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InDel molecular marker for identifying watermelon fusarium wilt and primer and application thereof

A watermelon wilt and molecular marker technology, applied in the field of molecular biology, can solve the problems of insufficient stability, cumbersome steps, and high cost, and achieve the effects of stable variation, low cost, and easy detection

Active Publication Date: 2016-02-17
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing developed watermelon fon1 resistance tightly linked molecular markers are CAPS / dCAPs markers, the detection process requires enzyme digestion, the cost is high, and the steps are cumbersome
The insertion / deletion (InDel) marker based on whole genome resequencing is a co-dominant marker, which is easy to detect and low in cost, but there are still some unsatisfactory points, such as less variation and insufficient stability, etc.

Method used

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  • InDel molecular marker for identifying watermelon fusarium wilt and primer and application thereof
  • InDel molecular marker for identifying watermelon fusarium wilt and primer and application thereof
  • InDel molecular marker for identifying watermelon fusarium wilt and primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Watermelon Fusarium wilt resistance identification, the specific steps are as follows:

[0034] (1) Preparation of bacteria soil: separate and cultivate the bacteria from the watermelon wilt plants cultivated in the incubator, multiply and cultivate with wheat grains before use, mix with sterilized sand in a ratio of 1:50, and set aside;

[0035] (2) Seed preparation: take 100 seeds of each variety, soak the seeds at room temperature for 24 hours, and sow seeds at 33°C for 36 hours;

[0036] (3) Inoculation identification: use the wheat grain sand fungus soil method, put the prepared fungus soil in a nutrient bowl, sow in a heated seedbed in the greenhouse, 5 seeds per nutrient bowl, repeat twice, and set a susceptible and disease-resistant control and protection lines;

[0037] (4) Disease investigation: 10 days after sowing, the disease begins, and the emergence and disease situation are recorded. After about 4 weeks, when the susceptible and disease-resistant contro...

Embodiment 2

[0047] The development method for identifying the InDel molecular marker of watermelon wilt, the specific steps are as follows:

[0048](1) Preliminary mapping of genome-wide QTL for Fusarium wilt resistance

[0049] F obtained by crossing the female parent "ZXG01478" (highly resistant to Fusarium wilt) and the male parent "14CB11" (highly susceptible to Fusarium wilt) 2 The population has constructed a high-density genetic linkage map (linkage grouping was carried out through the mLOD value between molecular tags, see Table 1), and the parents, F 1 and F 2 Fusarium wilt resistance identification was carried out on 93 individual plants of the population; combined genetic linkage map and F 2 Identification results of population Fusarium wilt resistance ( figure 1 ), using WinQTLcartographer2.5software (http: / / statgen.ncsu.edu / qtlcart / WQTLCart.htm) for initial QTL mapping, a QTL site fon1 related to Fusarium wilt resistance was identified in the LG1 linkage group, and its LO...

Embodiment 3

[0059] Watermelon F 2 Population molecular marker analysis, comprising the following steps:

[0060] (1) Extract the total DNA of leaves by CTAB method, the specific steps are as follows:

[0061] Take 1g of fresh leaves and put them into a mortar, add liquid nitrogen to grind them into powder, then transfer them into a centrifuge tube with 1ml of CTAB extraction solution, mix the two thoroughly, then place them in a constant temperature water bath at 65°C for 60 minutes, and mix them upside down for 2 minutes. -3 times;

[0062] After taking it out from the water bath, centrifuge at 8000rpm for 1min;

[0063] Take the supernatant and put it in another centrifuge tube, add an equal volume of chloroform: isoamyl alcohol (24:1, V / V), invert gently to mix well;

[0064] Centrifuge at 10000rpm for 5min, and take the supernatant (try not to get the middle sediment);

[0065] Add 0.7 times the volume of isopropanol (need to pre-cool for 30 minutes in advance), mix well...

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Abstract

The invention particularly relates to an InDel molecular marker for identifying watermelon fusarium wilt, a primer of the InDel molecular marker and an application of the InDel molecular marker in watermelon fusarium wilt resistance breeding. The nucleotide sequence of the InDel molecular marker is TTTATTTTTTATTTTTTATTT. The primer sequence of the InDel molecular marker comprises InDel1_fon1F:TTCCAAAAGTGCAGATTTC and InDel1_fon1R:CACATGGGGATTGACTAAG. According to the InDel molecular marker for identifying watermelon fusarium wilt and the primer and application thereof, main-effect QTL for resisting fon1 is positioned in a first chromosome in the similar way through QTL initial positioning, the InDel molecular marker which is tightly linked with the watermelon fusarium wilt resistance and the primer of the InDel molecular marker are prepared by combining parent re-sequencing, and the application of the InDel molecular marker in watermelon fusarium wilt resistance breeding is realized; novel means can be provided for watermelon fusarium wilt resistance, the improvement process of the watermelon fusarium wilt resistance character is accelerated, and the breeding accuracy and selecting efficiency are improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to an InDel molecular marker for identifying watermelon wilt, a primer and application thereof. Background technique [0002] Fusarium wilt (FW), caused by the parasitism of Fusarium subphylum Fusarium watermelon (fon), is a fungal soil-borne disease that causes the most serious reduction in yield and quality in watermelon production worldwide. At present, there are four fon races 0, 1, 2 and 3 that have been reported. Except for fon3, different commercial watermelon varieties have integrated the resistance of races fon0, fon1 and fon2 to a certain extent. However, most varieties are not resistant to Fusarium wilt, and the pathogen can quickly spread and spread in watermelon growing areas. Therefore, the development of easy-to-operate and low-cost molecular markers that are closely linked to Fusarium wilt resistance can not only quickly identify Fusarium wilt-resistant ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 李娜尚建立马双武王吉明李楠楠
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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