CN103103279B - Molecular marker primer combination for polymerization of wide-compatibility fertility site segments of indica-japonica subspecies and application thereof - Google Patents
Molecular marker primer combination for polymerization of wide-compatibility fertility site segments of indica-japonica subspecies and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker primer combination for the polymerization of wide-compatibility fertility site segments of indica-japonica subspecies and an application thereof. The primer combination consists of molecular marker primers W5, W7 and W17 respectively corresponding to fertility sites S5-n, S7-n and S17-n, wherein a left end primer sequence of the W5 is SEQ ID NO. 1, and a right end primer sequence of the W5 is SEQ ID NO. 2; a left end primer sequence of the W7 is SEQ ID NO. 3, and a right end primer sequence of the W7 is SEQ ID NO. 4; and a left end primer sequence of the W17 is SEQ ID NO. 5, and a right end primer sequence of the W17 is SEQ ID NO. 6. The application of molecular marker primer combination in crossbreeding is suitable for the breeding of wide-compatibility fertility site segment polymerization systems of hybridized descendants of rice indica and japonica, and is capable of solving the problem of infertility of intersubspecific hybrids between the indica and the japonica and promoting the utilization of superiority of the intersubspecific hybrids between the indica and the japonica.
Description
Technical field
The invention belongs to molecular genetics field, a kind of molecule marker combination of primers and application thereof for the site fragment polymerization of the wide affine fertility of indica and japonica subspecies is provided.
Background technology
In recent years, along with the continuous increase of China's size of population, and in the limited situation of cultivated area, improve rice yield significant to ensureing national food safety, and the utilization of indica-japonica heterosis is the important channel of improving rice yield.But subspecies indica and japonica hybrid F1 shows partial sterility conventionally, thereby limit the effective utilization of its powerful hybrid vigour on producing.Being found to be of wide compatibility gene S 5-n overcome between indica and japonica subspecies fertility obstacle provide approach (Japan.J.Breed., 1984,34:304-312).At present, wide compatibility gene S 5-n is widely used in (Sci Agric Sin1992,23:1-6) in Indica round-grained rice heterosis utilization.Expansion along with breeding parent scope, investigators find, although the wide affine kind of one of some cross combination parent for carrying S5-n, but still there is half sterile phenomenon (Japan J Breed1993 in its hybrid F1,43:507 – 516), therefore only depend on wide compatibility gene S 5-n can not solve the hybrid semisterility problem between indica and japonica subspecies completely.In the practices of breeding, expansion along with hybrid fertile evaluation scope, identified S7 (Japan J Breed1992, 42:793 – 801), S8 (Japan J Breed1993, 43:507 – 516), S9 (Theor Appl Genet, 1996, 92:183-190), S15 (Theor Appl Genet, 1996, 92:183-190), S16 (Breeding Science1995, 45:161-170), S17 (Breeding Science1995, 45 (2): 191-195), S29 (Plant Breeding, 2005, 124:440-445), S30 (Breed Science, 2005, 55:409-414), S31 (Euphytica, 2006, 151:331-337) and S32 (Theor Appl Genet, 2007, 114:515 – 524) the sterile site of hybrid megagamete such as.At present, how to utilize the fertility site having been found that, particularly wide affine fertility site is applied to japonica Cross molecular marker breeding or blank out.Mechanism based on japonica Cross hybrid dysgenesis is mainly unit point sporinite-gametophyte cooperating type (IRRIRice genetics.IRRI, Manila, 1984, pp119 – 130), and on some fertility site, the allelotrope in long-grained nonglutinous rice and japonica rice and wide affine kind is respectively SX-i, SX-j and SX-n.Genotype is that the heterozygote of SX-i/SX-j produces half sterile small ear owing to carrying the megagamete part abortion of SX-j.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, the molecule marking method of the wide affine fragment polymerization in a kind of indica-japonica hybrid fertility site is provided.
