SSR (Simple Sequence Repeat) marker for simultaneously identifying sterile cytoplasm of gossypium harknessii and gossypium hirsutum

A Hackney West cotton and cytoplasmic technology, applied in the field of SSR labeling, can solve the problem of inability to identify two different sterile cytoplasmic substances, achieve the effects of improving accuracy and efficiency, wide application range, and saving manpower

Pending Publication Date: 2021-12-07
INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that traditional conventional breeding methods cannot identify two different sterile cytoplasms, the present invention provides an SSR marker for simultaneously identifying the sterile cytoplasms of Hackneys cotton and cotton trifida

Method used

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  • SSR (Simple Sequence Repeat) marker for simultaneously identifying sterile cytoplasm of gossypium harknessii and gossypium hirsutum
  • SSR (Simple Sequence Repeat) marker for simultaneously identifying sterile cytoplasm of gossypium harknessii and gossypium hirsutum
  • SSR (Simple Sequence Repeat) marker for simultaneously identifying sterile cytoplasm of gossypium harknessii and gossypium hirsutum

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Using the leaves of cotton sterile lines and maintainer lines containing the male sterile cytoplasm of Harknessy cotton (CMS-D2) and cotton trilobes (CMS-D8) as research materials, 64 pairs of mitochondrial SSR markers (according to ncbi The design of the mitochondrial genome sequence of the Hackney cotton sterile line and maintainer line published in the Internet, the mitochondrial genome sequence number is JX536494.1, JX065074.1) screening, a total of 8 can distinguish the sterile line and the maintainer line Mitochondrial SSR marker, a mitochondrial SSR marker (named mSSR59) that can distinguish CMS-D2 from CMS-D8.

[0027] The PCR product of mSSR59 was purified, connected to the T vector, and sequenced in E. coli to obtain a PCR product of CMS-D2 (D2A-mSSR59) of 127bp, a PCR product of CMS-D8 (D8A-mSSR59) of 132bp, and a product of the maintainer line (B -mSSR59) is 158 bp. The PCR product of CMS-D2 is 5bp less "ATAAT" than the PCR product of CMS-D8, and the two ar...

Embodiment 2

[0031] (1) DNA extraction

[0032] The seeds of the cotton sterile line and maintainer line of Hackney West cotton and three-lobed cotton male sterile cytoplasm, and the cotton sterile line, maintainer line and F 1 Mitochondrial DNA was extracted using the CTAB method.

[0033] (2) PCR amplification

[0034] Using the above-mentioned mSSR59 molecular marker primer pair, the extracted DNA was amplified by Taq (5U / ul, Quanshijin Company) system. The PCR amplification system was as follows: 20μL reaction system, containing 10×Buffer (containing Mg 2+ ) 2 μL, dNTP (10 mM) 0.4 μL, Taq enzyme 0.2 μL, upstream and downstream primers (10 μM) each 0.8 μL, DNA template (300~500ng / μL) 0.5 μL and ddH 2 O15.3 μL.

[0035] PCR amplification program: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 10 s, 34 cycles; extension at 72°C for 5 min, storage at 4°C.

[0036] (3) Polyacrylamide gel electrophoresis

[0037] Aft...

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Abstract

The invention discloses an SSR (Simple Sequence Repeat) marker for simultaneously identifying sterile cytoplasm of gossypium harknessii and gossypium hirsutum. Primers of the SSR marker are shown as SEQ ID NO.1-2, and the SSR marker is not only linked with a gossypium harknessii cytoplasmic male sterility gene, but also linked with a gossypium hirsutum cytoplasmic male sterility gene. The simple repetitive sequence polymorphism of the nucleotide is presented on a polyacrylamide electrophoretogram, whether a single cotton plant contains a sterile gene or not can be identified according to an amplification band, and the type (CMS-D2 / CMS-D8) containing the sterile gene can be judged. By utilizing the same SSR marker, not only can the sterile line and the maintainer line of a cotton three-line material be identified, but also two sets of cytoplasmic male sterility genes of the cotton can be distinguished at the same time, and hybrid seeds matched with different three lines can be identified. The SSR marker plays an important role in breeding new cotton materials with different sterile genes and ensuring the purity of three-line hybrid seeds.

Description

technical field [0001] The invention relates to a molecular marker, in particular to an SSR marker for simultaneously identifying the sterile cytoplasm of Hackneys cotton (CMS-D2) and cotton trifida (CMS-D8), and belongs to the field of biotechnology. Background technique [0002] The utilization of heterosis can significantly increase cotton yield, improve cotton quality and enhance cotton resistance. Studies have shown that cotton hybrids yield about 15 percent more than conventional varieties. Hybrid seed production is an effective way to utilize heterosis in cotton. In production practice, the common methods of hybrid seed production are artificial emasculation and pollination, chemical detasseling and "three-line matching" method. The cost of artificial detasselling and pollination is increasing year by year, and the purity of the hybrids is low, so the quality of the seeds cannot be guaranteed; the chemical detasseling method can reduce the manpower and material input...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/6895C12Q1/686C12Q2600/13C12Q2600/156C12Q2525/151
Inventor 邢朝柱李永旗吴建勇张梦张学贤郭立平戚廷香唐会妮王海林乔秀琴
Owner INST OF COTTON RES CHINESE ACAD OF AGRI SCI
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