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Separation and cloning of alleles Sc-j and Sc-i of pollen fertility gene loca Sc of paddy rice hybrids, and application in seed breeding

A technology of alleles and fertility genes, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve problems such as hindering utilization, achieve the effect of improving fertility and overcoming Sc hybrid sterility

Inactive Publication Date: 2017-09-15
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The phenomenon of hybrid sterility severely hinders the conversion of rice heterosis into higher rice yield and the utilization of useful genes in wild rice

Method used

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  • Separation and cloning of alleles Sc-j and Sc-i of pollen fertility gene loca Sc of paddy rice hybrids, and application in seed breeding
  • Separation and cloning of alleles Sc-j and Sc-i of pollen fertility gene loca Sc of paddy rice hybrids, and application in seed breeding
  • Separation and cloning of alleles Sc-j and Sc-i of pollen fertility gene loca Sc of paddy rice hybrids, and application in seed breeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: sc Fine-positioned cloning and sequence structure analysis of

[0032] The inventor's previous studies have sc Mapped to the approximate region on rice chromosome 3 (Yang et al. 2004. Chinese Science Bulletin 49, 1718-1721). The present invention uses as figure 2 The map-based cloning method shown further clones sc Gene. According to the publicly available rice genome sequence database (http: / / www.ncbi.nlm.nih.gov), multiple molecular markers were developed in this region (Table 1). With T65 ( Sc-j / Sc-j genotype) and its near isogenic line E5 ( Sc-i / Sc-i ) to hybridize to produce F 1 and F 2 population, after linkage analysis 8689 strains of F 2 individual, put sc finely mapped to a 8.6 kb interval in japonica rice ( figure 2 A). According to the rice genome sequence database GenBank (http: / / www.ncbi.nlm.nih.gov), there is one annotation numbered in the 8.6 kb interval of the mapped Japonica rice Os03g0247300 The gene of the present inven...

Embodiment 2

[0036] Example 2: sc Functional verification of genes

[0037] Amplified from Japonica Rice by RT-PCR Sc-j cDNA fragment (located at 514-1284 bp of the coding region), ligated in reverse to maize Ubiquitin The gene promoter is connected to construct a binary vector for antisense gene expression. Transform T65 with this vector to obtain a transformant. Transgenic hemizygous (one chromosome has a transgene and the other homologous chromosome has no transgene) Sc-j The expression level decreased ( image 3 A), roughly half of the pollen was sterile ( image 3 B). prove Sc-j It is the pollen gamete sports gene.

Embodiment 3

[0038] Example 3: sc Mechanism Analysis of Hybrid Sterility

[0039] With primers P3 and P8 (amplified all sc alleles) for quantitative RT-PCR, for T65, E5, hybrid F 1 The expression analysis of Sc-j It is mainly expressed specifically in pollen in the early stage of dinuclei, and the expression level is low. Sc-i The repeated gene was expressed at different developmental stages from anther microspore mother cell to dinuclear early pollen, and the expression level was high ( Figure 4 A). by Sc-j Quantitative RT-PCR expression analysis with specific primers (P3, P4) showed that F 1 of Sc-j The expression level of each copy is only equivalent to about 1 / 5 of its parental T65 ( Figure 4 ), indicating that in the hybrid Sc-j Inhibition of expression results in pollen abortion.

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Abstract

The invention belongs to the technical field of plant gene engineering and particularly discloses separation and cloning of alleles Sc-j and Sc-i of a pollen fertility gene loca Sc of paddy rice hybrids, and an application in seed breeding. Sc-j encoding of japonica rice and Sc-i encoding of indica rice contain proteins in a DUF1618 structural domain, which are required in normal growth of pollen of paddy rice. The Sc-j only has one gene copy while the Sc-i, in different varieties, has two or three tandem gene copies. In a hybrid between the japonica rice and indica rice, high-level expression products of the Sc-i multi-copy gene can inhibit expression of the Sc-j, which causes hybrid pollen sterility. The invention discloses a hybridization seed breeding method, in which a part of copies of th Sc-i is mutated by utilizing a gene-editing technology, so that the gene dosage effect of the Sc-i is reduced and further fertility of Sc-j pollen in a japonica-indica paddy rice hybrid is recovered, thus overcoming male sterility of a japonica-indica paddy rice hybrid.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular, to rice hybrid pollen fertility loci sc alleles of Sc-j and Sc-i isolated clones and their application in breeding. Background technique [0002] Plants produce hybrids by crossing between different varieties. Hybrids have advantages in growth potential, adaptability, yield, etc., which are called heterosis. The use of heterosis can improve the yield, quality and resistance of crops. Asian cultivated rice is divided into the indica subspecies ( Oryza sativa ssp. indica ) and Japonica subspecies ( O. sativa ssp. japonica ), the hybrids among them have strong heterosis and great yield increasing potential. On the other hand, through breeding methods such as hybridization and backcrossing between cultivated rice and wild rice, some useful traits existing in wild rice, such as disease resistance, insect resistance, and high yield, can be introduced in...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H1/02
CPCA01H1/02C07K14/415C12N15/8213C12N15/8287
Inventor 刘耀光
Owner SOUTH CHINA AGRI UNIV
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