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Method for cultivating male sterile line and corresponding maintainer line of alfalfa and related biological material

An alfalfa and fertility technology, applied in chemical instruments and methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as low probability, high genome heterozygosity, and difficult genetic manipulation.

Active Publication Date: 2021-11-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most alfalfa cultivars are autotetraploid, with semi-self-incompatibility physiological characteristics, high heterozygosity in the genome, and difficult genetic manipulation; therefore, the genetic breeding of alfalfa is very difficult and the progress is slow
Alfalfa has obvious heterosis, and hybrids may increase yield by 30%. However, there is currently a lack of alfalfa male sterile line materials that can be used in production in my country, which greatly hinders the process of hybrid seed production
Natural mutation or mutagenesis to obtain male sterile material takes a long time and the probability is low

Method used

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  • Method for cultivating male sterile line and corresponding maintainer line of alfalfa and related biological material
  • Method for cultivating male sterile line and corresponding maintainer line of alfalfa and related biological material
  • Method for cultivating male sterile line and corresponding maintainer line of alfalfa and related biological material

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0122] Example 1. Cultivation of the male sterile line of Medicago truncatula without exogenous transgene fragments and its phenotypic identification

[0123] 1. Construction of vector p6401-MtNP1

[0124] 1.a. Gene editing target design

[0125] The coding sequence (CDS) of Medicago truncatula R108 is the MtNP1 coding gene shown in sequence 4 in the sequence listing, and encodes the protein MtNP1 whose amino acid sequence is shown in sequence 2 in the sequence listing.

[0126] Through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, select target 1 as 5′-GTGAATGTTGCGCCGCTAG-3′, and target 2 as 5′-GTCGATTACGCGCAATGCG-3′.

[0127] 1.b, sgRNA module construction

[0128] Primers were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.

[0129] MtNP1-BsF: 5′-ATATATGGTCTCGCTTG GTGAATGTTGCGCCGCTAG GTT-3', wherein the 18th to 36th nucleotides are the designed target 1 sequence;

[0130] MtNP1-F0: 5′-G GTGAATGTTGCGCCGCT...

Embodiment 2

[0172] Embodiment 2, cultivate the male sterile line of alfalfa

[0173] 1. Construction of vector p6401-MsNP1

[0174] 1.a. Gene editing target design

[0175] The coding sequence (CDS) of Medicago MsNP1 is the MsNP1 coding gene shown in sequence 3 in the sequence listing, which encodes the protein MsNP1 whose amino acid sequence is shown in sequence 1 in the sequence listing.

[0176] According to the alfalfa MsNP1 genome sequence, through the online target prediction website ( http: / / crispor.tefor.net / ) to generate a target list, select target 1 as 5′-GCTACATTGAAGCCGCTAG-3′, and target 2 as 5′-TCCTTTTTGAGAACCTGTGA-3′.

[0177] 1.b, sgRNA module construction

[0178] Primers synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd.:

[0179] MsNP1-BsF: 5′-ATATATGGTCTCGCTTG GCTACATTGAAGCCGCTAG GTT-3';

[0180] MsNP1-F0: 5′-G GCTACATTGAAGCCGCTAG GTTTTAGAGCTAGAAATAGC-3′;

[0181] MsNP1-R0: 5′-AAC TCACAGGTTCTCAAAGAGA CAATTTAATGGTTCGCTTGTA-3';

[0182] MsNP1-...

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Abstract

The invention discloses a method for cultivating a male sterile line and a corresponding maintainer line of alfalfa by using a gene editing technology. An efficient gene editing system is utilized to target medicago truncatula MtNP1 and a homolog MsNP1 of the medicago truncatula MtNP1 in medicago sativa, and agrobacterium tumefaciens-mediated genetic transformation is adopted, so that a material in which all alleles of the MtNP1 or the MsNP1 are mutated is obtained, and the phenotype of the material is male sterility; a material retaining a wild-type allele is obtained from the system, and the material is male fertile and can be used as a corresponding maintainer line. The method of cultivating the male sterile line mediated by the gene editing technology and a matched maintainer line is short in period, high in efficiency and good in targeting property, greatly saves the time and economic cost of traditional alfalfa sterile line screening and cultivation, and has important application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for cultivating male sterile lines of alfalfa and corresponding maintainer lines and related biological materials. Background technique [0002] Utilization of heterosis is an important idea to increase crop yield and improve agronomic traits. Hybrid breeding and seed production techniques have been widely used in field crops such as rice and corn. The bottleneck problem of hybrid breeding and seed production is female parent emasculation. Commonly used detasseling methods include artificial detasseling, mechanical detasseling and chemical detasseling, etc. These methods often consume a lot of manpower and material resources, but the effect is not ideal; and the cultivation of male sterile lines can effectively avoid the above problems. and key steps in seed production. [0003] Alfalfa is an important perennial leguminous forage crop with high protein content and rich nut...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/84C12N15/55A01H5/02A01H6/54
CPCC07K14/415C12N15/8218C12N15/8289C12N15/8205C12N9/22
Inventor 王涛董江丽叶沁怡
Owner CHINA AGRI UNIV
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