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Molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application

A molecular marker, kpni-1 technology, applied in the field of molecular biology, can solve the problems of low polymorphism, few molecular markers, narrow genetic distance of watermelon, etc., and achieve the effect of shortening breeding years and improving breeding efficiency.

Active Publication Date: 2015-06-17
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genetic distance of watermelon is relatively narrow, and the number of available molecular markers published in the literature is small and the polymorphism is low, which seriously restricts the gene mapping of main agronomic traits of watermelon and the development of related molecular markers.

Method used

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  • Molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application
  • Molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application
  • Molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Primer design and extraction of genomic DNA

[0037] Select the light yellow watermelon line Cream of Saskatchewan (abbreviated as "COS") and the red watermelon line LSW-177 and their crosses to obtain F 1 , F 2 The generation population is the experimental material to verify the CAPS marker to identify the presence of lycopene in watermelon pulp. The result is figure 1 Shown.

[0038] The two watermelon lines COS and LSW-177 were sequenced using high-throughput sequencing technology, and the cds region sequence of the watermelon lycopene β-cyclase gene was obtained through the NCBI website, and the sequence was compared with the sequencing materials. The chromosomal interval in which the cds region sequence of the watermelon lycopene β-cyclase gene exists is analyzed. Within this interval, the base section of the sequencing data with the SNP mutation site is excavated, and the restriction information of the SNP site is analyzed to obtain the existence For CAPS m...

Embodiment 2

[0039] Example 2 Obtaining PCR products

[0040] Using the obtained CAPS primers and genomic DNA to perform PCR reaction, the PCR reaction system is shown in Table 1 (20μl):

[0041] Table 1. PCR reaction system

[0042]

[0043] Using touchdown PCR (TD-PCR) for amplification, the procedure is: 94℃ pre-denaturation for 7 minutes, 94℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 0.5℃ per cycle, 72℃ extension for 40s, 30 cycles; 94 Denaturation at ℃ for 30s, annealing at 45℃ for 30s, extension at 72℃ for 40s, 10 cycles; extension at 72℃ for 10min, storage at 4℃.

[0044] Use 1% agarose gel electrophoresis to detect the obtained PCR products, the detection results are as follows figure 1 As shown, LSW-177, COS, F 1 And F 2 Each individual plant amplified a fragment of 517 bp, which was consistent with the expected fragment size.

Embodiment 3

[0045] Example 3 Enzyme digestion verification of PCR products

[0046] According to Thermo's restriction endonuclease operation guide, use restriction endonuclease to verify the obtained PCR product. The restriction enzyme digestion system is shown in Table 2: (15.3μl)

[0047] Table 2. Enzyme digestion reaction system

[0048]

[0049] Digestion in a water bath at 37°C overnight, and the digested product is detected by 1% agarose electrophoresis. The detection result is as follows figure 1 Shown. Since LSW-177 has no restriction enzyme site, it is still 517 bp without restriction endonuclease. Due to the restriction enzyme site in COS, it is cut by KpnI to obtain 389 and 128 bp fragments. F 1 The generations showed co-dominant features including not only a 517 bp fragment of LSW-177, but also 389 and 128 bp fragments. In the 18 strains tested F 2 Among the individual plants, 9 plants showed red in the field phenotype, and lycopene could be detected in the pulp tissue. The bands ...

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Abstract

The invention discloses a molecular marker KpnI-1 for indicating and identifying watermelon pulp color and application and belongs to the technical field of molecular biology. The fragment size of a PCR product amplified by the molecular marker is 517bp, and the nucleotide sequence is as shown in SEQ ID NO.1. The CAPS molecular marker has universality in different materials and can identify watermelons in the watermelon seedling stage, the operation is simple and convenient, and the result is definite. The molecular marker can be used for molecular marker-assisted selection of high lycopene of watermelon in future, and the breeding work efficiency is improved.

Description

Technical field [0001] The invention relates to a molecular marker KpnI-1 for predicting and identifying the color of watermelon pulp and its application, and belongs to the technical field of molecular biology. Background technique [0002] Molecular marker-assisted selection is a new technology produced with the rapid development of modern molecular biology technology. It can quickly and accurately analyze the genetic composition of individuals at the molecular level, thereby achieving direct selection of genotypes and molecular breeding. The key to the success of molecular marker-assisted selection lies in whether the obtained molecular marker is linked to the trait to be analyzed and whether it is universal among different materials of the same species. [0003] The color of watermelon pulp is an important indicator of the quality of watermelon. Watermelon fruit contains lycopene, β-carotene, lutein, zeaxanthin and other pigments. The composition and content of different pigmen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 朱子成马鸿艳栾非时李冰
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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