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Primer applied to rice photosensitive male nucleic male sterility gene lncR parting and application thereof

A rice and gene technology, applied in the field of agricultural bioengineering, can solve the problems of restricting the size of the selectable population, restricting breeding, short sunshine, etc., and achieve the effect of improving the efficiency of variety selection, accelerating the breeding process, and good primer specificity

Inactive Publication Date: 2013-06-12
ZHEJIANG UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are great differences in light and temperature conditions in different places and different seasons in the same place, which is very unfavorable for the selection of two-line CMS
For example: (1) Hainan is an important place for additional breeding of rice in winter and spring in my country. However, due to the short sunshine, PGMS rice generally does not show male sterility. Breeding
(2) When sowing in the early season in Guangdong and other southern regions, it is difficult to carry out the breeding of PGMS rice due to the short sunshine; It is sterile, and once the sterile plants are selected, they need to be stubbled and regenerated before they can bear fruit; and the rice piles need to be brought to Hainan to let them regenerate and tiller to obtain seeds, which is not only time-consuming and laborious, but also seriously restricts the selection of groups. size
So far, there is no public report on the development of functional molecular markers of rice PGMS genes, so there is no application of such markers in PGMS rice breeding

Method used

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  • Primer applied to rice photosensitive male nucleic male sterility gene lncR parting and application thereof
  • Primer applied to rice photosensitive male nucleic male sterility gene lncR parting and application thereof
  • Primer applied to rice photosensitive male nucleic male sterility gene lncR parting and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 Development of Rice Photosensitive Sterile Gene Functional Molecular Marker

[0067] 1. Functional CAPS molecular markers to distinguish photosensitive sterility from wild-type alleles

[0068] 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 mutation of base 789 C→G in a gene encoding long non-coding RNA (lncR).

[0069] 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 Primer Premier5.0 software to design a pair of The fragment amplified by PCR primers contains the C→G mutation site.

[0070] The base sequence of the primer is:

[0071] Upstream primer (LR-F): 5'-ATCCCACAAATCCTTTAGCA-3';

[0072]Downstream primer (LR...

Embodiment 2

[0082] Example 2 Using CAPS Molecular Markers to Distinguish Photosensitive Sterile and Non-photosensitive Sterile Rice Varieties

[0083] Different rice materials were collected in 2012, including homozygous photosensitive sterile lines, heterozygous materials containing photosensitive sterile genes, and non-photosensitive sterile materials. These materials were planted in the experimental farm of Zhejiang University in the summer of 2012, and the leaves were taken from the plants in the vegetative growth stage in the field for use.

[0084] 1. Extraction of rice genomic DNA

[0085] (1) Cut the rice leaves into pieces and put them in a 2.0mL centrifuge tube, put a steel ball into them at the same time, and grind them with a tissue mill;

[0086] (2) Add 800μL CTAB extraction buffer (Tris-HCl, 100mM, pH8.0; EDTA, 20mM, pH8.0; NaCl, 500mM; CTAB, 2%), 65℃ water bath for 40min, shake 3-4 times during the period;

[0087] (3) Add an equal volume of chloroform: isoamyl alcohol (...

Embodiment 3

[0101] Example 3 Using HRM Molecular Markers to Distinguish Photosensitive Sterile and Non-photosensitive Sterile Varieties

[0102] 1. Extraction of rice genomic DNA

[0103] For the method, refer to the extraction of rice genomic DNA in Example 2.

[0104] 2. PCR amplification

[0105] Using rice DNA as a template, using the primers LR-F, LR-R and probe primers designed in Example 1, asymmetric PCR amplification was performed.

[0106] 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 reaction mixture with mineral oil (Sigma), and , in order to avoid affecting the PCR amplification, it is also possible to add a saturated fluorescent dy...

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Abstract

The invention discloses a primer applied to rice photosensitive male nucleic male sterility gene lncR parting and an application thereof. The base sequence of the primer is as follows: an upstream primer, namely 5'-ATCCCACAAATCCTTTAGCA-3', and a downstream primer, namely 5'-CCGTTATAGATAGACCCGAGA-3'. The invention further provides an application of the primer in the rice photosensitive male nucleic male sterility gene lncR parting, and an application of the primer in photosensitive male nucleic male sterility rice seed selection. The primer for the rice photosensitive male nucleic male sterility gene lncR parting is good in specificity and is simple, effective and high in accuracy when being applied to a CAPS or an HRM molecular marker to part the rice photosensitive male nucleic male sterility gene lncR.

Description

technical field [0001] The invention belongs to the technical field of agricultural bioengineering, and in particular relates to a primer for typing of rice photosensitive male sterility gene lncR and an application thereof. 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 tw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 舒庆尧张华丽黄建中
Owner ZHEJIANG UNIV
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