Cloning and application of flowering period BnFLC.A2 and Bnflc.a2 genes of brassica napus

A technology of Brassica napus and genes, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve few problems such as

Active Publication Date: 2017-06-13
武汉联农种业科技有限责任公司
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  • 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 f...

Method used

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  • Cloning and application of flowering period BnFLC.A2 and Bnflc.a2 genes of brassica napus
  • Cloning and application of flowering period BnFLC.A2 and Bnflc.a2 genes of brassica napus
  • Cloning and application of flowering period BnFLC.A2 and Bnflc.a2 genes of brassica napus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0030] 1. Experimental materials

[0031] The materials used in this experiment are high-generation near-isogenic lines of Brassica napus: late-flowering material L04 and early-flowering material L06 ( figure 2 ). Source of material: Two pure lines of Brassica napus, HZ396E and Y106, were used as the original parents to cross, and HZ396E was used as the recurrent parent. After five generations of continuous backcrossing, a high-generation segregating population was obtained. It was found that the segregation at the flowering stage was selected. We selected an early-flowering homozygous type material and named L06 and a late-flowering homozygous material and named L04. Under the winter rapeseed environment, the NILs material has a stable flowering period for many years. The early-flowering material L06 blooms about 93 days after sowing, while the late-flowering p...

Embodiment 2

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

[0049] 1. Development of molecular markers

[0050] According to the initial mapping results, we developed 119 pairs of SSR markers for the physical interval of about 1Mb in the interval between the farthest markers SRA2-6 and SRA2-31 on both sides, using the cabbage genome A2 chromosome sequence as the reference sequence. Screening of polymorphic markers 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 these 28 pairs of SSR markers, 10 pairs of SSR markers evenly distributed in the target interval and with better banding patterns were selected for subsequent population analysis and identification of genotypes of exchanged individual plants. Sequence information for these markers is shown in Table 5.

[0051] Table 5. Fine-mapping marker sequences

[0052]

[0053]

[0054] 2. Fine m...

Embodiment 3

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

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

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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 rapeseed molecular breeding, and in particular relates to the isolation, cloning, functional verification and application of two DNA fragments comprising an early-flowering gene Bnflc.a2 of Brassica napus and its late-flowering allele BnFLC.A2. Background technique [0002] Brassica napus L. is an allotetraploid crop that was formed by the natural cross and natural doubling of the diploid crops B. rapa and B. oleracea about 7500 years ago (Chalhoub et al 2014). Brassica napus (B. napus) is an important oil crop that is widely grown 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 period is one of the important target traits in Brassica napus breeding. According to the vernalization demand of Brassica napus, it is divided into winter rape, semi-winter rape and spring rape, of which se...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/84A01H5/00
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
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