Another object of the present invention is to provide the molecule marker combination of primers for the site fragment polymerization of the wide affine fertility of indica and japonica subspecies.
Another object of the present invention is to provide the application of described molecule marker combination of primers in cross-breeding.
Object of the present invention can be achieved through the following technical solutions:
The molecule marking method of the wide affine site fragment polymerization in indica-japonica hybrid fertility site, with molecule marker primer W5, W7 and W17, respectively the wide affine fertility fragment in S5-n site, fertility site, S7-n site and S17-n site is selected polymerization by molecule marker, wherein
Use labeled primer W5
Left end primer sequence 5 '-AATGGAGTCGACCGTGTGTTCGTCG-3'(SEQ ID NO.1)
Right-hand member primer sequence 5'-CCCCAAACCTGAAATCACCAGTGTT-3'(SEQ ID NO.2)
The wide affine rice varieties DNA that increases, obtaining S5 site labeled fragment size is 105bp; Amplification rice variety DNA, obtaining weak site labeled fragment size is 145bp; Amplification japonica rice variety DNA, obtaining S5 site labeled fragment size is 131bp;
Labeled primer W7
Left end primer sequence 5 '-TTCAGCCTCGTATCGCTCCTA-3 ' (SEQ ID NO.3)
Right-hand member primer sequence 5 '-TGCTATCCTGGTTGCTCTGCT-3 ' (SEQ ID NO.4)
The wide affine kind DNA that increases, obtaining S7 site labeled fragment has 2 bands, and size is respectively 140bp and 175bp; Amplification rice variety DNA, obtaining S7 site labeled fragment size is 150bp; Amplification japonica rice variety DNA, obtaining S7 site labeled fragment has 2 bands, and size is respectively 140bp and 150bp;
Labeled primer W17
Left end primer sequence 5 '-GGTATCCATAATCTCCACAAC-3 ' (SEQ ID NO.5)
Right-hand member primer sequence 5 '-CTTTCTACATGACGATGAACA-3 ' (SEQ ID NO.6)
The wide affine kind DNA that increases, obtaining S17 site labeled fragment size is 200bp; Amplification rice variety DNA, obtaining S17 site labeled fragment size is 180bp; Amplification japonica rice variety DNA, obtaining S17 site labeled fragment has 2 bands, and size is respectively 180bp and 190bp.
A kind of molecule marker combination of primers for the site fragment polymerization of the wide affine fertility of indica and japonica subspecies, this combination of primers is comprised of molecule marker primer W5, W7 and W17, wherein, molecule marker primer W5, W7 and W17 are respectively to S5-n site, fertility site, S7-n site and S17-n site, the left end primer sequence of molecule marker primer W5 is SEQ ID NO.1, and right-hand member primer sequence is SEQ ID NO.2; The left end primer sequence of molecule marker primer W7 is SEQ ID NO.3, and right-hand member primer sequence is SEQ IDNO.4; The left end primer sequence of molecule marker primer W17 is SEQ ID NO.5, and right-hand member primer sequence is SEQ ID NO.6.
The application of molecule marker combination of primers of the present invention in cross-breeding.
Molecule marker combination of primers of the present invention is the application in the seed selection of the rice varieties of a plurality of wide compatibility genes of polymerization preferably.
The application of described molecule marking method, further comprises following method:
(1) with Indica Rice restorer Zhenhui 129 (hybrid rice, 2000, 15 (4): be 8-9) maternal, anti-63 (hybrid rice with high resistance to hoja blanca, 2005, 20 (1): 11-14, patent No. ZL92103511X, certificate number 30184, http://www.uast.com.cn/njnd/pzzs/pzzsdetail.asp Varid=108) hybridize to obtain F1 seed, add generation F1 seed belt Hainan winter, protect new variety R6547http with the R437(rice in China Information Network C.NRRI> with wide affine kind 02428 blood relationship of japonica rice again: //www.chinariceinfo.com/variety/baohu/20041008) reestablish diplomatic relations, in separated San Jiao colony, use respectively the mark W5 in S5 site, the mark W7 in S7 site, the mark W17 in S17 site detects.In segregating population, need only the fragment with these 3 sites, comprise heterozygosis fragment, all in this season sowing simultaneously.In each generation subsequently, all use the same method and detect, until the CF8 that reestablishes diplomatic relations obtains basicly stable Indica Rice Restorer Lines material W8733, i.e. a Zhenhui 129/anti-63//R437(CF8 from generation to generation).
