Molecular marker for removing wax powder properties on surface of grafted cucumbers in pumpkin rootstocks and application thereof

A technology of molecular markers and cucumbers, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of long screening time and large floor space, and improve the efficiency of breeding selection and Accuracy, reduced floor space, shortened breeding years

Active Publication Date: 2017-12-29
QINGDAO ACAD OF AGRI SCI +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that the screening method of wax-removing pumpkin rootstocks in the prior art takes a long time and occupies a large area, the present invention provides a single nucleotide polymorphism (SNP) related to the properties of wax powder removal on the surface of grafted cucumbers from pumpkin rootstocks. ) molecular markers, the present invention also provides the application of SNP molecular markers related to the removal of grafted cucumber surface wax powder traits from pumpkin rootstocks in pumpkin rootstock breeding, and the technology of the present invention can be used to judge the removal of grafted cucumber wax powder traits from pumpkin rootstocks at the seedling stage , which greatly saves breeding time and floor space

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  • Molecular marker for removing wax powder properties on surface of grafted cucumbers in pumpkin rootstocks and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Obtaining CmLsi1 Gene Markers Related to Cucumber Surface Wax Powder Removal from Pumpkin Rootstocks

[0026] Specific steps are as follows:

[0027] (1) Extraction of Pumpkin Genomic DNA

[0028] Sow the pumpkin rootstock seeds with and without the wax powder removed in the cultivation pot, and when the pumpkin seedlings grow two true leaves, take the true leaves of 4 plants with the wax powder removed and 4 plants without the wax powder removed. Stored in liquid nitrogen. Take 1cm 2Put the large and small leaves into a 1.5ml centrifuge tube, grind them into powder in liquid nitrogen, add 600μl CTAB buffer, and extract in a water bath at 65°C for 1 hour. Add an equal volume of chloroform, mix well, and centrifuge at 4°C (12000rpm, 10min), and take the supernatant. Repeat the previous step, transfer the supernatant to a new centrifuge tube, add an equal volume of precipitation buffer (0.28mol / L NaCl, 70% ethanol) and 200μL isopropanol, and let stand at -20...

Embodiment 2

[0040] Example 2 Rapid Screening of Individuals Carrying CmLsi1 Gene Markers Related to Cucumber Surface Wax Powder Removal from Pumpkin Rootstocks

[0041] Take 1 μl of pumpkin true leaf DNA as a template, use forward primer: 5'-ATGAGTTCTTCTCAGGATCCTCAGCT-3' and reverse primer: 5'-GCATAAATCATCACCGTCACAATAAGG-3' as amplification primers, and perform PCR amplification according to the following procedure: Denaturation at 94°C for 5 minutes; Denaturation at 94°C for 30 s, annealing at 58.5°C for 30 s, extension at 72°C for 1 min, 30 cycles; finally, incubation at 72°C for 10 min.

[0042] Take 35 μL of PCR amplification product for enzyme digestion, enzyme digestion system: 10× buffer 4.0 μL, HinP1Ⅰ 1.0 μL, PCR product 35 μL, mix well, centrifuge, and digest overnight in a 37°C water bath. Take 20 μL of the digested product, add 4 μL of 6×bromophenol blue loading buffer, and run electrophoresis on 1.5% agarose gel at 90V for 90min. Electrophoresis results refer to figure 1 , s...

Embodiment 3

[0043] Example 3 Pumpkin Rootstock Removal Application of Molecular Markers Related to Grafting Cucumber Surface Wax Powder Properties

[0044] The CmLsi genotype 360C / C, which occurs frequently in wax-removing squash populations, was used as a gene marker related to wax-removing traits. In the pumpkin breeding process, the pumpkins carrying the Lsi1 mutation marker are propagated, the offspring are bred, the Lsi1 gene in the offspring is cloned, and the polymorphism is studied; the genetic law of the Lsi1 gene marker and the removal of wax on the cucumber surface by the pumpkin rootstock are studied. In order to obtain more hybrids with excellent traits, the markers containing Lsi1 gene were screened out, and the marker individuals containing Lsi1 gene were crossed with other pumpkin parents with excellent traits.

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Abstract

The invention belongs to the technical field of preparation of plant molecular markers, in particular to preparation and application of an SNP molecular marker related to the removal of wax powder properties on the surface of grafted cucumbers in pumpkin rootstocks. A silicon transport protein CmLsi1 gene fragment related to wax powder formation is obtained through cloning the pumpkin rootstocks; a nucleotide sequence is shown as a SEQ ID NO. 1 in a sequence table; a C/T base mutation exists at 360 bp of the SEQ ID NO. 1, so that PCR-RFLP-HinP1 I polymorphism is caused. The invention discloses application of the polymorphism of the CmLsi1 gene fragment to the detection of the removal of the wax powder on the surface of the grafted cucumbers in the pumpkin rootstocks, so that a novel molecular marker is provided for the marker-assisted selection of the removal of the wax powder properties on the surface of the cucumbers in the pumpkin rootstocks.

Description

technical field [0001] The invention belongs to the technical field of plant molecular marker preparation, and in particular relates to the preparation and application of a SNP molecular marker related to the properties of wax powder on the surface of pumpkin stock removed from grafted cucumbers. Background technique [0002] Fruit surface wax powder is one of the important commercial properties of cucumber. In recent years, high-quality, less waxy cucumbers have become more and more popular with consumers. Studies have shown that the formation of cucumber fruit wax powder has a certain relationship with silicon absorption. The stronger the ability of roots to absorb silicon, the more wax powder may be formed (Namiki Mitani, 2011). The researchers will graft cucumbers on rootstocks without wax powder, and found that the silicon content in the roots, stems, leaves, and fruits of cucumbers grafted with very little wax powder rootstocks is less than that of cucumbers grafted w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 崔健李凤梅张洪亮梁妍娇刘素芹宋云云江志训
Owner QINGDAO ACAD OF AGRI SCI
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