CN102605074A - Molecular marker method for identifying BT type cytoplasmmale sterility restoring geneRfla of japonica rice - Google Patents

Abstract

The invention relates to a molecular marker method for identifying BT type cytoplasmmale sterility restoring gene Rfla of japonica rice, which belongs to the technical field of bioengineering. The molecular marker method includes: synthetizing a positive primer and a negative primer, amplifying different rice plant DNA (deoxyribonucleic acid), being a homozygote carrying restoring genes Rfla if a 1145 bp characteristic strip exists; being a homozygote without restoring genes Rfla if a 571 bp characteristic strip exists, and being a heterozygote carrying restoring genes Rfla if the 1145 bp characteristic strip and the 571 bp characteristic strip exist simultaneously. The molecular marker method is capable of identifying whether rice germplasm resources or other breeding colonies carry the BT type cytoplasm male sterility restoring gene Rfla of japonica rice or not quickly and accurately, and simultaneously further identifying the homozygotes and the heterozygotes carrying genes Rfla and predicting offspring gene types, and selecting efficiency of BT type japonica rice restorer is improved greatly and breeding process is accelerated.

Description

A kind of japonica rice BT type cytoplasmic male sterility of differentiating is recovered the molecule marking method of gene Rf1a

One, technical field

The present invention relates to a kind of discriminating japonica rice BT type cytoplasmic male sterility and recover gene Rf1aMolecule marking method, belong to biotechnology engineering field, be exclusively used in and contain Rf1aIt is that germ plasm resource is identified and breed breeding that gene BT type japonica rice recovers.

Two, background technology

In recent years, the Along with people's growth in the living standard, China urban and rural residents to rice consumption appear the trend that changes to polished rice by long-grained nonglutinous rice (open high and steep etc., agricultural prospect, 2007,1:9-14; Wu Le etc., agricultural techno-economy, 2011,5:87-96).Polished rice market is good, has also expanded more wide space for the production of japonica rice.At present, China japonica rice cultivated area is at 8,300,000 hm ²About, be main wherein with the conventional japonica rice kind, have only 330,000 hm and have than the Japonica Hybrid cultivated area of big yield potential ², proportion less than 4% (Deng Huafeng etc., hybrid rice, 2006,21 (1): 1-6).Therefore; Strengthen the seed selection of Japonica Hybrid (particularly triple crossing japonica rice) kind,, realize the great-leap-forward development of Japonica Hybrid to increase the competitive edge of Japonica Hybrid; To ensureing China's grain security, promote growth of agricultural efficiency, increasing peasant income to have crucial meaning.

The cultivation of excellent parent is the basis of apolegamy triple crossing japonica rice advantage combination.The male sterile cytoplasm of China's Japonica Hybrid application at present mainly is that the BT type is (from India spring Xian kind; Chinsurah Boro II), secondly be Yunnan I type (from the high height above sea level long-grained nonglutinous rice in Yunnan) (Yuan Longping etc., breeding of hybridized rice cultivation; 1988,83-84; Li Zhengyou, Yunnan type breeding of hybridized rice, 2000,1-15).These two kinds of sterile cytoplasms all belong to the sterile type of gametophyte, have similar extensive guarantor's relation, and its fertility restorer receives 1 pair of nuclear gene control (He Hechu, Yunnan Prov Agriculture University's journal, 1988,3 (1): 54-68).In breeding, the sterile line of Japonica Hybrid be mostly by produce blooming of promoting, different friendship habit preferably conventional japonica rice form through backcross transformation, itself just has economical character preferably; And recover be aspect; Owing to generally do not have corresponding recovery source in the japonica rice; It recovers gene is that method through artificial hybridization imports from long-grained nonglutinous rice, recovers inevitably also to have infiltrated some disadvantageous genetic constitution (Wang Cailin etc., Scientia Agricultura Sinicas in the long-grained nonglutinous rice in the gene introducing; 1989,22 (5): 8-13).Therefore, the good japonica rice of the seed selection key of recovering system just is to identify fast and accurately and select recovering gene.

The evaluation that early stage BT type japonica rice recovers gene mainly relies on test cross, and the solid situation of offspring plant is investigated, and this method not only needs two seasons of growth, also will hybridize a large amount of individual plants simultaneously, and its process is quite loaded down with trivial details.For simplifying the procedure of breeding, just came the seed selection homogeneity extensive afterwards, but must cause making up heterotic reduction (Li Jianhong etc., Acta Agronomica Sinica, 2005,31 (7): 851-857) owing to the plasma inheritance background is identical by means of sterile cytoplasm.Utilize and recover the gene close linkage or be total to isolating molecule marker then can overcome above-mentioned shortcoming, effectively reducing recovery is the time and intensity of seed selection, improves the foresight of breeding work greatly.