(2) with Indica Rice Restorer Lines material W8733, do the further selfing of breeding material, neat and consistent is stablized in discovery strain W107 performance, and plant type is good, uses respectively the mark W5 in S5-n site simultaneously; The mark W7 in S7-n site; The mark W17 in S17 site detects, all with the fragment in S5-n site, S7-n site and S17-n site.By the tentative restorer W107 by name of this strain.
With W107, make male parent and the paired test cross such as sterile line II-32A and Xieqingzao A, its corresponding cross-fertilize seed is stable and consistent also, and advantage is strong, and yielding ability is good.Excellent 107 cross-fertilize seed of the II excellent 107 that the W107 of take is male parent system and association, in the examination of indica Hybrid Rice district, than contrast Shanyou 63 volume increase 6.98%, increase production extremely remarkable.Xie You 107 indica Hybrid Rice high-quality group districts, participating nation man middle and lower reach of Yangtze River examination, than contrast Shanyou 63 volume increase 7.34%, extremely remarkable, Second Year continues test, breaks a rule simultaneously and enters production test.The powerful hybrid vigour of cross combination shows that W107 is the restorer of a strong advantage.
Restorer W107 can with the conventional variety preparing hybrid kind application that utilizing in long-grained nonglutinous rice, japonica rice and breeding.
The molecule marking method of the wide affine fertility of beneficial effect indica-japonica hybrid provided by the present invention site fragment polymerization, has the following advantages:
(1) by the closely linked molecule marker of acquisition of the present invention and the wide affine fertility of indica-japonica hybrid site, on S5-n site, develop molecule marker W5, on S7-n site, develop molecule marker W7, on S17-n site, develop molecule marker W17.
(2) by the present invention, can carry out wide affine fragment polymerization to S5-n, S7-n, S17-n fertility site and carry out molecule marker, the fragment locality specific of selection, it is convenient to identify.By detecting the molecule marker in wide affine fertility site, measurable Xian round-grained rice mingles the size of kind of fertility restorer, and then hands over rapid screening in segregating population to go out the offspring of Gao Yu for the breeding of hybrid rice at Xian round-grained rice.The method is easy to detect fast, not affected by environment; The method is suitable for the seed selection of the wide affine fertility of japonica Cross site fragment polymerization system, can solve Xian round-grained rice and hand over the sterile problem of inter subspecific hybrid, promotes the utilization of subspecies indica and japonica hybrid advantage.
(3) assistant breeding select target is clear and definite, cost-saving, restorer that can quick breeding powerful advantages.At Xian round-grained rice, hand in traditional breeding way, first will plant larger secondary segregating population, need to drop into a large amount of manpowers, material resources and financial resources.Secondly, the cycle of breeding is long, and the seed selection of a conventional variety needs 8-10, and not necessarily in the seed selection of fertility site to the stable plant of isozygotying.And select by the molecule marker in early stage fertility site, can in less segregating population, by detecting Xian round-grained rice, hand over the molecule marker in fertility site, in seedling stage, just identify the individual plant of polymeric segment, eliminate other plant, not only save production cost but also greatly improve efficiency of selection.By this method, superior restorer line W107 only used for 6 years just selected, and more conventional breeding method is 2 years in advance.Success is for combining with sterile line preparing hybrid, and Posterity phenotype goes out powerful Xian round-grained rice and hands over hybrid vigour, Ru Xieyou 107 indica Hybrid Rice high-quality group districts, participating nation man middle and lower reach of Yangtze River examination, than contrast Shanyou 63 volume increase 7.34%, extremely remarkable, Second Year continues test, breaks a rule simultaneously and enters production test.
