CN114427006A - Primer and method for molecular marker of photo-thermo-sensitive male sterile gene pms3 of rice - Google Patents
Primer and method for molecular marker of photo-thermo-sensitive male sterile gene pms3 of rice Download PDFInfo
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Abstract
The invention discloses a primer and a method for molecular marking of a photo-thermo sensitive male sterile gene pms3 of rice, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3. The molecular marker for the rice photo-thermo-sensitive male sterile gene pms3 can play a role in genotyping only by one-time conventional PCR and agarose gel electrophoresis technology, in addition, the primer can only amplify a target strip, the amplification result is clear at a glance, the recording of the result is convenient, in addition, in the whole operation process, the operation steps are simple, the operation time is short, the cost of reagents and consumables required by the experiment is low, the experiment process is accelerated, the experiment cost is saved, and the purpose of rapid, convenient and reliable molecular marker-assisted breeding is achieved.
Description
Technical Field
The invention relates to a molecular marker of a photo-thermo-sensitive male sterile gene of rice, in particular to a primer and a method for molecular marker of the photo-thermo-sensitive male sterile gene pms3 of rice.
Background
In order to improve the yield per unit of rice, the development of hybrid rice in China currently goes through two important stages, namely a three-line method mainly based on cytoplasmic male sterile line inheritance to a two-line method mainly based on photo-thermo-sensitive male sterile line inheritance. The three-line method is easily restricted by a restorer line and a maintainer line, has the problems of long breeding period and the like, and is not beneficial to seed production of rice. In recent years, two-line hybrid rice has been favored because of its advantages such as dual-purpose use and free combination, and has occupied an important position in the cultivation and popularization of hybrid rice.
With the deep research of a breeder on the molecular mechanism of the photo-thermo-sensitive genic male sterile line, genes of a large quantity of two-line sterile lines are gradually positioned and cloned, such as pms3, pms1, tms5 and the like, and a foundation is better laid for molecular marker assisted breeding.
The Pms3 gene is derived from rice farming 58S, transcribes a 1236bp long-chain non-coding RNA (lncRNA) -LDMAR, under the long-day condition, the sufficient transcription amount of the LDMAR can meet the normal development of pollen, but because a base difference from C to G exists on a 789 th site, the secondary structure of the LDMAR is changed, the transcription amount of the LDMAR is reduced, and finally, the pollen is sterile.
"the effect of the rice photo-thermo-sensitive genic male sterile gene tms5 and pms3 is initially detected" (proceedings of academic seminar in 2019 of genetics institute of Jiangsu province), according to SNP variation on pms3 transcript, a restriction enzyme site is arranged on the mutation site and can be recognized and cut by Csp6I, and then corresponding primers are synthesized for PCR amplification and restriction enzyme. The technical result can not be obtained in one step, an accurate result is obtained by enzyme digestion, the operation is complicated, the experiment cost is relatively high, the requirement on the quality of DNA recovered from a PCR product is high, and the large-batch detection takes long time and is not beneficial to the experiment process.
Disclosure of Invention
The invention aims to provide a primer and a method for molecular marking of a rice photo-thermo-sensitive male sterile gene pms3, which can play a role in genotyping only by one-time conventional PCR and agarose gel electrophoresis technology, can only amplify a target strip, and can achieve the aim of rapid, convenient and reliable molecular marking-assisted breeding.
In order to achieve the aim, the invention provides a primer for rice photo-thermo-sensitive male sterile gene pms3 molecular marker, wherein the nucleotide sequence of the primer is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
Another objective of the invention is to provide a kit for rice photo-thermo-sensitive male sterile gene pms3 molecular marker, wherein the primers in the kit are the primers for rice photo-thermo-sensitive male sterile gene pms3 molecular marker as claimed in claim 1.
Another purpose of the invention is to provide an application of the primer for molecular marking of the rice photo-thermo-sensitive male sterile gene pms3, wherein the application is any one of the following:
(1) the method is used for identifying or screening the rice with the rice photo-thermo-sensitive male sterile gene pms 3;
(2) is used for rice molecular marker assisted breeding.
