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Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof

A Fusarium wilt resistance and auxiliary identification technology is applied in molecular markers, special primers and application fields for auxiliary identification of Fusarium wilt resistance in cabbage, achieving the effect of early breeding and major application prospects

Inactive Publication Date: 2012-10-24
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on molecular markers closely linked to resistance genes, such as apples (Xu et al.2007), peppers (Arnedo-Andres et al.2002), and melons (Takahiro Tezuka et al.2009). However, the molecular markers closely linked to the resistance gene of cabbage wilt have not been reported yet.

Method used

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  • Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof
  • Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof
  • Molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Acquisition of AFLP Molecular Marker Closely Linked to Cabbage Fusarium Wilt Resistance Gene

[0029] Homozygous disease-resistant F2 individual plants (10 plants) and homozygous genotype susceptible F2 individual plants (10 plants) were randomly selected for 2 homozygous disease-resistant gene pools (R1, R2) and 2 Construction of homozygous susceptible gene pool (S1, S2). Two parallel BSA (Bulksegregant Analysis) resistant-susceptible pools R1 / S1 and R2 / S2 were constructed by taking the DNA of 5 homozygous resistant and 5 homozygous susceptible individuals.

[0030] AFLP molecular marker analysis: 256 pairs of AFLP primers were selected to amplify the disease-resistant gene pool, the susceptible gene pool and the parents.

[0031] Primer combinations: 16 pairs of EcoRI+ANN (N: A, G, C and T) and 16 pairs of MseI+ANN primer combinations, a total of 256 pairs of AFLP primers.

[0032]Pre-amplification program: 94°C for 5min; 94°C for 30s, 56°C for 30s, 72°C ...

Embodiment 2

[0038] Example 2. Obtaining the Sequence of a Specific Detection Gene Fragment Closely Linked to the Cabbage Fusarium Wilt Resistance Gene

[0039] After recovering and purifying the 2-item AFLP bands from the polypropylene gel, they were ligated with the pEASY-T1 vector. The ligation system: PCR products 4 μl, pMD-T vector 1 μl, solution I 5 μl, total volume 10 μl. Add the above reagents into a 0.5 centrifuge tube in an ice bath, flick to mix well, centrifuge briefly, and connect overnight (4°C). Take out the centrifuge tube of 50 μl of competent bacterial suspension from an 80°C refrigerator, put it on the ice box to thaw slowly, add about 10 μl of all the ligation products, flick the tube wall to mix well, and place it on the ice-water mixture for 30 minutes; Quickly heat-shock in a water bath at 42°C for 90s; quickly place it on ice for 3-5 minutes, do not shake the centrifuge tube during this period; add 700 μl LB liquid medium without AmP, transfer to 37°C, and culture o...

Embodiment 3

[0040] Example 3, Development and identification of SCAR primers for cabbage wilt resistance by specific gene fragment sequences

[0041] According to the sequencing results, the vector and linker sequences were removed, and SCAR primers were designed by using the remaining sequences combined with restriction sites. According to the AFLP primer numbering rules, this newly developed marker was named S46M48 198 , wherein S represents the SCAR marker, 46 represents E46 (E-ATT), 48 represents M48 (M-CAC), subscript 198 represents the length of the amplified fragment of the SCAR marker, and the primer sequence is:

[0042] S46M48 198 -F: 5'-ATTGGTGGCCTGCACTTTGAGCATC-3' (sequence 2 of the sequence listing);

[0043] S46M48 198 -R: 5'-ACAACAAACTAATCTCAAACATGTC-3' (SEQ ID NO: 3 of the Sequence Listing).

[0044] Using the resistant gene pool, the susceptible gene pool, the parental DNA and the DNA of individual plants that make up the resistant-susceptible pool, use the newly synth...

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Abstract

The invention discloses a molecular marker and specific primers for assisting in test of wilt disease resistance in brassica oleracea and use thereof. The invention provides a reagent for assisting in the test of wilt disease resistance in brassica oleracea and / or assisting in screening brassica oleracea with wilt disease resistance, which is a specific primer pair formed by nucleotides represented by a sequence 2 and a sequence 3 in a sequence table. The invention also provides a specific gene fragment which is at a genetic distance about 2.87cm to the wilt disease resistance gene in brassica oleracea and is formed by nucleotides represented by a sequence 1 in a sequence table. The result of the identification of wilt disease resistance in brassica oleracea and / or screening of the brassica oleracea with wilt disease resistance with assistance from the reagent (primer pair) or sequence characterized amplified region (SCAR) marker, which are provided by the invention, is 97 percent consistent with that of field identification. When used in breeding, the reagent, molecular marker or method has the advantages of accuracy, quickness, capability of realizing early breeding and the like and has a bright application prospect.

Description

technical field [0001] The invention relates to a molecular marker for auxiliary identification of cabbage wilt resistance, special primers and applications thereof. Background technique [0002] Cabbage (Brassica oleracea) is a vegetable crop of the genus Brassica (Cruciferae) commonly planted in the world. It is cultivated all over China. The main vegetables in South China and other places are also cultivated on a large scale in winter and spring. In most areas, suitable varieties are selected for row sowing and harvested in stages, which plays an important role in the annual supply of vegetables. In recent years, the annual planting area in my country has stabilized at about 1 million hectares. [0003] Cabbage wilt is a soil-borne vascular disease caused by Fusarium oxysporum f.sp.conglutinans. Infected cabbage diseased plants generally show typical symptoms 2 to 4 weeks after planting. The first symptom is yellowing of one side of the plant or the entire lower leaf, a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 康俊根许勇简元才姜明丁云花
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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