Accompanying drawing explanation
Fig. 1 detects long-grained nonglutinous rice, japonica rice and wide affine kind at S5 site molecule marker W5
M is Marker, and 1,7,8,9,10,11,12 swimming lanes represent the individual plant of parent's Zhenhui 129 fragment; 3,6 is the individual plant with anti-63 fragments of parental breed; 2,4,5 swimming lanes are the individual plant with wide affine kind R437 fragment.
Fig. 2 detects long-grained nonglutinous rice, japonica rice and wide affine kind at S7 site molecule marker W7
M is Marker, and 2,3,4,5,10 is the individual plant with Zhenhui 129 fragment; 6,9 swimming lanes are the individual plant with anti-63 fragments of parental breed; 1,7,8,11,12 swimming lanes are the individual plant with wide affine kind R437 fragment.
Fig. 3 detects long-grained nonglutinous rice, japonica rice and wide affine kind at S17 site molecule marker W17
M is Marker, and 2,5,6 is the individual plant with Zhenhui 129 fragment; 4,7,8,10 swimming lanes are the individual plant with anti-63 fragments of parental breed; 1,3,9,11,12 swimming lanes are the individual plant with wide affine kind R437 fragment.
Fig. 4 detects the wide affine site of W107 polymerization fragment with molecule marker W5, W7, W17 respectively in S5, S7, S17 site
M is Marker, 2,4,6,13,19,20,23,24, and 26,29,31,32 is the individual plant with W107 fragment.
Fig. 5 superior restorer line W107 and the comparison that contrasts Zhenhui 084 mature period fringe type, and with the comparison of parent's Zhenhui 129 particle shape.
Embodiment
Result of study shows that the mechanism of Xian round-grained rice friendship hybrid dysgenesis belongs to unit point sporinite-gametophyte cooperating type, and on some fertility site, allelotrope in long-grained nonglutinous rice and japonica rice is respectively SX-i and SX-j, and the heterozygote that genotype is SX-i/SX-j produces half sterile small ear owing to carrying the megagamete part abortion of SX-j.But wide affine kind and long-grained nonglutinous rice, japonica rice variety hybridization, genotype is respectively SX-n/SX-i and SX-n/SX-i, thus heterozygote can be educated owing to carrying the performance of SX-n compatibility gene.Therefore, a plurality of wide affine allelotrope of polymerization site on fertility site, so just can overcome the difficult problem that Xian round-grained rice is handed over hybrid dysgenesis.Wherein, the present invention is to S5, and Fine Mapping has been carried out in S7 and S17 fertility site, and corresponding molecule marker is also developed.If the left end primer sequence of molecule marker primer W5 is SEQ ID NO.1, right-hand member primer sequence is SEQ ID NO.2; The left end primer sequence of molecule marker primer W7 is SEQ ID NO.3, and right-hand member primer sequence is SEQ ID NO.4; The left end primer sequence of molecule marker primer W17 is SEQ ID NO.5, and right-hand member primer sequence is SEQ ID NO.6.
