RapMap method for rapid and high-throughput positioning and cloning of plant QTL gene

A high-throughput and fast technology, applied in genomics, bioinformatics, instruments, etc., can solve problems such as low efficacy and slow decay of local linkage disequilibrium, and achieve the goal of overcoming shortcomings and limitations, speeding up speed, and solving complexity Effect

Active Publication Date: 2020-08-04
HUAZHONG AGRI UNIV
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Problems solved by technology

However, there are three key issues to be addressed in GWAS research (Wallace et al., 2018; Huang & Han, 2014; Kover et al., 2009): First, due to the existence of a large population structure, researchers need to make a trade-off between reducing false positives and increasing false positives. Negative ratio, the second is that the efficacy of detecting rare alleles in natural populations is low, and the third is that the slow decay of local linkage disequilibrium causes multiple genes at one GWAS locus, which requires additional experiments and a large number of follow-up analyzes to determine true gene of interest

Method used

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  • RapMap method for rapid and high-throughput positioning and cloning of plant QTL gene
  • RapMap method for rapid and high-throughput positioning and cloning of plant QTL gene
  • RapMap method for rapid and high-throughput positioning and cloning of plant QTL gene

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Embodiment 1

[0032] Embodiment 1: The present invention is applied to the cloning of the QTL gene of rice grain length natural variation.

[0033] We selected 12 parental materials with representative grain length phenotypes ( figure 2 a), in Hainan in 2016, we prepared 8 hybrid combinations with parents with similar grain size, and the F of each combination 1 The seeds were propagated in Wuhan in 2016, and about 200 offspring and their genomic DNA were obtained from each gradient population in Hainan in 2017. Harvest F 2 After the seeds, in 2017, Wuhan carried out the test of grain length phenotype, and constructed a mixed pool with high value (last 15%) and low value (top 15%) ( figure 2 b). Mixed pools of 6 gradient populations (populations 1, 2, 3, 5, 7, 8) were genotyped and QTL mapped using the RICE6K commercial chip (China Seed Group Corporation), which was designed based on the above microcore collection , including four million SNP loci (Yu et al., 2014), the SNP detected by...

Embodiment 2

[0035] Embodiment 2: The present invention is applied to the cloning of QTL genes for natural variation of rice grain width.

[0036] While cloning the grain length gene, we selected 10 parental materials with representative grain width phenotypes from the above-mentioned rice micro-core germplasm (grain width variation range 1.99mm-3.43mm) ( image 3 a), In March 2016, 7 hybrid combinations were made in Hainan with parents with similar grain width. It should be noted here that as long as the difference in grain width between the two parents of the hybrid combination is not too large, it is suitable for RapMap, that is, the segregation population controlled by a single locus can be obtained as much as possible and a suitable near-isogenic line can be screened. f 1 The seeds were propagated in Wuhan in 2016 and planted in Jiadai, Hainan in 2017, and each group harvested 200 F 2 The seeds of a single plant were tested in Wuhan in 2017 for the grain width phenotype, and a singl...

Embodiment 3

[0044] The six genes cloned in the present invention contribute greatly to the inheritance of grain length and grain shape in the rice world micro-core collection.

[0045] Utilize the functional variation of 6 control grain length and grain width genes cloned in Example 1 and Example 2 in the genotype of the micro-core collection and the grain length, grain width, aspect ratio, thousand-grain weight, and grain number of the micro-core collection Multiple linear regression analysis was performed on the phenotypes of the six traits of yield per plant to evaluate the genetic contributions of these genes to the six traits, and it was found that three grain length genes could explain 49% of the grain length variation ( Figure 4 a), three grain width genes can explain 53% of the grain width variation ( Figure 4 c), 6 genes can explain 64%, 17%, 15%, and 13% of the phenotypic variation in aspect ratio, thousand-grain weight, grain number, and yield per plant, respectively ( Figu...

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Abstract

The invention discloses a RapMap method for rapid and high-throughput positioning and cloning of a plant QTL gene. The method comprises the steps of: selecting strains with relatively small characterdifferences from the core germplasm as gradient parents; constructing as many F2 gradient genetic populations as possible; preliminarily positioning QTL by performing chip detection or next-generationsequencing on two separated extreme phenotypic DNA pools of each group, verifying the QTL at the single plant level of each gradient group according to the co-separation criteria provided by the invention, and cloning a target gene by using a QTL heterozygous family as a near-isogenic line for fine positioning. The concept of gradient genetic populations and the idea of co-segregation criteria are the core of the invention. Six rice grain length and grain width genes are successfully cloned by applying the RapMap method, and the RapMap is indicated to be a rapid and high-throughput gene cloning RapMap method integrating QTL positioning, verification and near-isogenic line screening into a whole.

Description

technical field [0001] The invention belongs to the field of plant functional genome and genetic engineering, and in particular relates to the field of positioning, cloning and application of fast and high-throughput QTL genes. Background technique [0002] The large amount of natural genetic variation produced by crops or livestock in the process of natural and human selection is the basis and driving force for their domestication and improvement, their environmental adaptability and maintenance of genetic diversity, meeting human energy and health needs, and their production , quality, resistance, and formulation of molecular trait improvement strategies (Ellegren&Galtier, 2016; Rheenen et al., 2019; Mitchell-Olds, 2007). The regulation of multiple genes and the influence of the environment cause most economically important traits in biology, biomedicine and agriculture to be complex quantitative traits with continuous variation (Mauricio, 2001; Goddard & Hayes, 2009; Baza...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G16B20/40G16B20/20
CPCG16B20/40G16B20/20
Inventor 李一博张俊成张德建李翠翠许鹏坤樊亚伟李威苏小敏孙琪李星星宋盈盈
Owner HUAZHONG AGRI UNIV
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