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KASP molecular marker for detecting red bayberry fruit color and application of KASP molecular marker

A marking and coloring technology, applied in recombinant DNA technology, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of inaccurately finding SNP sites, difficulty in ensuring accuracy, and inability to distinguish red plums from dark plums, etc. , to achieve the effects of reducing time and labor costs, speeding up the selection process, and improving detection efficiency

Pending Publication Date: 2022-03-01
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CN113293227A discloses marker primers for identifying the color traits of red bayberry fruit, but it does not accurately find the SNP site that determines the color of red bayberry fruit, only amplifies the red bayberry gene through a forward primer and a reverse primer, and thinks that it can be amplified as white plum , if it cannot be amplified, it will be another color
This method can only be used to detect white plums, and cannot distinguish red plums from dark plums, and once the amplification fails, it is easy to mistake white plums for bayberry of other colors, and the accuracy is difficult to guarantee

Method used

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  • KASP molecular marker for detecting red bayberry fruit color and application of KASP molecular marker
  • KASP molecular marker for detecting red bayberry fruit color and application of KASP molecular marker
  • KASP molecular marker for detecting red bayberry fruit color and application of KASP molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 is used to detect the development of the KASP molecular marker of red bayberry fruit color

[0040] 1. Mining of red bayberry fruit color genes

[0041] Based on the previous survey of germplasm resources, the germplasm resources of bayberry production areas across the country were sorted out. A total of 173 germplasm resources were collected, genome resequencing was carried out, and the importance of fruit color of 136 germplasm resources was determined. Trait lightness (L), genome-wide association analysis was carried out between genome data and lightness, and a SNP was detected at the 4,691,249bp position of chromosome 6, and the gene located according to the SNP position was found to be MrChr6G813( figure 2 a and c), wherein a is the GWAS figure made by utilizing the TASSEL software calculation method, c is the GWAS figure made by the EMMAX software calculation method, a and c display are all associated with the SNP at the same position, as can be seen pr...

Embodiment 2

[0044] Embodiment 2 is used for the design of the primer combination of KASP detection molecular marker

[0045] In this embodiment, according to the SNP site obtained in Example 1, the upstream 50bp sequence (Seq ID NO.5) and the downstream 50bp sequence (Seq ID NO.6) of the SNP at position 4,691,249bp on chromosome 6 were searched, and the Cereals DB website was used. The KASP primer numbered KASP-Primer-813 was designed to detect the genotypes of Myrica rubra with different fruit colors. The primer sequences are shown in Table 1, wherein the 5' end of forward primer 1 has a FAM fluorescent signal label, and the 5' end of forward primer 2 has a HEX fluorescent signal label. The primer combination sequence can specifically combine with the corresponding sequences upstream and downstream of the SNP site.

[0046] Table 1 KASP-Primer-813 primer sequence

[0047]

Embodiment 3

[0048] Example 3 Verification of KASP molecular markers

[0049] 80 bayberry germplasms with different fruit colors were used to verify the accuracy of detection rate, among which 40 germplasms were black plums, 32 were red plums, and 8 were white plums (Table 3). The DNA of 80 parts of Myrica rubra germplasm materials was extracted by CTAB method, and the KASP primer genotype detection was carried out by using the KASP-Primer-813 primer provided in Example 2.

[0050] Using 1.6 μl PCR reaction system, containing 0.8 μl KASP master mix (LGC, BiosearchTechnologie), 0.05 μl each primer (Primer_AlleleFAM, Primer_AlleleHEX, Primer_Common) and 0.8 μl DNA (5-10ng / μl), according to IntelliQube instrument (LGC, Biosearch Technologies) instructions for PCR analysis, the PCR reaction program is: 95°C pre-denaturation, 15min; 95°C denaturation for 20s; 65°C annealing and extension for 25s, each cycle of annealing temperature decreased by 1°C, a total of 10 cycles; 95°C denaturation for 1...

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Abstract

The invention aims to provide the KASP molecular marker for detecting the color of the waxberry fruit and the application, the SNP site of the molecular marker is located at the 4th, 691st and 249th basic groups of the waxberry chromosome 6, and a KASP primer combination is designed by utilizing the difference of the single basic groups and can be used for quickly and accurately identifying the genetic typing of the site of the waxberry so as to predict the color of the waxberry fruit. The method can be used for selecting germplasm materials with required fruit colors, greatly quickens the selection progress of the myrica rubra germplasm materials, is simple, convenient, efficient and low in cost, and has a good application prospect in the aspects of myrica rubra fruit color identification and auxiliary breeding.

Description

technical field [0001] The invention relates to the technical fields of molecular marker development and molecular assisted breeding of crop morphological traits, in particular to a KASP molecular marker for detecting the color of red bayberry fruit and its application. Background technique [0002] Myrica rubra is a unique cultivated economic fruit tree in my country. It has a beautiful tree shape, is evergreen in all seasons, and has a delicious taste. The fruit is rich in functional substances such as anthocyanins and has a high nutritional value. According to the color of the fruit, it can be divided into white plums, red plums, and black plums. class and other types ( figure 1 ). [0003] At present, the breeding of bayberry is still based on conventional bud mutation breeding. However, the variation of bud mutation breeding traits is not oriented, and the excellent traits cannot be aggregated. Hybrid breeding can achieve the purpose of aggregating the excellent traits ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2531/113C12Q2563/107
Inventor 张淑文戚行江俞浙萍孙鹂梁森苗任海英郑锡良
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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