CN114672581A - Molecular marker of rice heterologous cytoplasmic fertility restoration QTL qRf5.1 and application thereof - Google Patents
Molecular marker of rice heterologous cytoplasmic fertility restoration QTL qRf5.1 and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of rice breeding and molecular biology, and particularly relates to an SSR molecular marker of a major QTLs locus for regulating and controlling rice heterogenous cytoplasm sterility restoration and application thereof (see figure 1). The sequences of the forward and reverse primers of the marker are 5'-GAGTGGAAATGGCACTTA-3' and 5'-ATTTCACCTCCCTCATCT-3' respectively, PCR amplification is carried out on the genomic DNA of the rice sample by using the primers, and electrophoresis detection is carried out on the amplified product, so that the rice variety with the amplified band size of 202bp has the function of restoring fertility. The invention has important application value for screening and breeding the rice heterogeneous cytoplasm sterile line restorer line.
Description
Technical Field
The invention relates to the technical field of water course breeding, in particular to a molecular marker of rice heterologous cytoplasmic fertility restoration QTL qRf5.1 and application thereof.
Background
The rice is one of the three most important grain crops in the world, and more than 50% of people in the world use rice as staple food, so that the high yield and the stable yield of the rice are closely related to the grain safety and the social stability in the world. Heterosis utilization can effectively improve crop yield, quality and resistance. The rice three-line successful matching greatly promotes the utilization of rice heterosis and the large-scale popularization and application of hybrid rice in China, and the planting area of the hybrid rice accounts for 1/2 (Yuanlongping, etc., 2010) of the rice planting area.
The discovery and utilization of rice cytoplasmic male sterility and restoring genes greatly improve the research of hybrid rice in China. The commercial application of the three-line hybrid rice needs to breed excellent sterile lines and select and breed matched excellent restorer lines. Therefore, in the course of the research on the three-line hybrid rice, the research on the restorer gene has been attracting much attention. With the rapid development of molecular biology technology, the research on the molecular mechanism of cytoplasmic male sterility and fertility restoration of rice is greatly promoted. Since the 70 s of the 20 th century, researchers at home and abroad performed fertility restorer QTL analysis for CMSs of different types, such as wild-type (CMS-WA), Honglian-type (CMS-HL), Baotai-type (CMS-BT) and dwarf-type (CMS-DA), and detected over 75 different cytoplasmic-type fertility restorer QTLs on 12 chromosomes of Rice, in which 7 restorers, such as wild-type restorer Rf4, Honglian-type fertility restorer Rf5 and Rf6, Baotai-type restorer Rf1(Rf1a and Rf1b), Ifr1, Lead-Rice-type restorer Rf2 and CW-type restorer Rf17, were cloned (Seikagaku et al, 2022). For example, Zhuangjiyun et al (2001) mapped wildtype restorer gene qRf1 in the interval RG532-RG472 chromosome 1 with a phenotypic contribution rate of 43.0%; ngangkham et al (2010) F constructed using Pusa6A and PRR78 2The population finely locates wild-abortive fertility restorer QTL Rf4 to the interval RM6737-RM6100, and the physical position is about 104 kb. Most of the reported fertility restorer QTLs/genes of different cytoplasmic sterility types come from cultivated rice and local varieties, and research and positioning research on restorer genes of wild rice is weak.
