pollen cannot be formed on the mutant plants.

[0035] wild type allele MS26 Compared to the mutant allele ms26 3102 bases were missing. The genotype of the locus can be determined by designing PCR primers to amplify the two by using the nucleotide sequence difference that causes the functional difference between the two. The specific method is as follows. exist ms26 Design a reverse primer Cyp-test-3 for the gene deletion sequence, the primer sequence is 5′-AAGGTGTAGAAGCGGAAGAGGATGG-3′; design a pair of primers on both side...

Embodiment 2

[0036] Example 2: Restoration of mutants ms26 Fertility

[0037] The wild-type allele corresponding to the mutant gene in Example 1 MS26 Cloned in the plant transformation vector, then introduced into the Agrobacterium strain AGLO by electric shock method, and then transformed into the callus induced by the rice sterile line in Example 1, and then differentiated into transgenic rice plants.

[0038] Transgenic rice by PCR ( image 3 ) and Southern blot detection ( Figure 4 ),the result shows MS26 The gene has been integrated into the rice mutant ms26 in the genome of the plant. The fertility of the transgenic rice was restored, and the seed setting rate reached 90.7% (attached Figure 5 ).

Embodiment 3

[0039] Example 3: Design and Synthesis of Molecular Markers for Prospect Selection of Sterile Genes and Wide-Affinity Genes

[0040] Three primers Cyp-test-3, Cyp-test-5 and Cyp-WT in Example 1 were used to select ms26. A 262bp fragment was amplified by primers Cyp-test-5 and Cyp-test-3 in the mutant, and a 567bp fragment was amplified by primers Cyp-test-5 and Cyp-WT in its corresponding wild-type rice. Both fragments can be amplified in the fusion.

[0041] S5-n The gene marker primer sequence can refer to the published literature (Yang Jie, 2009), according to the indica and japonica S5-n Sequence differences, that is, primers were designed for the sequences on both sides of the deletion position, the marker name was S5136, the forward primer sequence was 5′-ATC AACCCATTTCCTTTCCT-3′; the reverse primer sequence was 5′-ATACGCTCGATCGGATTAAC-3′. broad affinity gene S5-n The genomic DNA of the rice variety Lemont can amplify a 441 bp fragment without S5-n mutant ms26 O...

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Abstract

The invention provides a method for accurately performing rice germplasm resource transformation by using molecular marker assistant selection, belongs to the field of rice molecular breeding, and particularly relates to backcross transformation and molecular marker assistant selection. The method comprises the following steps of: performing foreground selection by using DNA sequence difference causing gene function difference as a molecular marker (or using a marker tightly interlocked with two sides of a target gene), simultaneously performing background selection by using high-density simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) molecular markers, and performing one-time three-way cross, two-time backcross, one-time selfing, one-time backcross and one-time selfing to quickly and accurately polymerize nuclear recessive male sterile genes and wide compatible genes in an excellent receptor parent so that the recovery rate of the recurrent parent reaches 99.5 percent and a middle material of a wide compatible rice recessive nucleic male sterile line is created. The method not only enriches the male sterile germplasm resource of rice, but also can promote the development of heterosis utilization potential among indica and round-grained subspecies. By the invention, the breeding cycle can be greatly shortened and the breeding efficiency and accuracy can be improved.

Description

technical field [0001] The invention belongs to the field of rice molecular breeding, and specifically relates to the establishment of a technical system for efficiently creating wide-compatibility rice recessive genetic male sterile lines through rice polymeric backcross breeding and high-throughput molecular marker-assisted selection (marker assistant selection, MAS) methods . Background technique [0002] my country is a large agricultural country with a large population. Ensuring food security has always been an important task of agricultural science and technology. At present, the most widely used and most effective technology in crop breeding is hybrid breeding technology. At present, "three-line" and "two-line" hybridization techniques are commonly used in rice hybrid breeding. "Three-line" hybridization requires specific restorer lines and maintainer lines. The breeding procedures and production links are complicated. The cycle of breeding new sterile lines and new ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 邓兴旺王海洋万向元周君莉
Owner BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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