Recover gene for improving BT type cytoplasmic male sterility Rf-1Efficiency of selection, existing investigator utilizes its chain molecule marker to attempt assistant breeding (Akagi Et al., Genome, 1996,39 (6): 1205-1209; Ichikawa Et al., Molecular Breeding, 1997,3 (3): 195-202; Komori Et al., Euphytica, 2003,129 (2): 241-247; Akagi Et al., Theor. Appl. Genet., 2004,108 (8): 1449-1457; Tan Et al., Plant breeding, 2004,123 (4): 338-341; Cheng Baoshan etc., Agricultural University Of Nanjing's journal, 2011,34 (1): 1-7).This has improved the safety of selecting really to a certain extent.Yet,, therefore have many defectives because these marks are mostly just chain with goal gene.On the one hand, these marks all are through the location Rf-1Obtain during gene, in actual breeding population, often lack polymorphum, on the other hand, owing to be linked marker, it is right to guarantee Rf-1The screening of gene reaches 100% accuracy.

Along with the development of modern molecular biology, people begin that the genetic mechanism of rice fertility restorer has been had new understanding.Wang Et al.( The Plant cell, 2006,18 (3): 676-687) utilize the method for map based cloning to resolve BT type cytoplasmic male sterility recovery gene Rf-1Fertility-restoration Mechanism, this gene is by two relevant fertility restore genes Rf1aWith Rf1bForm, the directed mitochondrial PPR albumen of encoding, wherein Rf1aEncoded protein RF1A cuts off with internal-cutting way B-atp6/orf79MRNA stops the proteic generation of ORF79 to make fertility restorer, and Rf1bEncoded protein RF1B is then through degraded B-atp6/orf79MRNA makes fertility restorer, and when RF1A and RF1B existed simultaneously, RF1A had preferential interaction.With the near isogenic line that can recover cytoplasmic male sterilty ( Rf-1Rf-1) compare, can not recover cytoplasmic male sterilty near isogenic line ( Rf-1rf-1) Rf1aThere are 1 bp and 574 bp, two places disappearance on the site.

Therefore; If recover in BT type cytoplasmic male sterility on the basis of gene clone; Can further design obtain with target gene altogether isolating PCR molecule marker come to identify fast and accurately that japonica rice BT type cytoplasmic male sterility recovers the different genotype of gene and will help the seed selection that good japonica rice recovers system, thereby promote the state of the art of triple crossing japonica rice breeding.

Three, summary of the invention

Technical problem:The present invention is directed to the recovery of japonica rice BT type cytoplasmic male sterility is to recover the comparatively loaded down with trivial details technical barrier of gene identification in the seed selection process, exists according to recovering system and sterile line (with the dyskaryosis maintenance line) Rf1aThe base difference of 574 bp that the site exists designs, synthesizes and goal gene is total to isolating functional label, and through simple detection method, Rapid identification contains Rf1aIt is germ plasm resource that the japonica rice of gene recovers, and can also further be applied to molecular mark simultaneously.

Technical scheme:

1, a kind of evaluation japonica rice BT type cytoplasmic male sterility is recovered gene Rf1aMolecule marking method, it is characterized in that:

Use Rf1aRecover gene-specific primer InDel- Rf1a

Forward primer InDel- Rf1a-F sequence is 5'-CTGATGATCGAGGAGGAGGTA-3'

Reverse primer InDel- Rf1a-R sequence is 5'-TAACGCGTCTTCCATCCTACT-3'

Utilize above-mentioned primer to distinguish japonica rice BT type cytoplasmic male sterility fast and recover gene Rf1aThe PCR molecule marking method of different genotype is:

(1) extraction of rice plant genomic dna (SDS method), with reference to Dellaporta S L, Et al., Plant Mol B iol Rep, 1983,1 (1): 19221.

(2) with described two molecule marker primer I nDel- Rf1a-F and InDel- Rf1a-R adds the PCR reaction system, and the DNA of rice germplasm resource or breeding population plant is increased; 20 μ L PCR reaction systems comprise: the DNA 2.0 μ L of 10 ng/ μ L, the primer 2 .0 μ L of 4 pmol/ μ L, wherein primer I nDel- Rf1a-F and InDel- Rf1aEach 1.0 μ L of-R, 10 * PCR contains 25 mmol/L MgCl ₂Buffer 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 13.1 μ L;

The PCR response procedures comprises: 95 ℃ of preparatory sex change 5 min; 95 ℃ of sex change 30 s, 55 ℃ of renaturation 30 s, 72 ℃ of extension 1 min circulate 35 times then; 72 ℃ are extended 7 min then, behind 10 ℃ of cooling 10 min, amplified production are added a kind damping fluid termination reaction.

(3) reaction product is electrophoresis on 1.0% the sepharose than concentration in quality, observes record down through ethidium bromide staining and in gel imaging system.If having the characteristic band of 1145 bp to be contains Rf1aRecover the homozygote of gene; If having the characteristic band of 571 bp to be does not contain Rf1aRecover the homozygote of gene; If have two characteristic bands of 1,145 bp and 571 bp simultaneously, then do Rf1aRecover the heterozygote of gene.

