A SNP marker for identification of high stone cell content in pear pulp based on high-resolution melting curves and its application

A technology of cell content and melting curve, applied in the field of molecular genetics and breeding, can solve the problems of development and application research that have not yet been reported

Active Publication Date: 2020-07-24
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on QTL mapping of quantitative traits in pears is still in its infancy. The existing researches are mainly aimed at some diseases or growth traits. There is no report on the development and application of SNP molecular markers for high stone cell content in pear pulp.

Method used

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  • A SNP marker for identification of high stone cell content in pear pulp based on high-resolution melting curves and its application
  • A SNP marker for identification of high stone cell content in pear pulp based on high-resolution melting curves and its application
  • A SNP marker for identification of high stone cell content in pear pulp based on high-resolution melting curves and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 The mensuration of stone cell content

[0022] Stone cells were separated and weighed according to the method of Tao et al. (2009) [6]. Peel and remove the core of the fruit, take 100g of pulp and add distilled water to beat, then dilute the homogenate with distilled water, stir and let it stand at room temperature for 30 minutes, discard the floating matter, resuspend the sediment in 0.5mol / L hydrochloric acid for 30 minutes, discard Floats were washed with distilled water. Repeat the above steps until the stone cells are separated from other cell fragments, and weigh after drying. Table 1 shows the content of stone cells in the flesh of 56 pear natural populations at 35 days after flowering.

[0023] Table 1 Stone cell content in the pulp of 56 pear natural populations at 35 days after flowering

[0024]

[0025]

[0026] Note: Serial numbers 1-30 are varieties with high stone cell content, and 31-56 are varieties with low stone cell content.

Embodiment 2

[0027] Example 2 Development of HRM-specific primers using the SNP marker site Psc397-15

[0028] The DNA sequence of the SNP marker Psc397-15 on the fifth chromosome was searched from the whole genome database of Dangshan Suli, and the 300 bp sequence before and after this site was selected, and the SNP marker primers were developed and designed according to the primer design principles (Table 2). Using the designed SNP marker primers to carry out PCR amplification on the genomic DNA of Dangshansu pear, the primers were amplified normally, and the PCR product was in line with the predicted size.

[0029] Table 2 SNP marker primers

[0030]

Embodiment 3

[0031] Example 3 Using HRM (high-resolution melting, high-resolution melting curve) technology to type the stone cell content of pear pulp

[0032] The HRM reaction system was performed according to the instructions in the LightCycler 480 High Resolution Melting Master kit, and the HRM analysis was performed on a LightCycler480II fluorescent quantitative PCR instrument. 20μL reaction system: containing 30ng pear genomic DNA template, 1×Master Mix, 2.5mmol / L MgCl 2 , 200nM of the primer described in claim 1 or 2; the amplification program adopts touchdown PCR (touchdown PCR): 95°C pre-denaturation for 10min, then 95°C denaturation for 10s, 65-55°C (0.5°C per cycle) annealing for 10s, The 10 s extension program at 72°C was performed for 45 cycles. Melting was performed after the PCR cycle was completed. The program was: 95°C for 1 min, 40°C for 1 min, 65°C for 1 s, and then the temperature was continuously raised from 70°C to 95°C. Fluorescence was collected once for each incre...

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Abstract

The invention discloses an SNP (single nucleotide polymorphism) marker for examining high sclereid content in pear pulp based on a high resolution melting curve and application thereof. The SNP marker is an SNP marker Psc397-15 closely related to the sclereid content in pear pulp; and primers are as shown SEQ ID NO.1, and SEQ ID NO.2. With the adoption of the special primers, the high sclereid content in the pear pulp can be typed based on the high resolution melting curve. The molecular marker and the primers for the sclereid content in the pear pulp are applied to assisting selecting and breeding of the molecular marker for the high sclereid content in the pear pulp, and important theoretical significance and practical guiding significance are shown on quickening the genetic improvement of pear varieties and increasing breeding selecting efficiency.

Description

technical field [0001] The invention belongs to the field of molecular genetic breeding, and relates to a SNP marker for identifying high stone cell content in pear pulp based on a high-resolution melting curve and an application thereof. Background technique [0002] Pear has a long history of cultivation in my country, and it is also an important deciduous fruit tree in the world. Pear fruit has high nutritional value, crisp and juicy meat, and is deeply loved by people. The edible quality of pear fruit has always been a concern of people, and it is an important factor determining the economic value of pear fruit, so it is of great significance to improve the edible quality of pear fruit. The eating quality of pear fruit is affected by many factors, among which stone cells are one of the important factors affecting the quality of pear fruit. Stone cells are a special type of thick-walled cells in pear fruit, which not only affect the eating quality of pear fruit, but als...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/6895C12Q1/686
CPCC12Q1/686C12Q1/6895C12Q2600/13C12Q2600/156C12Q2527/107C12Q2563/107
Inventor 吴俊薛程张绍铃张明月殷豪秦梦帆陶书田齐开杰
Owner NANJING AGRICULTURAL UNIVERSITY
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