After the three-line hybrid rice in China is successfully matched, a group of king brand backbone indica type restorers such as IR24, IR26, IR30, Minghui 63, Yanggao No. 6 (9311) and Huazhan are emerged, however, the backbone restorers have single genetic background, most of the family spectrum of the backbone restorers contain genetic components of a farmyard variety Peta in southeast Asia, and belong to indica ecotypes in south Asia and southeast Asia, so that the cultivation of high-yield and high-quality hybrid combinations is greatly limited. Therefore, the genetic diversity of the restorer line needs to be enriched in the breeding of the three-line hybrid rice. The Dongxiang wild rice is the ordinary wild rice (Oryza rufipogon Griff) which is distributed in the north of the world so far, is a specific wild resource in Jiangxi, and researches show that the Dongxiang wild rice has the recovery property on various cytoplasmic male sterility (Zhang jin et al, 2011). The discovery and utilization of cytoplasmic male sterility restoring source from Dongxiang wild rice has profound significance in promoting the development of hybrid rice. The invention locates and verifies the heterogenous cytoplasm fertility restorer gene qRf5.1 of Dongxiang wild rice on the No. 5 chromosome of rice, obtains the molecular marker which is tightly linked with the heterogenous cytoplasm fertility restorer gene qRf5.1 and has important significance for promoting the breeding of the restorer line and the molecular marker-assisted breeding.
Traditional rice breeding is mainly through phenotypic selection, which not only requires researchers to have rich experience, but also has breeding years as long as years of years or even decades. Therefore, by combining the research means and method of molecular biology and genomics, the target gene/QTL for restoring rice fertility is quickly detected and auxiliarily selected, the molecular breeding of the restorer is improved, the breeding process is accelerated, and a new breakthrough is realized. The technical personnel in the field are dedicated to develop a molecular marker which can rapidly detect the rice fertility restorer gene qRf5.1 of the rice material, and further realize the molecular breeding application of the fertility restorer gene qRf5.1.
Disclosure of Invention
The invention aims to solve the technical problem of providing a main QTL for regulating and controlling the fertility restoration of the rice heterogenous cytoplasm and a molecular marker tightly linked with the QTL, which is used for breeding a restorer line variety matched with a heterogenous cytoplasm sterile line and can improve the screening efficiency and the variety breeding process.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: a molecular marker of rice heterologous cytoplasmic fertility restoration QTL qRf5.1 comprises a main QTL for regulating and controlling rice heterologous cytoplasmic fertility restoration, wherein the main QTL is positioned on a No. 05 chromosome of rice and is named as qRf5.1 (shown in figure 1); the genetic distance of the interval is 104.8cM-110.6cM, and the physical distance is 21,378,343-22,671,210 bp;
The SSR primer pair RF01 for detecting the major QTLs with the restoration of the heterogenous cytoplasmic sterility fertility is also included, and the primer pair RF01 has one of the following nucleotide sequences 1) to 3):
1) the nucleotide sequence of the upstream primer in the primer pair RF01 is the nucleotide sequence shown by SEQ ID NO.1 in the sequence table, and the nucleotide sequence of the downstream primer in the primer pair RF01 is the nucleotide sequence shown by SEQ ID NO.2 in the sequence table;
2) a nucleotide sequence which can be hybridized with a DNA sequence limited by SEQ ID NO.1 or SEQ ID NO.2 in a sequence table under high-stringency conditions;
3) the sequence has more than 90 percent of homology with the DNA sequence limited by 1) or 2) and can amplify the sequence of the gene related to the rice fertility restoration;
RF01-F：5'-GAGTGGAAATGGCACTTA-3'(SEQ ID NO.1)
RF01-R：5'-ATTTCACCTCCCTCATCT-3'(SEQ ID NO.2)。
further, the application of the primer pair RF01 for detecting the SSR marker in preparing a kit or a PCR reagent for detecting or auxiliarily selecting the rice fertility restorer gene qRf5.1; the kit or PCR reagents comprise the primer pair RF01 of claim 1.
Further, the kit or PCR reagent also comprises reagents commonly used in PCR technology.
Furthermore, the kit or the PCR reagent is applied to detection of the rice heterologous cytoplasm fertility restorer gene qRf5.1 and/or application in rice restorer line molecular marker-assisted selective breeding.