Beneficial effect

A kind of discriminating japonica rice BT type cytoplasmic male sterility provided by the invention is recovered gene Rf1aMolecule marking method, have the following advantages:

(1) molecule marker provided by the invention is compared with the molecule marker that existing japonica rice BT type cytoplasmic male sterility is recovered gene; It is the functional mark of the employing pcr amplification of the insertion/deletion segment design according to gene; Its genotype can directly reflect the phenotype of plant; Not only do not exist the mistake that causes owing to the heredity exchange to identify, and efficient in operation, quick.；

(2) molecule marking method provided by the invention can realize japonica rice BT type cytoplasmic male sterility is recovered the Rapid identification of genetic resources, can from a large amount of japonica rice, accurately filter out to contain the recovery gene Rf1aKind, these japonica rice varieties with fertility restorer ability can be used as excellent parent and come the new recovery of seed selection system in breeding, effectively widening existing the recovery is hereditary basis, strengthens the combination advantage of Japonica Hybrid.

(3) molecule marking method provided by the invention can be effective to the assistant breeding that japonica rice BT type cytoplasmic male sterility is recovered system.After early recovery is tied to form type, also must recovers gene to it and carry out the test cross evaluation, this method not only needs two seasons of growth, also will hybridize a large amount of individual plants simultaneously, and its process is quite loaded down with trivial details.Though for simplifying the procedure of breeding, came the seed selection homogeneity extensive afterwards, must cause making up heterotic reduction owing to the plasma inheritance background is identical by sterile cytoplasm.Utilization is total to isolating molecule marker with the recovery gene then can overcome above-mentioned shortcoming, just can carry out genotype screening in seedling stage, and it is the time and intensity of seed selection that effective reduction recovers, and improves the foresight of breeding work greatly.

Four, description of drawings

Fig. 1 detects japonica rice BT type cytoplasmic male sterility and recovers gene Rf1aThe indicia designs strategy

(dash area representes not contain the recovery gene Rf1a1 bp and 574 bp two places base deletions that paddy rice exists; Black surround is partly represented the primer position)

Fig. 2 InDel- Rf1aTo the conventional rice variety in different areas sources, recover the Molecular Detection of system and maintenance line

(M:DNA molecular weight standard, 100-2,000 bp; 1-11: conventional rice variety Nanjing 11, the wax in Nanchong, middle morning 39, Huang Huazhan, Yunnan are auspicious 408, Guizhou Ma Gu, Tetep, Basmati 370, IR24, close positive 23, Dular; 12-19: long-grained nonglutinous rice recovers that the system town is extensive 084,93-11, another name for Sichuan Province are extensive 527, spoke is extensive 838, wide extensive 998, No. 1, boat, China are extensive 451, in extensive 8006; 20-24: the fragrant 29B in river, II-32B, Wu Xiang 2B, ridge 46B, middle lotus 1B)

Fig. 3 InDel- Rf1aMolecular Detection to the conventional japonica rice kind in different areas sources

(M:DNA molecular weight standard, 100-2,000 bp; 1-24: southern round-grained rice 44, southern round-grained rice 46, precious farming 34, good 33, good 48, holy rice No. 13, holy rice No. 14, No. 6, Henan round-grained rice, Zheng rice No. 18, Tianjin rice 263, No. 1, river, Tianjin, salt are rich 47, No. 9, distant round-grained rice, lucky round-grained rice 88, long white No. 9, No. 21, imperial round-grained rice, No. 9, loose round-grained rice, her No. 12, round-grained rice, new rice No. 32, No. 32, Chu's round-grained rice, No. 41, syzygy, the Northeast 194, more light, like to know 106)

Fig. 4 InDel- Rf1aThe japonica rice in source, different areas is recovered the Molecular Detection of system, sterile line and Japonica Hybrid kind

(M:DNA molecular weight standard, 100-2,000 bp; 1-14: japonica rice BT type cytoplasmic male sterility is recovered system peaceful extensive No. 8, C-25, C-53, R9511, Hunan are fine, R161, Shen are extensive 254, R192, extensive No. 9 of Anhui, HP121, LR27, C418, samsara 422, R187; 15-20: japonica rice BT type cytoplasmic male sterile line 95122A, No. 3 A of Xu rice, force are transported No. 7 A of round-grained rice, 863A, safe round-grained rice 985A, are connected No. 6 A of round-grained rice; 21-24: Japonica Hybrid make up 9 excellent 418, normal excellent No. 1,86 excellent No. 8,95 excellent 161)

Fig. 5 InDel- Rf1aTo peaceful extensive No. 8 F of 863A/ ₂The Molecular Detection of the different individual plants of colony

(M:DNA molecular weight standard, 100-2,000 bp; 1: peaceful extensive No. 8; 2:863A; Peaceful extensive No. 8 F of 3:863A/ ₁Hybrid is individual; 4-24: part F ₂Separate individual plant)

Five, embodiment

Recover gene for fully disclosing a kind of discriminating of the present invention japonica rice BT type cytoplasmic male sterility Rf1aMolecule marking method, explain below in conjunction with method validation and embodiment.Its practical implementation step is following:

(1) test materials

Conventional rice variety: Nanjing 11 (Jiangsu), Nanchong cured (Sichuan), middle morning 39 (Zhejiang), Huang Huazhan (Guangdong), auspicious 408 (Yunnan), Yunnan, Guizhou Ma Gu (Guizhou), Tetep (Vietnam), Basmati370 (Thailand), IR24 (Philippines), close positive 23 (Korea S), Dular (India).