(1) label screening: hybridize to obtain F1 seed with rice variety Zhenhui 129 with anti-63, and make male parent with restorer R437(R437) hybridization, restorer R437 is with wide affine kind 02428 blood relationship of japonica rice (Chinese rice, 2004).In separated San Jiao colony, use the mark W5 in S5 site simultaneously; The mark W7 in S7 site; The mark W17 in S17 site detects.In segregating population, need only the fragment (comprising heterozygosis fragment) with this wide affine site, 3 sites, all separated for the selfing in next season in this season sowing.While increasing with molecule marker W5, can obtain the fragment of S5 site mark 105bp size, while increasing with molecule marker W7, obtain S7 site labeled fragment and have 2 bands, be respectively the fragment of 140bp and 175bp size, while increasing with molecule marker W17, can obtain the fragment (in Table 1) of the 200bp size of S17 site mark.In each generation subsequently, all use the same method and detect, take into account other economical characters such as yield traits, resistance and quality simultaneously, up to the CF8 that reestablishes diplomatic relations, obtain from generation to generation a basicly stable Indica Rice Restorer Lines material W8733, i.e. Zhenhui 129/anti-63//R437(CF8).(2) with Indica Rice Restorer Lines material W8733, do the further selfing of breeding material, neat and consistent is stablized in discovery strain W107 performance, plant type is good, with 3 wide affine sites, detect simultaneously, all, with the fragment in S5-n site, S7-n site and S17-n site, the tentative restorer W107(by name of this strain is shown in to Fig. 4).With restorer W107 and long-grained nonglutinous rice, japonica rice and other conventional variety of producing upper application, hybridize, its cross combination all shows good affinity (in Table 2).In addition, with W107, make male parent and the paired test cross such as sterile line II-32A and Xieqingzao A, its corresponding cross-fertilize seed is stable and consistent also, and advantage is strong, and yielding ability is good.Excellent 107 cross-fertilize seed of the II excellent 107 that the W107 of take is male parent system and association, in the examination of indica Hybrid Rice district, than contrast Shanyou 63 volume increase 6.98%, increase production extremely remarkable.Xie You 107 indica Hybrid Rice high-quality group districts, participating nation man middle and lower reach of Yangtze River examination, than contrast Shanyou 63 volume increase 7.34%, extremely remarkable, Second Year continues test, breaks a rule simultaneously and enters production test.The powerful hybrid vigour of cross combination shows that W107 is the restorer (in Table 3) of a strong advantage.The PCR product size that table 1 molecule marker increases in different types of varieties
(2) materials and methods:
2.1 kinds: Zhenhui 129 (hybrid rice; 2000; 15 (4): 8-9); anti-63 (hybrid rice; 2005; 20 (1): 11-14; patent No. ZL92103511X, certificate number 30184), R437(rice in China Information Network C.NRRI> protection new variety R6547http: //www.chinariceinfo.com/variety/baohu/20041008); Zhenhui 084 (hybrid rice; 2005,20 (2): 31-32), wide affine kind 02428 (Science Bulletin; 2005,50:32-37).
At clip rice leaf in tillering phase, and frozen in-70 ℃ of refrigerators.Utilize SDS method (Plant Mol Bioi Rep, 1983,1:19-21) extract genomic DNA.The DNA extracting is dissolved in (10mM Trisbase, 0.1mMEDTA) in 200ulTE damping fluid.With MBA2000UV/VISSpectrometer, detect the quality of sample DNA.Then with distilled water, be diluted to the working fluid of 20ng/ul, be stored in 4 ℃ of refrigerators, during analysis, be used as template.PCR reaction is with reference to the method for (Theor Appl Genet, 1997,95:553 – 567).95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, carry out altogether 35 circulations, and last 72 ℃ are extended 7min, 10 ℃ of preservations.PCR reaction is carried out in MJ Reseach PTC-225 thermal cycler.Reaction system comprises: the about 10ng of DNA profiling, 10 * LA buffer1 μ l, LA Taq (5u/ μ l) 0.1 μ l, MgCl2 (25mM) 1 μ l, dNTPs (2.5mM) 1 μ l, justice, each 1.5 μ l of antisense primer (2mM), then add ddH2O and make volume arrive 10 μ l, and PCR program is: 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min.