An auxiliary breeding method for detecting rice fertility restorer molecular markers, which comprises the following steps:
(1) extracting the genome DNA of a rice sample to be detected;
(2) performing PCR amplification on the RF01 by using the genomic DNA extracted in the step (1) as a template and adopting the primer in claim 1 to obtain a PCR amplification product;
(3) and (3) carrying out non-denaturing polyacrylamide gel electrophoresis detection on the PCR amplification product obtained in the step (2) at the concentration of 6%, and if the rice sample to be detected is amplified to form a strip with the size of 202bp, carrying the rice strain of the heterologous fertility restoring gene qRf5.1 or candidate fertility restoring gene rice strains on the rice to be detected.
Further, the reaction system of the PCR amplification in the step (2) is: 2 XTolo FastTaq Premix 5.0. mu.l, 10 pmol/. mu.l of the primer set of claim 1 RF 011. mu.l, 300 ng/. mu.l of the rice genomic template DNA 1. mu.l, and sterilized ultrapure water was added to 10. mu.l.
Further, the reaction procedure of the PCR amplification in the step (2) is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and circulating for 35 times; finally, extending for 10min at 72 ℃; the amplification product was stored at 4 ℃.
Furthermore, the rice carries a fertility restorer gene qRf5.1.
Compared with the prior art, the invention has the advantages that: (1) the molecular marker which is closely linked with the rice fertility restoration is obtained by screening, and a foundation is laid for the qRf5.1 molecular marker-assisted selective breeding. The genomic DNA of the rice to be detected is used as a template, the primer pair disclosed by the invention is used for amplification, and if the size of an obtained amplification product is 202bp, the rice to be detected is judged to have a fertility restorer gene.
(2) The molecular marker disclosed by the invention is high in accuracy and good in stability. Because the marker is closely linked with the rice fertility restorer gene qRf5.1, the marker can be used for quickly, conveniently and accurately selecting the single plant carrying the target gene in the rice seedling stage, the labor cost of the test cross work is reduced, the number of the sterile line test cross single plants is reduced, the sterile line test and matching work is arranged in advance before the rice heading and flowering, and the breeding work process is accelerated.
(3) The molecular marker provided by the invention can be widely applied to molecular detection of rice fertility restorer genes in molecular assisted breeding (the marker is positioned between 21,378,325-21,378,343 bp of No. 5 chromosome of a rice reference genome), is a codominant marker, can detect homozygous and heterozygous individuals, shortens the breeding period, and improves the breeding efficiency and progress.
Drawings
FIG. 1 shows the chromosome position of major QTLs qRf5.1 for rice heterologous cytoplasmic fertility restoration and its linked marker RF 01;
FIG. 2 shows that the molecular marker RF01 is used to detect the Bc of the Junqingzao B/Dongxiang wild rice BC in 2019 in the embodiment of the present invention4F5The genotype of the population;
FIG. 3 shows the detection of Coqing early B/Dongxiang wild rice BC in 2020 by using molecular marker RF01 in the example of the present invention4F6The genotype of the population;
FIG. 4 is a cross-testing anther microscopic examination of east 2021B 11A and Xieqingzao B and backcross populations in an example of the present invention;
FIG. 5 and FIG. 6 show the grass A/(Xiqingzao B/Dongxiang wild rice BC in 2 sets of test cross F1 population in the example of the present invention4F5) And Dong B11A/(Xieqingzao B/Dongxiang wild rice BC)4F6) And (5) a firmness rate distribution graph.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention only, but not to limit the scope of the invention.
Example 1 construction and development of the introgression line of Xiqingzao B/Dongxiang wild rice
Dongxiang wild rice has restorer properties to various cytoplasmic male sterility. The method takes Dongxiang wild rice as a donor parent and further takes a dwarf male sterile maintainer line Xiqingzao B as a recurrent parent for hybridization, backcross and selfing to obtain 1 set of BC 1F10The Xieqingzao B// Dongxiang wild rice/Xieqingzao B backcross recombination inbred population. Detecting fertility restoration QTL qRf5.1 on the 5 th chromosome of rice by using the group of the group, selecting a strain (strain number 5339) carrying a target QTL qRf5.1 to backcross with the Xiqingzao B, combining molecular marker assistance to perform foreground selection and background selection, and respectively constructing 2 sets of BC by adopting a single seed transmission method4F5And BC4F6The generation-assisted early green B/Dongxiang wild rice introgression line population.