Long-grained nonglutinous rice recovers system: press down extensive 084 (Jiangsu), 93-11 (Jiangsu), extensive 527 (Sichuan), another name for Sichuan Province, extensive 838 (Sichuan) of spoke, wide extensive 998 (Guangdong), boat No. 1 (Fujian), extensive 451 (Hunan) of China, in extensive 8006 (Zhejiang).

Long-grained nonglutinous rice maintenance line: fragrant 29B (Sichuan, river; The wild-abortive cytoplasmic male sterility maintenance line), II-32B (Zhejiang; Seal water type cytoplasmic male sterility maintenance line), Wu Xiang 2B (Hubei; Horse association type cytoplasmic male sterility maintenance line), ridge 46B (Sichuan, ridge type cytoplasmic male sterility maintenance line), middle lotus 1B (Jiangsu, HL cytoplasm male sterile maintenance line).

Conventional japonica rice kind: southern round-grained rice 44 (Jiangsu); South round-grained rice 46 (Jiangsu); Precious farming 34 (Shanghai); Good 33 (Zhejiang); Good 48 (Zhejiang); Holy rice No. 13 (Shandong); Holy rice No. 14 (Shandong); No. 6, Henan round-grained rice (Henan); Zheng rice No. 18 (Henan); Tianjin rice 263 (Tianjin); No. 1, river, Tianjin (Tianjin); Rich 47 (Liaoning) of salt; The Liao Dynasty's No. 9, round-grained rice (Liaoning); Lucky round-grained rice 88 (Jilin); Long white No. 9 (Jilin); Dragon No. 21, round-grained rice (Heilungkiang); Pine No. 9, round-grained rice (Heilungkiang); Her No. 12, round-grained rice (Xinjiang); New rice No. 32 (Xinjiang); No. 32, Chu's round-grained rice (Yunnan); No. 41, syzygy (Yunnan); The Northeast 194 (Japan); Light (Japan) more; Like to know 106 (Japan).

Japonica rice recovers system: peaceful extensive No. 8 (Jiangsu), CR-25 (Jiangsu), C-53 (Jiangsu), R9511 (Jiangsu), Hunan fine (Zhejiang), R161 (Shanghai), extensive 254 (Shanghai), Shen, R192 (Shanghai), Anhui extensive No. 9 (Anhui), HP121 (Anhui), LR27 (Tianjin), C418 (Liaoning), samsara 422 (Hunan), R187 (Hubei).

BT type japonica rice sterile: 95122A, No. 3 A of Xu rice, force are transported No. 7 A of round-grained rice, 863A, safe round-grained rice 985A, are connected No. 6 A of round-grained rice, and above sterile line forms by Jiangsu improved variety (strain) transformation.

BT type Japonica Hybrid combination: 9 excellent 418 (9201A/C418, Jiangsu), normal excellent No. 1 (No. 7 A/R254 of military fortune round-grained rice, Jiangsu), 86 excellent No. 8 (peaceful extensive No. 8 of 863A/, Jiangsu), 95 excellent 161 (95122A/ evening 161, Jiangsu)

BT type cytoplasmic male sterility is recovered gene F ₂Segregating population: 200 F that 86 excellent No. 8 (peaceful extensive No. 8 of 863A/) obtain through selfing ₂Individual plant.