Another object of the present invention is to provide a method for identifying or screening rice having the rice photo-thermo-sensitive male-sterility gene pms3, which comprises: taking the genome of rice to be detected as a template, and respectively carrying out PCR amplification by adopting a primer group with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and a primer group with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO. 3; if only the PCR product amplified by the primer group with the nucleotide sequence shown as SEQ ID NO.1 and SEQ ID NO.2 has a strip at 280bp, the rice to be detected is pms3 homozygous genotype; if only the PCR product amplified by the primer group with the nucleotide sequence shown in SEQ ID NO.2 and SEQ ID NO.3 has a band at 281bp, the rice to be detected is the PMS3 homozygous genotype; if the PCR product amplified by the primer group with the nucleotide sequence shown as SEQ ID NO.1 and SEQ ID NO.2 has a strip at 280bp, and the PCR product amplified by the primer group with the nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO.3 has a strip at 281bp, the rice to be detected is the heterozygous genotype.
The primer and the method for molecular marking of the photo-thermo sensitive male sterile gene pms3 of the rice have the following advantages:
(1) the molecular marker for the rice photo-thermo-sensitive male sterile gene pms3 can play a role in genotyping only by one-time conventional PCR and agarose gel electrophoresis technology, in addition, the primer can only amplify a target strip, the amplification result is clear at a glance, the recording of the result is convenient, in addition, in the whole operation process, the operation steps are simple, the operation time is short, the cost of reagents and consumables required by the experiment is low, the experiment process is accelerated, the experiment cost is saved, and the purpose of rapid, convenient and reliable molecular marker-assisted breeding is achieved;
(2) the invention relates to a primer for rice photo-thermo-sensitive male sterile gene PMS3 molecular marker, which is designed according to the principle of four-primer amplification hindered mutation system PCR technology, wherein the primer is formed by combining an inner primer and an outer primer in pairs to form a two-primer molecular marker combination, mismatch is added at the 3' end of the inner primer to enhance the specificity of the primer, 10 primer markers are developed according to the difference of bases on the 789 th site of PMS3, 8 primer combinations are arranged, and the molecular marker of AS-PMS3 is obtained after optimized screening.
Drawings
FIG. 1 is a diagram of the sequence differences between allelic PMS3 and PMS 3.
FIG. 2 is a schematic diagram of the design of primers P1/P3 and P4/P4 according to the present invention.
FIG. 3 shows the result of the amplification of 33 typical photo-thermo-sensitive genic male sterile materials of the present invention with XP and DP primers.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 molecular marker of photo-thermo sensitive male sterility gene pms3 of Rice
(1) DNA extraction of rice material
A representative 33 parts of photo-thermo-sensitive nuclear sterile line materials are selected for testing rice materials, N5088S, long grain round-grained non-glutinous rice S (Fangguan), N95076S, NC228S, agricultural reclamation 58S, N202S, N550S, Shanghai-Han 2S, N74S, 1892S, Xi08S, 7-163S, Shen 08S, Tai 1S, strain 1S, crystal 4155S, Longke 638S, Guanxiang 24S, Y58S, Guangzhan 63-4S, Lu 18S, E, nong 1S, EK2S, Huangxiang S, Hua6421S, Hua 448S, Hua 5113S, Rui 18S, N252S, Zhi 08S, N25S and N418S, wherein the materials comprise 9 round-grained non-glutinous rice materials and 24 non glutinous rice materials. All the above materials were subjected to CTAB extraction of total DNA from leaves, and used for genotyping pms 3.
(2) Design of primers
According to the published gene sequences (NCBI accession numbers JQ317785.1 and JQ317784.1) of rice photosensitive genic male sterile genes PMS3 and PMS3 on NCBI, primers (see table 1) are designed according to the principle of PCR technology of a four-primer amplification hindered mutation system, each primer is formed by combining an inner primer and an outer primer in pairs to form a two-primer molecular marker combination, and mismatch is added to the 3' end of the inner primer, so that the specificity of the primers is enhanced.