All the materials are planted in the Nanchang experimental base of the institute of Paddy, agricultural sciences, Jiangxi province in 2019 and 2020 respectively, sown in late 5 th ten days, transplanted in middle 6 th ten days, and the Xiqingzao B/Dongxiang wild rice BC is selected 15 days after transplantation4F5And BC4F6Extracting DNA from leaves of individual plant of colony, detecting genotype, hybridizing with sterile lines of different cytoplasm types such as Heza sativa A and Dong wild sterile line Dong B11A in the field-abortive sterile line in heading stage (selecting homozygous individual plant of parent), harvesting F0Hybrid and planting test cross F in 2020 and 2021 years respectively1And (4) inspecting the natural fruit setting rate of the population.
Example 2, BC4F5And BC4F6Generation-assisted early-green B/Dongxiang wild rice test crossing population maturing rate investigation
2020. In 2021, test cross population was planted in Nanchang experimental base of Rice institute of agricultural sciences, Jiangxi province, and random block design was carried out for 3 times, 32 plants were planted in each block, transplanting specification was 16.5cm × 20cm, and water and fertilizer management was carried out conventionally. And in the mature period, 5 plants in the middle of the strain are taken every time each test cross material is repeated, indoor natural air drying is carried out, indoor artificial seed test is carried out, and the rice maturing rate and other related traits are inspected.
Example 3 location and verification of QTL qRf5.1 for fertility restoration of heterologous cytoplasmic sterile line
192 BC's are paired using Mapmaker 3.0 software4F5Genetic distance of the Xieqing early B/Dongxiang wild rice population, converting recombination rate into genetic distance by using Kosambi function, and constructing BC4F5Linkage genetic map of the population. Adopting software Windows QTL Cartograter 2.5 to BC4F5QTL analysis is carried out on genotype data of the Xieqing Zao B/Dongxiang wild rice population and the setting-rate table data of the tested cross population. A Composite Interval Mapping (CIM) method is adopted, a LOD value of 2.5 is used as a detection threshold value to judge whether a QTL exists or not, and finally a fertility restoration main effect QTL is positioned between RF01-RM188 on a chromosome 5, the genetic distance of the QTL is 104.8-110.6 cM, the physical distance of the QTL is 21,378,343-22,671,210 bp, and the QTL is named as qRf 5.1.
Example 4 application of major QTL qRf5.1 linkage marker for rice heterologous cytoplasmic fertility restoration
Planting Zhonghe A/(Xiqingzao B/Dongxiang wild rice BC) in experiment base of agricultural academy of sciences of Jiangxi province in 2020 and 2021 respectively4F5) And Dong B11A/(Xieqingzao B/Dongxiang wild rice BC)4F2) Test cross F1 population. In the flowering period of the ear-drawing poplar, young ears of F1 single plants which are subjected to test cross of the synechocystis sinensis B type, the eastern wild type and the synechocystis sinensis B and sterile line east B11A are selected according to genotypes, after I2-KI solution is used for dyeing, 1 visual field is randomly selected under a 10-fold microscope to observe pollen fertility of the F1 single plants which are subjected to test cross of the synechocystis sinensis B type, the synechocystis sinensis B homozygote type (single plant number 103) and the Dongxiang wild rice homozygote type (single plant number 2) and the Dong B11A (specific results are shown in figure 4), and the pollen fertility rates are 0.0%, 13.45% and 78.83% respectively.
