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Cloning and application of flowering gene bnflc.a2 and bnflc.a2 in Brassica napus

A technology of bnflc.a2, Brassica napus, which can be applied in application, genetic engineering, plant genetic improvement and other directions, and can solve very few problems.

Active Publication Date: 2020-06-12
武汉联农种业科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the research on flowering period of Brassica napus is still focused on the mapping of QTL for flowering period, and there are few reports on the successful cloning of flowering period genes in Brassica napus

Method used

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  • Cloning and application of flowering gene bnflc.a2 and bnflc.a2 in Brassica napus
  • Cloning and application of flowering gene bnflc.a2 and bnflc.a2 in Brassica napus
  • Cloning and application of flowering gene bnflc.a2 and bnflc.a2 in Brassica napus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of a near-isogenic line of the BnFLC.A2 gene and preliminary positioning of the gene

[0030] 1. Experimental materials

[0031] The materials used in this experiment are brassica napus high-generation near-isogenic lines: late-flowering material L04 and early-flowering material L06 ( figure 2 ). Source of material: Two Brassica napus pure line materials HZ396E and Y106 were used as the original parents to cross, and HZ396E was used as the recurrent parent to backcross for five generations and then self-crossed to obtain a high-generation segregation population. We found that the flowering period was segregated, and we selected an early-flowering homozygous Type material, named L06 and a late-flowering homozygous type material, named L04. The flowering period of the NILs material was stable under the winter rapeseed environment for many years. The early-flowering material L06 flowered about 93 days after sowing, while the late-flowering parent...

Embodiment 2

[0048] Example 2: Fine mapping of late flowering gene BnFLC.A2

[0049] 1. Development of Molecular Markers

[0050] According to the preliminary mapping results, we developed 119 pairs of SSR markers for a total physical interval of about 1Mb for the interval between the farthest markers SRA2-6 and SRA2-31 on both sides, using the A2 chromosome sequence of the Chinese cabbage genome as a reference sequence. The polymorphic markers screened by parents L06 and L04 can be used for large population genotype analysis. Through marker screening, a total of 28 pairs of polymorphic SSR markers were screened. From the 28 pairs of SSR markers, 10 pairs of SSR markers that were uniformly distributed in the target interval and had better band patterns were selected for subsequent population analysis and genotype identification of exchanged individual plants. See Table 5 for the sequence information of these markers.

[0051] Table 5 Fine positioning marker sequence

[0052]

[0053]...

Embodiment 3

[0056] Example 3: Comparative sequencing to determine natural variation among BnFLC.A2 alleles

[0057] In order to further confirm the candidate gene, we designed specific primers according to the candidate gene BnaA02g00370D gene in the target interval of Brassica napus A2 chromosome and its upstream and downstream reference sequences to amplify the coding region of the target candidate gene BnFLC.A2 and its upstream promoter region and downstream interval. We used these primers to analyze the parents L04 and L06, in which the marker STA2-6L / STA2-6R amplifies the upstream of the start codon of the BnFLC.A2 gene to the last exon of the previous gene, the theoretical size is about 2.3kb ; STA2-8L / STA2-8R amplifies the downstream of the stop codon of the BnFLC.A2 gene until the first exon of the next gene, the theoretical size is about 2.4kb ( Figure 6 A); For the BnFLC.A2 coding region, we designed two pairs of primers to amplify, namely STA2-55L / STA2-1R (theoretical size is...

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Abstract

The invention provides cloning and application of flowering period BnFLC.A2 and Bnflc.a2 genes of brassica napus, and particularly provides isolation cloning, functional verification and application of two DNA fragments of an early flowering Bnflc.a2 gene and a later flowering BnFLC.A2 allele of rape. The early flowering Bnflc.a2 allogene is a loss-of-function mutant of the BnFLC.A2 gene. A functional marker of the early flowering gene is developed; the Bnflc.a2 gene has remarkable influence on the flowering period in a natural population under seven environments through the natural population genotype and flowering period phenotypic analysis of 495 portions of brassica napus, which shows that the copy function loss caused by the insertion of long fragments in the Bnflc.a2 is an important factor influencing the natural variation of the flowering time of the brassica napus. A serial design functional marker of the Bnflc.a2 itself is used for carrying out assisted selection of molecular markers and breeding early-flowering materials, thereby breeding early-flowering and early-maturing rape. The invention has the characteristics of accuracy, high efficiency and economy.

Description

technical field [0001] The invention belongs to the technical field of molecular breeding of rapeseed, and specifically relates to the separation and cloning, functional verification and application of two DNA fragments comprising early flowering gene Bnflc.a2 and late flowering allele BnFLC.A2 of Brassica napus. Background technique [0002] Brassica napus (Brassica napus L.) is an allotetraploid crop formed about 7500 years ago by natural crossing of the diploid crops Chinese cabbage (B. rapa) and cabbage (B. oleracea) and by natural doubling (Chalhoub et al 2014). Brassica napus (B.napus) is an important oil crop, which is widely planted in my country. Timely flowering not only affects the yield and quality of Brassica napus, but also determines its adaptability to ecological regions. Therefore, flowering time is one of the important target traits in Brassica napus breeding. According to the vernalization requirements of Brassica napus, it is divided into winter rape, s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/02A01H6/20
CPCC07K14/415C12N15/8205C12N15/827
Inventor 杨光圣董发明陈磊洪登峰万丽丽辛强
Owner 武汉联农种业科技有限责任公司