Above material is public material, and agricultural germ plasm resource storehouse in mid-term, Jiangsu Province can provide free.Concrete reference is: Lin Shicheng etc., rice in China kind and pedigree thereof, Shanghai: Shanghai science tech publishing house, 1991:318; Li Xin etc., paddy rice semi-dwarf mutant gene Sd-nChromosomal localization research, Yangzhou University's journal (agricultural and life science version), 2002,23 (1):: 40-44; Mao Yonggen etc., 39 performance and high-yield culture techniques early among the conventional Xian early, modern agriculture science and technology, 2010,20:75-77 in the Jiangshan City; Zhou Shaochuan etc., the yellow China of grain quality rice Core Germplasms accounts for and the research of flavor idealized model rice in China science, 2009,29 (3): 153-159; Wan Jianmin etc., rice in China genetic breeding and kind pedigree, Beijing: Chinese agriculture press, 2009,272; Wu Junsheng etc., the evaluation and the utility value of wide affine kind zero expensive 40, China seed industry, 2000,5:28; Gan Daiyao etc., the seed selection of the anti-short Tetep in source and anti-pest are identified, Fujian agriculture science and technology, 1992,6:4; Bughio H., R., Improvement of grain yield in rice variety Basmati-370 ( Oryza sativaL.), through mutagenesis, Pak. J. Bot., 2007,39 (7): 2463-2466; Imbe T. Et al., A new gene for blast resistance in rice cultivar, IR24, A new gene for blast resistance in rice cultivar, IR24, Rice Genetics Newsletter, 1997,60; Masao Et al., Use of a Korean rice cultivar Milyang23 for improving Japanese rice, Breeding Science, 1994,44,219-222; Yan Changjie etc., the rflp analysis of autumn rice varieties Dular wide compatibility gene, Acta Genetica Sinica, 2000,27 (5): 409-417; Sheng Shenglan etc., long-grained nonglutinous rice recover the seed selection and the utilization in system town extensive 084, hybrid rice, 2002,17 (2): 6-7; Xu Maolin etc., the Xian new variety were raised seed selection and the utilization of rice No. 6, Chinese rice, 2001,1:24-26 during good quality and high output was disease-resistant; Wang Yu equality, it is the seed selection and the utilization in another name for Sichuan Province extensive 527 that the high-combining ability high-grade rice recovers, hybrid rice, 2004,19 (4): 12-14; Deng Dasheng etc., rice restorer spoke extensive 838 and the seed selection and the application of deriving and being, nuclear agricultural science newspaper, 2009,23 (2): 175-179; Li Shuguang etc., broad spectrum recover the feature and the seeding technique of system wide extensive 998, guangdong agricultural science, 2006,2:20-21; Thank to Huaan etc., super hybridized rice recovers the seed selection and the application of system's " navigating No. 1 ", Scientia Agricultura Sinica, 2004,37 (11): 1688-1692; Yang Yuanzhu etc., ideotype high-grade rice recover the seed selection and the application of system China extensive 451, hybrid rice, 2009,24 (5): 7-11; Wan Jianmin etc., rice in China genetic breeding and kind pedigree, Beijing: Chinese agriculture press, 2009,500-501; Qin Dynasty brocade etc., odor type long-grained nonglutinous rice sterile line river fragrant 29A high yield breeding technology, hybrid rice, 2007,22 (1): 27-29; Wu Chuangen etc., the development and use research of new germplasm source sterile line II-32A, hybrid rice, 1992,3:24-25; Zhang Wanchun etc., hybridisation rice newly make up military fragrant 988 plantation performance and high-yield culture techniques in Hanzhong, 2010,56 (1): 251-252; Li Shigui etc., the paddy rice excellent sterile is the seed selection and the applied research of Gang No.46A, Sichuan Agricultural University's journal, 1995,4:432-436; Zhong Liang etc., excellent 950 features of lotus and cultivation technique in the red lotus type hybridisation rice, Jiangsu agricultural sciences, 2011,3:120; Wang Cailin etc., the feature and the cultivation technique of anti-stripe virus disease new rice variety south round-grained rice 44, Jiangsu agricultural sciences, 2007,2:43-44; Wang Cailin etc., the feature and the cultivation technique of anti-stripe virus disease excellent flavour japonica rice new variety in evening south round-grained rice 46, Jiangsu agricultural sciences, 2008,2:91-92; Zhu Yifeng etc., high quality japonica " precious farming 34 " feature and cultivation main points, Shanghai Agricultural science and technology, 2007,4:24; Yu Zhihua etc., the good quality and high output new rice variety praises 33, China seed industry, 2007,6:65; Wan Jianmin etc., rice in China genetic breeding and kind pedigree, Beijing: Chinese agriculture press, 2009,417; Gong Deying etc., seed selection and the corresponding cultivation techniques of the holy rice 13 of new rice variety, Shandong agricultural sciences, 2007,5:113-114; Chen Feng etc., seed selection and the corresponding cultivation techniques of the holy rice 14 of new rice variety, 2007,6:109-110; Wang Shuyu, No. 6, super high-yielding high-grade rice new variety Henan round-grained rice, crop varieties resource, 1999,3:52; Yin Haiqing etc., seed selection that japonica rice new variety Zheng rice is No. 18 and cultivation main points, Chinese rice, 2007,, 1:20-24; Http:// www.ricedata.cn/variety/varis/611531.htm; Ding Deliang etc., the superior quality cultivation technique that No. 1, river, excellent flavour rice Tianjin, China seed industry, 2009,11:69; Ren Yongquan etc., rich 47 high yields of salt are optimized cultivation technique research, rice in north china, 2008,38 (3): 70-71; Liu Xian equality, new rice variety the Liao Dynasty's No. 9 features of round-grained rice and cultivation main points, cultivation and rice do, and 2004,1:12-13; Zhang Junguo etc., the excellent specific property of the lucky round-grained rice 88 of super hybridization rice and apply Chinese rice, 2008,3:42-44; Wan Jianmin etc., rice in China genetic breeding and kind pedigree, Beijing: Chinese agriculture press, 2009,329-330; Chu Guojiang etc., super hybridization rice dragon round-grained rice 21 features and high-yield culture technique, rice in north china, 2009,39,4:56-57; 2. Tao Yong celebrating etc., No. 9 features of loose round-grained rice and high-yield culture technique comment are recommended, Chinese rice, 2006,6:25-26; Tu Er does river etc., No. 12, high yield and high quality new rice variety-her round-grained rice, the land-reclaimable science and technology in Xinjiang, 2006,1:40-41; Tu Er does river etc., fragrant No. 32 features of the new rice of rice new variety of high-quality and cultivation technique main points, rice in north china, 2011,41 (3): 59-60; Liu Ji is new etc., the seed selection that No. 41, the cold-resistant disease-resistant japonica rice new variety syzygy of Yunnan eurytropy high yield, southwestern agriculture journal, 2002,15 (4): 5-9; Http:// ineweb.narcc.affrc.go.jp/; Sun Juying etc., normal excellent No. 4 seed selection and the application of the new combination of good-quality high-yield hybrid japonica rice, hybrid rice, 2010,25 (5): 17-19; Peak, Huajian etc., the normal excellent No. 2 high purity high-yield seeds production techniques of Japonica Hybrid, Jiangsu agricultural sciences, 2008,3:45-46; Wang Jianping etc., it is fine feature in Hunan and effect that japonica rice recovers, Jiangsu agricultural sciences, 2008,3:50-51; Wan Jianmin etc., rice in China genetic breeding and kind pedigree, Beijing: Chinese agriculture press, 2009,570-574; Zhang Huilian etc. utilize samsara 422 selection cross wide-congenial restoring lines and survey thereof to join performance, hybrid rice, 1991, (5): 29-33; Wang Changyi etc., it is seed selection and the utilization of R187 that double-line hybrid japonica rice recovers, hubei agricultural science, 1997,3:6-9; Liu is superfine, round-grained rice 9 excellent 418 main characteristiies and cultivation strategy in the hybridization, hybrid rice, 2002,17 (2): 35-37; He Jianhua etc., the normal excellent No. 1 high yield and high quality seeding technique of Japonica Hybrid, Chinese rice, 2003, (2): 24-25; Paddy forint etc., the new combination of high-quality hybrid japonica rice 86 excellent No. 8, hybrid rice, 2001,16 (2): 59-60; Wang Cailin etc., the seed selection and the utilization thereof of the new combination 95 excellent 161 of good-quality high-yield hybrid japonica rice, hybrid rice, S1:23