Setting a molecular marker RF01 at the downstream of the QTL locus qRf5.1,
the primer pair of the molecular marker RF01 is as follows:
the upstream primer GAGTGGAAATGGCACTTA is used to make the primer,
a downstream primer ATTTCACCTCCCTCATCT;
taking parent Xiqingzao B, Dongxiang wild rice and introgression line group BC thereof4F5And BC4F6The rice leaf of (1) is subjected to the following operations:
extracting genome DNA, and carrying out PCR amplification on the genome DNA by using the molecular marker, wherein a PCR reaction system comprises: 2 XTolo FastTaq Premix 5.0. mu.l, 10 pmol/. mu.l of the primer set described in claim 1. mu.l, 300. mu.l of 500 ng/. mu.l of the rice genomic template DNA, and sterilized ultrapure water was added to 10. mu.l. Pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and circulating for 35 times; finally, extension is carried out for 10min at 72 ℃. PCR amplification products were detected by 6% polypropylene gel electrophoresis. The presence of the corresponding marker was determined by banding pattern analysis, indicating that the line does not carry the heterologous cytoplasmic restorer qRf5.1 if the band is trending towards the parent synephrine promo B and indicating that it carries the heterologous cytoplasmic restorer qRf5.1 if it is trending towards Dongxiang wild rice (FIG. 2, FIG. 3).
The method comprises the following specific steps:
firstly, when the primer pair of the molecular marker RF01 is subjected to PCR amplification, as shown in FIG. 2 and FIG. 3, the band of the Xiqingzao B is the lower band; the strip of the Dongxiang wild rice is an upper strip;
Secondly, selecting a introgression line group BC4F5And BC4F6The middle RF01 marker strip type is a Xieqing early B type/east wild type homozygous single plant, and is subjected to test crossing with the sterile line middle standing grain A and the sterile line east B11A respectively;
thirdly, comparing the maturing rate of the tested strain with a prediction result, and respectively keeping the test crossing maturing rates of the Xiqingzao B of the line and the Heza A and Dong B11A in the sterile line to be 21.3 percent and 13.5 percent; the single plant test cross seed setting rate without RF01 marker is mostly 10-30%, and the single plant test cross F1 carrying RF01 marker has seed setting rate over 50%.
Fourth, the results show that the predicted results match the actual detection results (fig. 5 and 6).
In conclusion, the regulation and control of the major QTL site for the rice heterologous cytoplasm sterility restoration can effectively accelerate the rice restorer line variety breeding process optimization. The rice restorer line for restoring the heterology cytoplasm fertility of the rice can be cultivated in the process of rice molecular assisted breeding. The method is simple, convenient, feasible, safe and effective, and is suitable for large-scale popularization and application.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
The present invention and the embodiments thereof have been described above, and the description is not intended to be limiting, and the embodiments shown in the drawings are only a part of the embodiments of the present invention, not all of the embodiments, and the actual configuration is not limited thereto. In summary, those skilled in the art should be able to conceive of the present invention without creative design of the similar structural modes and embodiments without departing from the spirit of the present invention, and all such modifications should fall within the protection scope of the present invention.
Claims (8)
1. A molecular marker of rice heterologous cytoplasmic fertility restoration QTL qRf5.1 is characterized by comprising a main QTL for regulating and controlling rice heterologous cytoplasmic fertility restoration, wherein the main QTL is positioned on a No. 05 chromosome of rice and is named as qRf 5.1; the genetic distance between the sections is 104.8-110.6 cM, and the physical distance is 21,378,343-22,671,210 bp;
the SSR primer pair RF01 for detecting the major QTLs with the restoration of the heterogenous cytoplasmic sterility fertility is also included, and the primer pair RF01 has one of the following nucleotide sequences 1) to 3):
1) the nucleotide sequence of the upstream primer in the primer pair RF01 is the nucleotide sequence shown by SEQ ID NO.1 in the sequence table, and the nucleotide sequence of the downstream primer in the primer pair RF01 is the nucleotide sequence shown by SEQ ID NO.2 in the sequence table;
2) A nucleotide sequence which can be hybridized with a DNA sequence limited by SEQ ID NO.1 or SEQ ID NO.2 in a sequence table under high-stringency conditions;
3) a sequence which has more than 90 percent of homology with the DNA sequence limited by 1) or 2) and can amplify genes related to fertility restoration of paddy rice;
RF01-F：5'-GAGTGGAAATGGCACTTA-3'(SEQ ID NO.1)
RF01-R：5'-ATTTCACCTCCCTCATCT-3'(SEQ ID NO.2)。
2. the molecular marker of the rice heterologous cytoplasmic fertility restorer QTL qRf5.1 in accordance with claim 1, wherein the primer pair RF01 for detecting the SSR molecular marker is applied to the preparation of a kit or a PCR reagent for detecting or assisting in the selection of the rice fertility restorer gene qRf 5.1; the kit or PCR reagents comprise the primer pair RF01 of claim 1.
