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Construction method and application of ssr molecular fingerprint of "Shenyang" strawberry

A technology of molecular fingerprinting and molecular fingerprinting, which is applied in the construction of SSR molecular fingerprinting, can solve the problems of somatic cell variation and degeneration, and achieve the effects of stable genetic traits, strong discrimination, and good polymorphism

Active Publication Date: 2021-07-13
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In the prior art, although in sterile conditions and limited space, a large number of plant seedlings can be rapidly propagated through tissue culture, and strawberry tissue culture micropropagation seedling technology is also relatively common, but in production, tissue culture sources are often encountered. The problem of somatic cell variation of seedlings will cause many problems in production and application, so that many growers would rather use the field propagation route that is susceptible to viral diseases than take the risk of adopting tissue culture micropropagation seedlings. The reason is that somatic cell variation caused by inappropriate tissue culture and micropropagation process will lead to changes in various traits of plants, such as: flowering and fruiting time, fruit appearance, flavor, yield and other traits may change, among them, flowering delay, in production During this period, no flowers or fruit quality degradation is the most common phenomenon

Method used

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  • Construction method and application of ssr molecular fingerprint of "Shenyang" strawberry
  • Construction method and application of ssr molecular fingerprint of "Shenyang" strawberry
  • Construction method and application of ssr molecular fingerprint of "Shenyang" strawberry

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Effect test

Embodiment 1

[0065] The specific process of obtaining the 8 pairs of SSR primer sequences of the present invention and constructing the SSR fingerprint of "Shenyang" strawberry is as follows:

[0066] First, SSR markers were selected from the Rosaceae genome database website (www.rosaceae.org) in the whole genome and followed the principle of even distribution, and a total of 140 pairs of SSR marker primers were selected and synthesized.

[0067] Afterwards, using the genomic DNA of five strawberry germplasms including "Shenyang", "Jiuxiang", "Hongyan", "Zhangji" and "Sweet Charlie" as templates, electrophoresis after PCR amplification confirmed that each pair of SSR marker primers The optimal PCR conditions for amplification and the size range of amplified product fragments; according to the three repeated PCR amplification and electrophoresis analysis of 140 pairs of SSR marker primers in 5 strawberry samples, the non-amplified or overly complicated amplified band patterns, Amplify prime...

Embodiment 2

[0087] Using the SSR specific molecular fingerprint of "Shenyang" strawberry to carry out molecular identification of the strawberry sample to be tested includes the following steps:

[0088] 1) DNA extraction

[0089] For the strawberry population to be identified, after random sampling, every 5 plants are mixed, and at least 5 mixed samples are set up, with deionized water as the negative control and "Shenyang" strawberry as the positive control, 3 replicates are set, and the strawberries are extracted by the CTAB method Genomic DNA, the specific method refers to the master's thesis "Strawberry Anthracnose Resistance Genetic Map Construction and the Cloning and Expression Analysis of Resistance-related Genes" (Li Jing. Nanjing Agricultural University, 2012). After the DNA is analyzed by colorimetry and electrophoresis, it is diluted to 25ng / ul Standby.

[0090] 2) PCR amplification

[0091] Using the DNA extracted in step 1) as a template, use 3 to 8 pairs of SSR primer s...

Embodiment 3

[0100] A micropropagation method for strawberry hybrid "Shenyang", comprising the steps of:

[0101] 1) Disinfection of explants

[0102] Rinse the stolons of the strawberry hybrid "Shenyang" with running water for more than 2 hours, soak them in 75% ethanol solution for 5 seconds, rinse them with sterile water for 3 times, then disinfect them with 5% sodium hypochlorite solution for 7 minutes, and then rinse them with sterile water 5 times;

[0103] 2) Shoot tip stripping and bud induction

[0104] The growth point of the stem tip is stripped from the sterilized stolon, and the growth point is inoculated in the induction medium at a culture temperature of 23-25° C., cultivated in a shading environment for 2 days, and then cultured under light for 5 days to induce bud germination. Illumination time 12h / d, light intensity 30~50μmol / m 2 s, after 30 days, sterile buds with a length of 0.5-1.0 cm were obtained; the induction medium included: MS basic medium, sucrose 30 g / L, BA ...

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Abstract

A construction method and application of SSR molecular fingerprint of "Shenyang" strawberry, screened 8 pairs of SSR primer sequences from 140 pairs of SSR primers covering the whole strawberry genome, and used them to establish SSR molecules of strawberry hybrid "Shenyang" Fingerprints can be used to identify the purity of strawberry hybrid "Shenyang" and its close relatives and sisters, non-close relatives and excellent lines from the genetic nature. It has high specificity, stable polymorphism, and band type combination. Easy to identify, good dominant characteristics, can accurately and quickly identify strawberry hybrids, and screen culture conditions, control the risk of somatic cell variation that tissue culture may bring, prevent vitrification, prevent callus formation, and limit tissue culture Multiplication algebra and multiplication coefficient, establish the micropropagation method of the strawberry hybrid "Shenyang", and quickly micropropagate the original strawberry seedlings of "Shenyang" with complete variety purity.

Description

technical field [0001] The invention relates to the field of construction of molecular fingerprints of fruit trees, in particular to a construction method and application of SSR molecular fingerprints of strawberry hybrids. Background technique [0002] Strawberry is a perennial plant of the Rosaceae Fragaria genus, an important horticultural economic crop, and ranks second in the world's berry production. Modern cultivated strawberries are allo-octoploid, highly heterozygous, and hybrid crops. Asexual reproduction is necessary to maintain the characteristics of the variety. However, long-term asexual reproduction in the field has buried hidden dangers for the production of strawberries caused by viral diseases. At the same time, it also brings Variety confusion in production. [0003] Strawberry anthracnose is a devastating fungal disease in strawberry seedlings. Through molecular marker-assisted screening, indoor inoculation of suspended spores of pathogenic bacteria and ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12N15/11C12N15/10A01H4/00
CPCA01H4/008C12Q1/6806C12Q1/6895C12Q2600/156
Inventor 高清华段可张玲孙佳欣张丽勍
Owner SHANGHAI ACAD OF AGRI SCI
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