Molecular marker for controlling flowering phase of cabbage type rape and applications of molecular marker

A technique for molecular markers and flowering stages, applied in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc.

Inactive Publication Date: 2018-02-23
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cloning photoperiod pathway genes in rapeseed and using them for assist

Method used

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  • Molecular marker for controlling flowering phase of cabbage type rape and applications of molecular marker
  • Molecular marker for controlling flowering phase of cabbage type rape and applications of molecular marker
  • Molecular marker for controlling flowering phase of cabbage type rape and applications of molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Cloning of Brassica napus BnaA10.CO gene

[0032] (1) Use specific primers (as shown below) to test the winter rape variety Tapidor (provided by the Rape Research Laboratory of Huazhong Agricultural University, see Table 1 for the original source) and the spring rape variety Westar (provided by the Rape Research Laboratory of Huazhong Agricultural University, see the original source in Table 1) 1) and an additional 11 winter rapeseed, 7 spring rapeseed and 9 semi-winter rapeseed (provided by Huazhong Agricultural University Rapeseed Research Laboratory, which is a conventional rapeseed resource, see Table 1 for the original source) of the Open-reading frame of BnA10.CO (ORF) were amplified separately. The primers for the amplification of the BnA10.CO gene (the gene ID in the rapeseed reference genome "Darmor-bzh" is BnaA10g18430D) are BnA10.CO-F (ACCATCACACTTAATTACTACATCA) and BnA10.CO-R (TACAGGTTAGCAATTCTAGTATTCTT).

[0033] (2) KOD-plus-standard reaction s...

Embodiment 2

[0042] Example 2: Identification of Brassica napus BnaA10.CO haplotype

[0043] (1) Using Clustal Omega (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ) to compare the sequences of the cloned BnaA10.CO gene coding regions of winter rape, spring rape and semiwinter rape.

[0044] (2) The results are shown in Table 2. There are base mutations and InDel mutations in exons, of which there are 7 non-synonymous mutations. Winter rapeseed only contains haplotype 1, and spring rapeseed only contains haplotype 2. But semiwinter rape contains two haplotypes. See Table 2.

[0045] Table 2 Nucleotides in the coding region that cause amino acid variation in BnaA10.CO

[0046]

Embodiment 3

[0047] Example 3: Identification of functional differences of BnaA10.CO alleles in Brassica napus

[0048] (1) Use specific primers (as shown below) to detect winter rape Tapidor (provided by the Rape Research Laboratory of Huazhong Agricultural University, see Table 1 for the original source) and spring rape Westar (provided by the Rape Research Laboratory of Huazhong Agricultural University, see Table 1 for the original source) The genomic DNA of the gene was amplified to obtain a PCR product containing a promoter and a 4kb fragment of the full length of the gene. The amplification product is as follows: EcoRI_Pro_CO.A10-F (CCG GAATTC AAATGTAAGAGCCACTTGAATGC) and PstⅠ_Ter_CO.A10-R (AA CTGCAG GGTAAGGCAAGTAACCAGTCTC); wherein, the sequence marked in bold is the protected base, and the sequence marked underlined is the restriction site.

[0049] (2) KOD-plus-standard reaction system (TOYOBO) was used for gene amplification. The 50μl PCR reaction system included: 100ng genom...

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Abstract

The invention belongs to the field of molecular marker screening, and relates to a molecular marker for controlling the flowering phase of cabbage type rape and applications of the molecular marker. The molecular marker is cloned from a BnaA 10.CO gene, is located on the A10 chromosome of the cabbage type rape, and is a key gene of a photoperiod route in a flowering phase regulation network. BnaA10. CO gene segments are cloned from winter rape and spring rape, and the sequences of the segments are shown as SEQ ID NO: 1 and SEQ ID NO: 3. Primers of the molecular marker related to the floweringphase are shown as SEQ ID NO: 5 and SEQ ID NO : 6. The transformation proves that the gene is derived from an allele of winter rape, and the effect of promoting flowering of the gene is stronger thanthat of the allele of the spring rape. The allele derived from the winter rape can shorten the growth period of half-winter rape. The molecular marker developed according to the InDel differences ofthe 25th-27th sites of the BnA 10.CO gene region can be used for early-maturing breeding of rape.

Description

technical field [0001] The invention belongs to the field of rapeseed genetic engineering, and in particular relates to a molecular marker for controlling the flowering stage of Brassica napus and the preparation and application of a molecular marker. The molecular marker of the present invention is derived from the BnaA10.CO gene, which is located on the A10 chromosome of Brassica napus and is a key gene of the photoperiod pathway in the flowering period regulation network. The invention discloses BnaA10.CO obtained by isolating and cloning from winter, semi-winter and spring rapeseed. The invention also relates to the development of a molecular marker using the gene fragment and its application in breeding for early maturity traits in rapeseed. Background technique [0002] Brassica napus is an important oil crop widely grown in the world. Rapeseed oil can not only be used as edible oil, but also industrial raw materials such as lubricating oil and biodiesel. In the near...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895C12Q2600/13
Inventor 王晶刘克德李海涛李娟娟郭涛殷帅
Owner HUAZHONG AGRI UNIV
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