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Method for identification of purity of two-line hybrid rice seed

A hybrid rice and seed technology, which is applied in the field of agricultural bioengineering, can solve the problem that a general two-line hybrid seed purity identification method has not been established, and achieve the effect of accurate identification.

Active Publication Date: 2013-10-02
ZHEJIANG UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A universal two-line hybrid seed purity identification method based on PGMS or TGMS gene differences has not yet been established

Method used

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  • Method for identification of purity of two-line hybrid rice seed
  • Method for identification of purity of two-line hybrid rice seed
  • Method for identification of purity of two-line hybrid rice seed

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Purity identification of photosensitive hybrid rice seeds, taking the hybrid combination derived from DS550 as an example

[0080] Ding et al. (Ding et al. PNAS, 2012, 109: 2654-2659) and Zhou et al. Dwarf 64S photosensitive nuclear male sterility (PGMS) is caused by a C→G mutation at base 789 of a gene encoding a long non-coding RNA (lncR).

[0081] The lncR allele carried by the photosensitive sterile rice derived from Nongken 58S was named lncR g , the non-photosensitive rice allele is named lncR c . Accordingly, download the corresponding nucleotide sequence from the Gramene website (http: / / www.gramene.org / ), as shown in SEQ ID NO.1, and use the Primer Premier5.0 software to design a pair of The fragment amplified by PCR primers contains the C→G mutation site.

[0082] The base sequence of the primer is:

[0083] Upstream primer (LR-F): 5'-ATCCCACAAATCCTTTAGCA-3';

[0084] Downstream primer (LR-R): 5'-CCGTTATAGATAGACCCGAGA-3';

[0085] Since this mu...

Embodiment 2

[0107] Embodiment 2 Utilizes HRM method to identify the purity of photosensitive hybrid rice seeds

[0108] Utilize the samples of the above-mentioned Example 1 and simultaneously use the HRM method to identify the purity.

[0109] 1. Extraction of Genomic DNA from Rice Seeds

[0110] Refer to Example 1 for the method.

[0111] 2. PCR amplification

[0112] Using rice seed genomic DNA as a template, using the primers LR-F, LR-R and probe primers designed in Example 1 (the probe primer sequence is: 5'-GTGCATTGTTTGTGTACCATCCATC-3'), asymmetric PCR amplification was performed.

[0113] When used to develop HRM molecular markers, asymmetric PCR amplification is generally carried out in a volume of 10 μl, and the reaction solution includes PCR buffer, MgCl 2 , dNTP, Taq DNA polymerase, primers, total DNA of rice samples, sterile water, etc. In addition to conventional components, it is necessary to add saturated fluorescent dyes (such as LC Green) before PCR, and cover the react...

Embodiment 3

[0119] Example 3 Purity identification of temperature-sensitive hybrid rice seeds, taking the hybrid combination derived from Guangzhan 63S as an example

[0120] 1. dCAPS marker for RNaseZ typing of thermosensitive male sterility gene in rice seeds

[0121] In 2011, a hybrid F of thermosensitive male sterile line 1S and wild type 08EZ01 was constructed 2 Progeny groups, and in the field survey segregation ratio, it was found that at 1260 F 2 Among the individual plants, there were 286 completely sterile individual plants, and the phenotype segregation ratio was 3:1, indicating that the temperature-sensitive traits were controlled by a single recessive gene.

[0122] Microscopically observed and sampled 549 completely sterile single plants at the flowering stage, used to locate the genes controlling the 1S thermosensitive sterility traits of the plants, and constructed a gene pool, using 348 pairs of SSR markers evenly distributed on each chromosome for preliminary analysis ...

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Abstract

The invention discloses a method for identification of purity of a two-line hybrid rice seed. The method comprises the following steps of extracting a genomic DNA of a seed needing to be detected or a plant sample growing from the seed, designing primers according to functional single nucleotide polymorphic sites of a genic male-sterile gene and carrying out PCR amplification, carrying out characteristic analysis of the PCR amplification products to determine a sample genotype and calculating purity of the seed sample. The method utilizes the functional single nucleotide polymorphic sites of the functional gene to develop a molecular marker. Any parents subjected to cross combination necessarily have polymorphism and in hybrid seed purity identification, analysis of two-line cross combination adopting the two-line hybrid rice seed as a female parent can be directly carried out without research on parents. Through conventional indoor tests, hybrid rice seed purity can be simply, fast, economically and accurately determined.

Description

technical field [0001] The invention belongs to the technical field of agricultural bioengineering, in particular to a method for identifying the purity of two-line hybrid rice seeds. Background technique [0002] Plant male sterility can be caused by cytoplasmic genes, called cytoplasmic male sterility (CMS), or by nuclear gene mutations, also known as nuclear gene male sterility (GMS). Rice is a self-pollinating crop, and its utilization of heterosis must depend on male sterility. At the beginning of hybrid rice research and application, the male sterile lines used were all CMS types. In 1973, Shi Mingsong discovered a spontaneously mutated photoperiod-sensitive genic male sterile (PGMS) strain in the Hubei late japonica rice variety Nongken 58 population, which was later named Nongken 58S (Shi Mingsong, Chinese Agricultural Sciences, 1985 , 2:44-48), opened the prelude to the use of photothermosensitive male sterility to breed two-line hybrid rice. [0003] After decad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 舒庆尧张华丽黄建中
Owner ZHEJIANG UNIV
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