10 primer markers were developed based on the difference in bases at position 789 in pms3 (FIG. 1), and 8 primer combinations were set, P1/P3, P2/P3, P4/P3, P5/P3, P8/P6, P8/P7, P8/P9, and P8/P10, respectively. As shown in FIG. 2, the two primer combinations of P1/P3 and P4/P3 are taken as an example, and the other primer combinations are designed according to the same principle as P1/P3 and P4/P3. Then, after the optimization screening, the PMS3 homozygous genotype (PMS3PMS3, PP) can only be effectively amplified by the P4/P3 primer combination, the length of the amplified product is 281bp, and the primer combination is named as DP; the pms3 homozygous genotype (pms3pms3, pp) was only amplified efficiently by the P1/P3 primer combination, which had a theoretical product length of 280bp and was named XP. The primer combination DP and XP was therefore named AS-PMS3 (see Table 1).
Table 1 shows primers AS-PMS3 designed for molecular markers of genes PMS3 and PMS3
Note: bases marked with bold lowercase letters indicate mismatched bases, and bases marked with underlined and bold letters indicate bases at functional mutation sites.
(3) PCR amplification experiment
The genomic DNA of rice was extracted from the above-mentioned material by the CTAB method and used as a template.
The PCR reaction system comprises: DNA 2. mu.L, 2 XEs Taq MasterMix (Dye) 10. mu.L, and each of the upstream and downstream primers 0.5. mu.L, and make up to 20. mu.L with sterilized water.
The amplification procedure for PCR was as follows: pre-denaturation at 94 deg.C for 5 min; denaturation at 94 ℃ for 30s, annealing at 52-54 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extending for 10min at 72 ℃; the temperature was kept at 25 ℃ for 30 s.
The PCR products were electrophoresed 180V on a 2% agarose gel containing a nucleic acid dye for 15min and then recorded by photography using a gel imaging system.
(4) PCR amplification results
33 typical photo-thermo-sensitive nuclear sterile materials were amplified with XP and DP primer combinations, see FIG. 3, where M in FIG. 3 is DL500 marker, lanes 1-33 are 1892S, xi 08S, 7-163S, Shen 08S, Rui 18S, Tai 1S, N5088S, N252S, Shanghai Han 2S, N74S, Long grain nong rice (Fangguan), S284, N95076S, NC228S, Strain 1S, Crystal 4155S, Longke 638S, Guangxiang 24S, Y58S, Guangxian 63-4S, Lu 18S, Zhonghua 08S, Nongnao 58S, N202S, N418S, N25S, N550S, E Non 1S, EK2S, Huangxiang S, Hua6421S, Huahua and Huahua 5113S, respectively.
The amplification results were as follows:
N5088S, N252S, Shanghania drought 2S, N74 3874 74S, S284, N95076S, NC228S, Guangxiang 24S, Lu 18S, Nongsu 58S, N202S, N550S and Hua6421S, which are 13 materials, can be amplified by XP to form bright and clear bands, wherein the length of the amplified product is 280bp, and the amplified product is pms3 homozygous genotype.
And the DP can amplify 22 materials of 1892S, xi 08S, 7-163S, deep 08S, Rui 18S, Tai 1S, Long grain Japonica S (Fangguan), strain 1S, Crystal 4155S, Longke 638S, Y58S, Guangzhan 63-4S, Lu 18S, Ji 08S, N418S, N25S, E nong 1S, EK2S, Huangxiangzhan S, Hua6421S, Hua448S and Hua5113S into clear bands, the length of the amplified product is 281bp, and the amplified product is a PMS3 homozygous genotype.
