KASP molecular marker for predicting soluble solids of waxberry fruits and application of KASP molecular marker

A soluble and solid technology, applied in the field of KASP molecular markers, can solve the problems of prolonging the breeding cycle, inability to aggregate good traits, increase breeding workload, etc., and achieve the effects of improving detection efficiency, good taste, and reducing time and labor costs

Active Publication Date: 2022-05-10
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] At present, the breeding of bayberry is still dominated by conventional bud mutation breeding. However, the variation of bud mutation breeding traits is not directional, and excellent traits cannot be aggregated. Hybrid breeding can achieve the purpose of aggregation of the excellent traits of both parents, but the hybrid offspring has a childhood period of up to 10 years. The fruit quality traits cannot be detected during the childhood period, which increases the breeding workload and prolongs the breeding cycle

Method used

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  • KASP molecular marker for predicting soluble solids of waxberry fruits and application of KASP molecular marker
  • KASP molecular marker for predicting soluble solids of waxberry fruits and application of KASP molecular marker
  • KASP molecular marker for predicting soluble solids of waxberry fruits and application of KASP molecular marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Development of KASP molecular markers for predicting soluble solids in bayberry fruit

[0039] 1. Excavation of TSS gene in red bayberry fruit

[0040] Based on the previous investigation 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 fruit TSS content of 136 germplasm resources was determined. Genome-wide association analysis was performed on the genomic data and TSS, and a SNP was detected at the 9,667,472bp position of chromosome 5, and the target gene was found to be MrChr5G1084( figure 1 a and b), the target gene MrChr5G1084 has the sequence shown in Seq ID NO.4 in the sequence listing.

[0041] 2. Function prediction of gene MrChr5G1084

[0042] The CDS sequence of MrChr5G1084 was compared by Blast at NCBI, and it was found that the similarity with the gene reported by the previous sequence n...

Embodiment 2

[0043] Embodiment 2 is used for the design of the primer combination of KASP detection molecular marker

[0044] 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 9,667,472bp on chromosome 5 were searched, and the Cereals DB website was used. The KASP primer numbered KASP-Primer-1084 was designed to detect the genotypes of red bayberry fruit soluble solids. 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.

[0045] Table 1 KASP-Primer-1084 primer sequence

[0046]

Embodiment 3

[0047] Example 3 Application of KASP Molecular Markers

[0048] 78 germplasms of Myrica rubra were used as test materials, and the TSS content in fruit ripening period was determined (Table 2). The DNA of 78 germplasm materials of Myrica rubra was extracted by CTAB method.

[0049] Utilize the KASP-Primer-1084 primer that embodiment 2 provides to carry out germplasm genotype detection, the reaction system is as follows:

[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) using instructions for PCR analysis, the PCR reaction program is: 95 ° C pre-denaturation, 15 min; 95 ° C denaturation 20 s; Annealing at 57°C for 60s, a total of 30 cycles.

[0051] Primer KASP-Primer-1084 detected two genotypes, namely CT and CC ( image 3 ). Depend on image 3 It can b...

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Abstract

The invention provides a KASP molecular marker for predicting soluble solids of waxberry fruits, SNP sites of the KASP molecular marker are located at 9th, 667th and 472nd basic groups of waxberry chromosome 5, a KASP primer combination is designed by utilizing the difference of the single basic groups, the KASP molecular marker can be used for rapidly and accurately identifying the genotype of the site of waxberry, the genotype with higher soluble solids and better taste of the waxberry fruits is selected, and the molecular marker has the advantages that the molecular marker is used for predicting the soluble solids of the waxberry fruits; therefore, the soluble solid content of the fruits is predicted in the germplasm seedling stage, time and labor cost are reduced, meanwhile, multiple samples can be detected in a high-throughput mode, detection efficiency is greatly improved, and the method plays an important role in breeding of high-quality varieties.

Description

technical field [0001] The present invention relates to the technical fields of molecular marker development and molecular assisted breeding for crop fruit character prediction, in particular to a KASP molecular marker for predicting soluble solids of red bayberry fruit and its application. Background technique [0002] Red bayberry is a unique economic fruit tree in my country. It has a unique taste and rich nutrition. It is rich in vitamin C, flavonoids, and polyphenols. It has the functions of lowering blood sugar, lowering fat, and inhibiting various tumor activities. According to the data of the National Bureau of Statistics, in 2020, the cultivated area of ​​bayberry in Zhejiang Province alone will reach 1.35 million mu, with an output value of nearly 4.6 billion yuan. It has significant economic benefits and is an advantageous tree species to promote rural revitalization. [0003] Fruit soluble solids (The soluble solids, TSS) refers to the general term for compounds ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6895C12Q1/6858C12Q2600/13C12Q2600/156C12Q2600/172C12Q2531/113C12Q2563/107Y02A50/30
Inventor 张淑文戚行江俞浙萍孙鹂
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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