Amplified production is electrophoresis on 8% non-denaturing polyacrylamide gel, then according to the method for (Biotechniques1994,17:915 – 919), analyzes and reading after cma staining.Fig. 1 is the electrophoresis photo that S5 site molecule marker W5 selects individual plant, and Fig. 2 detects the electrophoresis photo of long-grained nonglutinous rice, japonica rice and wide affine kind at S7 site molecule marker W7, and Fig. 3 detects the electrophoresis photo of long-grained nonglutinous rice, japonica rice at S17 site molecule marker W17
2.2 pollen, small ear and the investigation of blastular fertility
Pollen fertility investigation adopts I
2-KI staining: 1-2 days before blooming for examination material, 2 grain husk flowers are respectively got in middle part, middle part and middle and lower part from each individual plant small ear, are placed in ethanol: glacial acetic acid (3:1) solution is fixing, 4 ℃ of Refrigerator stores.During microscopy, from 6 grain husks of every strain are spent, get 1 flower pesticide, mixed sample is observed pollen fertility, uses 1%I
2-KI dyeing, compressing tablet, 10 * 20 power microscopes are observed.Each slice, thin piece is observed 4 visuals field, guarantees that pollen granule sum is more than 200.According to pollen staining and morphological specificity judge its fertility (Oryza, 1981,18:140-142): the pollen granule of circular chocolate is for educating, and out-of-shape, yellow and achromophil pollen granule are sterile.Pollen fertility represents with the percentile mean value of fertile pollen.
Spikelet fertility investigation:
In the ripening stage, often select good strains in the field for seed and get 3 single fringes that setting percentage is suitable, examine or check the Spikelet fertility of its first half; During statistics, using the average fertility of 3 single fringes as the Spikelet fertility of this strain; Spikelet fertility is the percentage that fertilization grain number accounts for total number.From each material, get 5 strains and carry out fertility investigation, using the pollen of 5 strains and Spikelet fertility mean value fertility value (Theor Appl Genet, 1996,92 (1): 183-190) as this material.
(3) results and analysis:
The molecular selection of the temperature sensitive restorer W107 fertility of 3.1 light site polymerization
With high yield restorer Zhenhui 129 (hybrid rice, 2000, 15 (4): be 8-9) maternal, anti-63 (hybrid rice with high resistance to hoja blanca, 2005, 20 (1): 11-14, patent No. ZL92103511X, certificate number 30184, http://www.uast.com.cn/njnd/pzzs/pzzsdetail.asp Varid=108) hybridize to obtain F1 seed, add generation F1 seed belt Hainan winter, protect new variety R6547http with the R437(rice in China Information Network C.NRRI> with wide affine kind 02428 blood relationship of japonica rice again: //www.chinariceinfo.com/variety/baohu/20041008) reestablish diplomatic relations, in separated San Jiao colony, use respectively the mark W5 in S5 site, the mark W7 in S7 site, the mark W17 in S17 site detects.In segregating population, need only the fragment with these 3 sites, comprise heterozygosis fragment, all in this season sowing simultaneously.In each generation subsequently, all use the same method and detect, until the CF8 that reestablishes diplomatic relations obtains basicly stable Indica Rice Restorer Lines material W8733, i.e. a Zhenhui 129/anti-63//R437(CF8 from generation to generation).From restorer material W8733, select 5 strains and II-32A and Xieqingzao A etc. to produce the paired test cross of sterile line of upper large-area applications, the positive season plantation CF8 in Nanjing and corresponding hybrid, wherein neat and consistent is stablized in strain W107 performance, plant type is good, its corresponding cross-fertilize seed is stable and consistent also, advantage is strong, and yielding ability is good; With 3 wide affine sites, detect simultaneously, all, with the fragment in S5-n site, S7-n site and S17-n site, this strain is fixed tentatively to restorer W107 by name.W107 is taken to Hainan and plant and intersect the combo production of hybrid seeds, male parent W107 performance is consistent with separated.The II excellent 107 that the W107 of just take in season is male parent system and association excellent 107 participate in the our units' indica Hybrid Rices of 2 years comparation and assessment.II in 2004 excellent 107 participates in the examination of indica Hybrid Rice districts, Anhui, than contrast Shanyou 63 volume increase 6.98%, increases production extremely significantly, ranked third, and within 2005, continues to participate in the experiment, and participates in production test simultaneously.Xie You107Yu 2004 indica Hybrid Rice high-quality group districts, participating nation man middle and lower reach of Yangtze River examination, than contrast Shanyou 63 volume increase 7.34%, extremely remarkable, within 2005, continue test, break a rule simultaneously and enter production test.