(2) molecular markers development

(1) japonica rice BT type cytoplasmic male sterility is recovered gene Rf1aThe nucleotide sequence analysis of variant sites

According to Wang ( The Plant cell, 2006,18 (3): the result of study that 676-687) waits, utilize japonica rice to recover gene locus Rf1aGenomic information, the design primer carries out pcr amplification and directly order-checking to this site sequence, obtains Rf1aThe genome sequence in site.Confirm that through sequential analysis the japonica rice BT type cytoplasmic male sterility that has checked order is recovered system and sterile line (with the dyskaryosis maintenance line) exists Rf1aThe base difference of 574 bp that the site all exists.

(2) design of primers

Utilize Rf1aThe nucleotides sequence of gene is listed in rice genome website (Gene Bank; Www.ncbi.nlm.nih.gov/) download it and be positioned at the long-armed place of paddy rice the 10th karyomit(e) pac clone (P1-derived artificial chromosome; PAC) nucleotide sequence (OSJNBa0017E08; AC068923), and to correlated series analyze; Then, utilize Primer Premier 5.0 (http://www.premierbiosoft.com) near 574bp base insertion disappearance zone, design is insertion/disappearance mark accordingly, and with synthetic labeled primer called after InDel- Rf1aIts forward primer sequence is 5'-CTGATGATCGAGGAGGAGGTA-3', and the reverse primer sequence is 5'-TAACGCGTCTTCCATCCTACT-3', and annealing temperature is 55 ℃ (Fig. 1).

(3) molecule marker checking

(1) extraction of rice plant genomic dna

The extraction of rice plant genomic dna with reference to the SDS method (Dellaporta S L, et al., Plant Mol B iol Rep, 1983,1 (1): 19221.).Concrete steps are: the water intaking rice seedling phase blade, in the mortar of-20 ℃ of precoolings with the liquid nitrogen grinding and the 1.5 mL centrifuge tubes of packing into; Add 600 uL extracting solutions (20%SDS, 1M Tris-HCl, 0.5M EDTA, 5M NaCl, 65 ℃ of preheatings), shake up, 65 ℃ of temperature are bathed 30 min, middle vibration 3 ~ 4 times; Add 1/4 volume 5M KAC, shake up postposition 30 min on ice; Add chloroform-primary isoamyl alcohol (24:1) 300 ~ 400 uL, fully vibration on shaking table, 120 rpm, 30 min; 8,000 ~ 10, centrifugal 15 minutes of 000rpm, the liquid level layering, lower floor's color is darker, and the little band yellow-green colour in upper strata is got supernatant (about 400 uL) to another centrifuge tube; Add equal-volume chloroform-primary isoamyl alcohol (24:1), fully vibration on the shaking table, 80 ~ 90 rpm, 30 min; Centrifugal 15 minutes of 8,000 rpm shift supernatant (about 400 uL) to new centrifuge tube; The absolute ethyl alcohol that adds 2 times of volume-20 ℃ precoolings shakes up up to there being floss to produce the centrifugal 6min of 12,000 rpm gently; Abandon absolute ethyl alcohol, add 4 ℃ of 70% ethanol, place 10 min, abandon supernatant, air-dry 1h on the Bechtop; Add 100 ~ 200 uL TE ,-20 ℃ of preservations.