3. The molecular marker of the rice heterologous cytoplasmic fertility restoration QTL qrf5.1 as claimed in claim 2, wherein the kit or the PCR reagents further comprise reagents commonly used in PCR technology.
4. The molecular marker of the rice heterologous cytoplasmic fertility restorer QTL qRf5.1 of claim 3, wherein the kit or the PCR reagent is applied to detection of the rice heterologous cytoplasmic fertility restorer gene qRf5.1 and/or rice restorer molecular marker assisted selective breeding.
5. A molecular marker assisted breeding method for detecting rice fertility restoration is characterized by comprising the following steps:
(1) Extracting the genome DNA of a rice sample to be detected;
(2) performing PCR amplification on RF01 by using the genomic DNA extracted in the step (1) as a template and adopting the primer in claim 1 to obtain a PCR amplification product;
(3) and (3) carrying out non-denaturing polyacrylamide gel electrophoresis detection on the PCR amplification product obtained in the step (2) at the concentration of 6%, and if the rice sample to be detected is amplified to form a strip with the size of 202bp, carrying the rice strain of the heterologous fertility restoring gene qRf5.1 or candidate fertility restoring gene rice strains on the rice to be detected.
6. The method for detecting molecular markers for rice fertility restoration according to claim 5, wherein the reaction system for PCR amplification in step (2) is: 2 XTolo FastTaq Premix 5.0. mu.l, 10 pmol/. mu.l of the primer set RF 011. mu.l, 300 and 500 ng/. mu.l of the rice genomic template DNA 1. mu.l, and sterilized ultrapure water was added to 10. mu.l.
7. The rice fertility restorer molecular marker auxiliary breeding method according to claim 5, wherein the PCR amplification reaction program in the step (2) is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 45s, and circulating for 35 times; finally, extending for 10min at 72 ℃; the amplification product was stored at 4 ℃.
8. The molecular marker assisted breeding method for detecting rice fertility restoration according to any one of claims 5 to 7, wherein the rice carries a fertility restoration gene qRf5.1.
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| CN105483217A (en) * | 2015-12-08 | 2016-04-13 | 江西省农业科学院水稻研究所 | Molecular marker method for identifying paddy rice Dongxiang wild cytoplasmic male sterility sources |
| CN108866232A (en) * | 2018-08-15 | 2018-11-23 | 江西省农业科学院水稻研究所 | Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)11 of Dongfu-type rice, application and detection method |
| CN108950050A (en) * | 2018-08-15 | 2018-12-07 | 江西省农业科学院水稻研究所 | Detection primer and kit for Dongfu type rice cytoplasmic male sterility restoring gene Rf (DW)10, application and detection method |
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| CN105483217A (en) * | 2015-12-08 | 2016-04-13 | 江西省农业科学院水稻研究所 | Molecular marker method for identifying paddy rice Dongxiang wild cytoplasmic male sterility sources |
| CN108866232A (en) * | 2018-08-15 | 2018-11-23 | 江西省农业科学院水稻研究所 | Detection primer and kit for cytoplasmic male sterility restoring gene Rf (DW)11 of Dongfu-type rice, application and detection method |
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