In addition, the land 18S and Hua6421S can be effectively amplified by P1/P3 and P4/P3, and belong to a heterozygous genotype.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
<110> institute of food crops of academy of agricultural sciences of Hubei province
<120> primer and method for molecular marker of photo-thermo-sensitive male sterile gene pms3 of rice
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Claims (4)
1. A primer for molecular marking of a photo-thermo sensitive male sterile gene pms3 of rice is characterized in that the nucleotide sequence of the primer is shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
2. A kit for rice photo-thermo-sensitive male sterile gene pms3 molecular marker, wherein the primer in the kit is the primer for rice photo-thermo-sensitive male sterile gene pms3 molecular marker as claimed in claim 1.
3. The application of the primer for rice photo-thermo-sensitive male sterility gene pms3 molecular marker according to claim 1, which is any one of the following:
(1) used for identifying or screening rice with rice photo-thermo sensitive male sterility gene pms 3;
(2) is used for rice molecular marker assisted breeding.
4. A method for identifying or screening rice having a rice photo-thermo-sensitive male sterility gene pms3, comprising:
taking the genome of rice to be detected as a template, and respectively carrying out PCR amplification by adopting a primer group with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 and a primer group with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO. 3;
if only the PCR product amplified by the primer group with the nucleotide sequence shown as SEQ ID NO.1 and SEQ ID NO.2 has a strip at 280bp, the rice to be detected is pms3 homozygous genotype;
if only the PCR product amplified by the primer group with the nucleotide sequence shown in SEQ ID NO.2 and SEQ ID NO.3 has a band at 281bp, the rice to be detected is the PMS3 homozygous genotype;
if the PCR product amplified by the primer group with the nucleotide sequence shown as SEQ ID NO.1 and SEQ ID NO.2 has a strip at 280bp, and the PCR product amplified by the primer group with the nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO.3 has a strip at 281bp, the rice to be detected is the heterozygous genotype.
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Citations (6)
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|---|---|---|---|---|
| WO2013023623A1 (en) * | 2011-08-18 | 2013-02-21 | Huazhong Agricultural University | Isolation, cloning and application of pms3, a gene for photoperiod-sensitive genic male-sterility in rice |
| CN103789419A (en) * | 2014-01-13 | 2014-05-14 | 浙江省嘉兴市农业科学研究院（所） | Co-dominance tag primer group for identifying allele type of rice photo-thermo-sensitive genic male-sterile gene p/tms12-1, and applications thereof |
| CN104531693A (en) * | 2014-12-31 | 2015-04-22 | 广西壮族自治区农业科学院水稻研究所 | Specificity functional marker for rice sterility gene pms3 and application of specificity functional marker |
| CN106676179A (en) * | 2017-01-19 | 2017-05-17 | 海南波莲水稻基因科技有限公司 | Molecular marker for paddy recessive genic male sterility gene cyp704b2 and application thereof |
| CN108048598A (en) * | 2018-01-25 | 2018-05-18 | 华智水稻生物技术有限公司 | For detecting the SNP marker of rice sterile gene pms3 |
| CN111926102A (en) * | 2020-08-25 | 2020-11-13 | 海南波莲水稻基因科技有限公司 | Molecular marker of rice photo-thermo-sensitive male sterility gene pms3 and application thereof |
-
2022
- 2022-02-24 CN CN202210177997.2A patent/CN114427006A/en active Pending
Patent Citations (6)
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| WO2013023623A1 (en) * | 2011-08-18 | 2013-02-21 | Huazhong Agricultural University | Isolation, cloning and application of pms3, a gene for photoperiod-sensitive genic male-sterility in rice |
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| CN111926102A (en) * | 2020-08-25 | 2020-11-13 | 海南波莲水稻基因科技有限公司 | Molecular marker of rice photo-thermo-sensitive male sterility gene pms3 and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| YONGBIN QI等: "Development and validation of a functional co-dominant SNP marker for the photoperiod thermo-sensitive genic male sterility pms3 (p/tms12-1) gene in rice", 《BREEDING SCIENCE》 * |
| 李香花等: "水稻光敏核不育基因pms3的精细定位", 《作物学报》 * |
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