(2) amplification of molecule marker and electrophoresis detection

Utilize japonica rice BT type cytoplasmic male sterility to recover gene InDel- Rf1aMark, its primer sequence is:

Forward primer (InDel- Rf1a-F) sequence is 5'-CTGATGATCGAGGAGGAGGTA-3'

Reverse primer (InDel- Rf1a-R) sequence is 5'-TAACGCGTCTTCCATCCTACT-3'

To different water rice varieties (strain or combination) and Rf1aThe genotype segregating population carries out pcr amplification and electrophoresis detection.

(3) 20 μ L PCR reaction systems comprise: the DNA 2.0 μ L of 10 ng/ μ L, the primer 2 .0 μ L of 4 pmol/ μ L, wherein primer I nDel- Rf1a-F and InDel- Rf1aEach 1.0 μ L of-R, 10 * PCR Buffer (MgCl that comprises 25 mmol/L ₂) 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 13.1 μ L;

The PCR response procedures comprises: the PCR response procedures comprises: 95 ℃ of preparatory sex change 5 min; 95 ℃ of sex change 30s, 55 ℃ of renaturation 30s, 72 ℃ of extension 1min circulate 35 times then; 72 ℃ are extended 7min then, behind 10 ℃ of cooling 10min, amplified production are added a kind damping fluid termination reaction.

(4) reaction product is electrophoresis on 1.0% the sepharose than concentration in quality, observes record down through ethidium bromide staining and in gel imaging system.If having the characteristic band of 1145 bp to be contains Rf1aRecover the homozygote of gene; If having the characteristic band of 571 bp to be does not contain Rf1aRecover the homozygote of gene; If have two characteristic bands of 1145 bp and 571 bp simultaneously, then do Rf1aRecover the heterozygote of gene.

(5) interpretation of result

1) InDel- Rf1aMark recovers system and maintenance line to conventional rice variety, long-grained nonglutinous rice Rf1aThe genotype detection in site

Utilize molecule marker InDel- Rf1a24 parts of long-grained nonglutinous rice materials to from different areas carry out pcr amplification and electrophoresis detection.The result shows: No. 11,11 conventional rice variety Nanjing, the wax in Nanchong, middle morning 39, Huang Huazhan, Yunnan are auspicious 408, Guizhou Ma Gu, Tetep, Basmati 370, IR24, close positive 23, Dular; 8 long-grained nonglutinous rices recover that the system town is extensive 084,93-11, another name for Sichuan Province are extensive 527, spoke is extensive 838, wide extensive 998, No. 1, boat, China are extensive 451, in the fragrant 29B in extensive 8006 and 5 long-grained nonglutinous rice male sterility maintainer line rivers, II-32B, Wu Xiang 2B, ridge 46B, middle lotus 1B all can stablize the band (Fig. 2) that amplifies 1 about 1,145 bp of length.This shows that above material exists Rf1aThere is not the disappearance of 574 bp in the site, and its genotype does Rf1aRf1a, they all have restitution to japonica rice BT type cytoplasmic male sterile line.

2) InDel- Rf1aMark is to the conventional japonica rice kind Rf1aThe genotype detection in site

Utilize molecule marker InDel- Rf1a24 parts of japonica rice varieties to from different areas carry out pcr amplification and electrophoresis detection.The result shows: 22 conventional japonica rice kind south round-grained rice 44, southern round-grained rice 46, precious farming 34, good 33, good 48, holy rice No. 13, holy rice No. 14, No. 6, Henan round-grained rice, Zheng rice No. 18, Tianjin rice 263, No. 1, river, Tianjin, salt are rich 47, No. 9, distant round-grained rice, lucky round-grained rice 88, long white No. 9, No. 21, imperial round-grained rice, No. 9, loose round-grained rice, new rice No. 32, No. 32, Chu's round-grained rice, No. 41, syzygy, the Northeast 194 and more light all can stablize the band that amplifies 1 about 571 bp of length, these kinds exist Rf1aThere is the disappearance of 574 bp in the site, and its genotype does Rf1arf1a, so these materials have the effect that keeps sterile to BT type cytoplasmic male sterile line; And other 2 rice varieties like to know 106 with No. 12, her round-grained rice owing to can amplify Rf1aRf1aThe band of distinctive 1,145 bp of genotype, they have restitution to BT type cytoplasmic male sterile line, and these japonica rice varieties with fertility restorer ability can be used as excellent parent and come the new recovery system (Fig. 3) of further seed selection in breeding.Therefore, through InDel- Rf1aThe pcr amplification of mark can identify accurately that it is germ plasm resource that BT type japonica rice recovers.

