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Molecular marker for early identification of sex of A. arguta young seedlings and application of molecular marker

A technology of kiwifruit and molecular markers, which is applied in the field of early identification of molecular markers for the sex of kiwifruit jujube seedlings, can solve the problems of high labor and time cost, poor versatility, no gender specificity, etc., and achieves good application prospects and stability. Good, universal effect

Active Publication Date: 2019-04-02
ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above-mentioned markers found in the current research have only been verified in the research material samples involved in the report. We have tested on varieties or individual plants of Actinidia jujube with known sex in the early stage, and the above-mentioned markers are not sex-specific.
[0007] Although studies on sex markers of Actinidia jujube have also been reported (Li Xu, Wu Songquan, Jiang Mingliang et al.: Sex-related SRAP molecular markers of Actinidia jujube. Jiangsu Agricultural Sciences, 2016,44(8):69-71.), but related No gender-specific bands were found in the study, and obtaining the test results required complex cluster analysis of multiple unstable polymorphic bands, and the results of the cluster analysis and the gender matching rate of the samples were not consistent. only reach 70%
Not only that, the above-mentioned test methods have poor versatility, the test process is complicated to operate, and the cost of manpower and time is high, and the stability of the results is easily affected by the test operation, resulting in low accuracy of the test results

Method used

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  • Molecular marker for early identification of sex of A. arguta young seedlings and application of molecular marker
  • Molecular marker for early identification of sex of A. arguta young seedlings and application of molecular marker
  • Molecular marker for early identification of sex of A. arguta young seedlings and application of molecular marker

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Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Development of Actinidia jujube-specific sex marker primers

[0045] (1) Using 〔Ruby Star (Actinidia jujube variety)×11-17 (Male Jujube)] hybrid seed population with 15 male and female plants plus 2 male and female plant parents, extract DNA to establish a BSA mixed pool; apply Illumina HiSeq sequencing technology The platform performs sequencing and bioinformatics analysis, including genome assembly and reference genome comparison statistics, SNP detection and annotation, Small InDel detection and annotation, sex-determining gene association analysis and primer sequence development.

[0046] A total of 31Gbp Raw Data was obtained by sequencing, and the Clean Data obtained after filtering was 30Gbp, with Q30 reaching 92.67%, and the average sequencing depth of each individual was 15X. The average comparison rate between the sample and the reference genome was 87.42%, the average coverage depth was 15X, and the genome coverage was 74.81% (at least 1X coverage)...

Embodiment 2

[0064] Example 2: Early sex identification method of Actinidia jujube

[0065] (1) Extraction of Genomic DNA from Actinidia jujube leaves

[0066] (1) Sampling and storage: young leaves of Actinidia jujube were taken, frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0067] (2) Extraction and preservation of leaf genomic DNA: take 100-200 mg of frozen leaf tissue, put it into a 2ml EP tube with 1 steel ball, freeze it again in liquid nitrogen, and grind it into a fine powder with a mill. Using the Plant Genomic DNA Rapid Extraction Kit (full name: DH08CTAB Plant Genomic DNA Rapid Extraction Kit) produced by Beijing Pulehai Biotechnology Co., Ltd., the CTAB method was used to extract the genomic DNA of the sample, and the specific extraction steps were carried out according to the kit instructions. The template DNA concentration was diluted to 20ng / μL, and stored in a -20°C refrigerator for later use.

[0068] (3) Main instrument models:

[0069] The centrifug...

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Abstract

The invention belongs to the fields of artificial cultivation and molecular biology of A. arguta, and particularly relates to a molecular marker for early identification of sex of A. arguta young seedlings and an application of the molecular marker. Most of A. arguta belongs to dioecious plants, the economic value of female plants is apparently higher than that of male plants, in the breeding course, the juvenile stage of tree seedlings is excessively long, and male plants and female plants can be distinguished out by naked eyes after 5-7 years, so that if the sex of the young seedlings can beidentified at the early stage, massive breeding cost can be saved. The invention provides the molecular marker for early sex identification of tree seedlings of the A. arguta, and based on the molecular marker, a reagent kit for auxiliary breeding for the sex of the A. arguta and an early identification method for the sex of the A. arguta are established. Through detection on 95 A. arguta singleplants of which the sex is known, the testing accuracy rate can reach 95.7%. The molecular marker and the method disclosed by the invention are good in generality, simple to operate, good in stabilityand good in application prospects.

Description

technical field [0001] The invention belongs to the fields of artificial cultivation and molecular biology of Actinidia jujubae, and specifically relates to a molecular marker for early sex identification of Actinidia jujube seedlings and an application thereof. Background technique [0002] The development of the world's kiwifruit industry originated in the early 20th century, while the development of my country's kiwifruit industry began in the late 1970s. The varieties cultivated and planted in the early days mainly came from Actinidia chinensis Planch.var.chinensis and Actinidia chinensis Planch.var.chinensis and A.chinensis Planch.var.deliciosaA.Chev .). Actinidia jujuba [A.arguta (Siebold&Zucc.) Planch.ex Miq.] is the most rapidly developing new cultivar in recent years. [0003] From the morphological point of view, the flower type of Actinidia plants is complete flower, and most of them belong to functional dioecious plants. Actinidia plants generally have a child...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6879C12N15/11
CPCC12Q1/6879C12Q1/6895C12Q2600/13
Inventor 齐秀娟方金豹钟云鹏郭丹丹林苗苗孙雷明王然陈锦永顾红
Owner ZHENGZHOU FRUIT RES INST CHINESE ACADEMY OF AGRI SCI
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