3) InDel- Rf1aMark recovers system, sterile line and Japonica Hybrid kind to japonica rice Rf1aThe genotype detection in site

Utilize molecule marker InDel- Rf1aCarry out pcr amplification and electrophoresis detection to recovering system, 6 parts of male sterile lines and 4 parts of Japonica Hybrid kinds from 14 parts of japonica rice BT type cytoplasmic male sterilities of different areas.The result shows, the band of recover system peaceful extensive No. 8, C-25, C-53, R9511, Hunan are fine, R161, Shen are extensive 254, R192, extensive No. 9 of Anhui, HP121, LR27, C418, samsara 422 and R187 can increase 1 about 1,145 bp of length; Sterile line 95122A, No. 3 A of Xu rice, force are transported No. 7 A of round-grained rice, 863A, safe round-grained rice 985A and are connected the band that No. 6 A of round-grained rice can amplify 1 about 571 bp of length; Japonica Hybrid kind 9 is excellent 418, normal excellent No. 1,86 excellent No. 8 and 95 excellent 161 then can amplify about 1,145 bp of length and 571 bp simultaneously two bands (Fig. 4).Therefore, exist Rf1aThe site, the genotype that japonica rice recovers system does Rf1aRf1a, the genotype of sterile line does Rf1arf1a, and the genotype of Japonica Hybrid combination does Rf1arf1a

4) InDel- Rf1aMark is to peaceful extensive No. 8 F of 863A/ ₂The different individual plants of segregating population Rf1aThe genotype detection in site

For further verifying InDel-<i >Rf1a</i>To the detection effect of different genotype, the Sheng phase of tillering to peaceful extensive No. 8 F of 863A/<sub >2</sub>200 individual plant DNA of segregating population carry out pcr amplification, and its electrophoresis product presents 3 types band, promptly recover system peaceful extensive No. 8 (<i >Rf1aRf1a</i>, 1145 bp), japonica rice sterile 863A (<i >Rf1arf1a</i>, 571bp) and hybrid F<sub >1</sub>(<i >Rf1arf1a</i>, 1145 bp and 571 bp) banding pattern (Fig. 5).Through statistics, at F<sub >2</sub>Detect altogether in the colony<i >Rf1aRf1a</i>41 of genotype individual plants,<i >Rf1arf1a</i>111 of genotype individual plants,<i >Rf1arf1a</i>48 of genotype individual plants, three kinds of genotype meet 1:2:1 (χ<sup >2</sup>=2.91,0.25<i ><P<</i>0.50).Therefore, through InDel-<i >Rf1a</i>The pcr amplification of mark can accurately be distinguished<i >Rf1a</i>The different genotype in site reaches the purpose of assistant breeding.

SEQUENCE?LISTING

 

 

< 110>Jiangsu Province Agriculture Science Institute

 

 

< 120>a kind of japonica rice BT type cytoplasmic male sterility of differentiating is recovered the molecule marking method of gene Rf1a

 

 

<130> 0

 

 

<160> 2

 

 

<170> PatentIn?version?3.1

 

 

<210> 1

<211> 21

<212> DNA

< 213>manual work

 

 

<220>

< 221>forward primer InDel-Rf1a-F

<222> (1)..(21)

<223>

 

 

 

<400> 1

ctgatgatcg?aggaggaggt?a 21

 

 

<210> 2

<211> 21

<212> DNA

< 213>manual work

 

 

<220>

< 221>reverse primer InDel-Rf1a-R

<222> (1)..(21)

<223>

 

 

 

<400> 2

taacgcgtct?tccatcctac?t 21

Claims (2)

1. identify japonica rice BT type cytoplasmic male sterility recovery gene for one kind Rf1aMolecule marking method, it is characterized in that:

Use Rf1aRecover gene-specific primer InDel- Rf1a

Forward primer InDel- Rf1a-F sequence is 5'-CTGATGATCGAGGAGGAGGTA-3'

Reverse primer InDel- Rf1a-R sequence is 5'-TAACGCGTCTTCCATCCTACT-3'

The genomic dna of amplifying rice plant contains if there is the characteristic band of 1,145 bp to be Rf1aRecover the homozygote of gene; If having the characteristic band of 571 bp to be does not contain Rf1aRecover the homozygote of gene; If have two characteristic bands of 1,145 bp and 571 bp simultaneously, then do Rf1aRecover the heterozygote of gene.

2. method according to claim 1 is characterized in that:

The extraction of rice plant genomic dna;

With described two the molecule marker primer I nDel-of claim 1 Rf1a-F and InDel- Rf1a-R adds the PCR reaction system, and the DNA of rice germplasm resource or breeding population plant is increased; 20 μ L PCR reaction systems comprise: the DNA 2.0 μ L of 10 ng/ μ L, the primer 2 .0 μ L of 4 pmol/ μ L, wherein primer I nDel- Rf1a-F and InDel- Rf1aEach 1.0 μ L of-R, 10 * PCR contains 25 mmol/L MgCl ₂Buffer 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 13.1 μ L;

The PCR response procedures comprises: 95 ℃ of preparatory sex change 5 min; 95 ℃ of sex change 30 s, 55 ℃ of renaturation 30 s, 72 ℃ of extension 1 min circulate 35 times then; 72 ℃ are extended 7 min then, behind 10 ℃ of cooling 10 min, amplified production are added a kind damping fluid termination reaction;

Reaction product is electrophoresis on 1.0% the sepharose than concentration in quality, observes record down through ethidium bromide staining and in gel imaging system.

Images