The pollen fertility of 3.2 superior restorer line W107 and conventional variety hybridization F1 and setting percentage investigation and analysis
Be bred as superior restorer line W107 S5 site with wide compatibility gene S 5-n; S7 site with wide compatibility gene S 7-n, S17 site with wide compatibility gene S 17-n.With the conventional variety preparing hybrid combination (in Table 3) utilizing in superior restorer line W107 and typical long-grained nonglutinous rice, japonica rice and breeding.From table, result is known, and the pollen fertility of restorer W107 and japonica rice Testers Barilla, autumn light hybridization hybrid is normal, is respectively 97.62 ± 0.93 and 88.18 ± 4.31; Spikelet fertility also shows as Gao Yu, only has respectively 55.64 ± 2.42 and 62.01 ± 3.13.The pollen fertility of restorer W107 and long-grained nonglutinous rice Testers Nanjing 11, IR36 hybridization hybrid is normal, is respectively 99.49 ± 0.77 and 91.98 ± 0.27; Spikelet fertility is also acted normally, and reaches respectively 92.77 ± 0.61 and 85.35 ± 0.77.Meanwhile, restorer W107 is normal with most of wide affine mixing breed hybrid fertile.This result hint restorer W107 major part on sterile site has all been polymerized to a plurality of wide affine fragments, therefore, acts normally with japonica rice and indica hybrid hybrid fertile.
The molecule marking method that the invention provides the site fragment polymerization of the wide affine fertility of indica-japonica hybrid, belongs to molecular genetics field.The molecule marker primer W5 of development and Design, W7 and W179 can be respectively at S5, S7, and these 3 Xian round-grained rice of S17 hand over the wide affine site of educating to amplify differential fragment.Molecule marker by fertile gene site carries out the polymerization of wide affine fertility fragment, can accurately and fast import needed fragment, greatly improved Xian round-grained rice and handed over the efficiency of selection of wide affine fertility site fragment polymerization, and utilized this method to select superior restorer line W107(in Table 3).
Material involved in the present invention is public material: Zhenhui 129 (hybrid rice; 2000; 15 (4): 8-9); anti-63 (hybrid rice; 2005; 20 (1): 11-14; patent No. ZL92103511X, certificate number 30184), R437(rice in China Information Network C.NRRI) and protection new variety R6547http: //www.chinariceinfo.com/variety/baohu/20041008); Zhenhui 084 (hybrid rice; 2005,20 (2): 31-32), wide affine kind 02428 (Science Bulletin; 2005,50:32-37).
Pollen fertility and the Spikelet fertility of table 2 superior restorer line W107 and long-grained nonglutinous rice, japonica rice variety hybridization hybrid F1.
Table 3 superior restorer line W107 does parent and produces the performance situation that the upper sterile line preparing hybrid utilizing is combined in different location output, utilizes the cross combination of restorer W107 preparation to show stronger hybrid vigour.
Claims (4)
1. the molecule marking method of the wide affine fertility of indica and japonica subspecies site fragment polymerization, is characterized in that, uses molecule marker primer W5, W7 and W17 respectively to fertility site
s5-nsite,
s7-nsite and
s17-nthe wide affine fertility fragment in site selects polymerization by molecule marker, wherein
With molecule marker primer W5: left end primer sequence SEQ ID NO.1, the right-hand member primer sequence SEQ ID NO.2 wide affine rice varieties DNA that increases, obtains
s5-nsite labeled fragment size is 105bp; Amplification rice variety DNA, obtains
s5-isite labeled fragment size is 145bp; Amplification japonica rice variety DNA, obtains
s5-jsite labeled fragment size is 131bp;
With molecule marker primer W7: left end primer sequence SEQ ID NO.3, the right-hand member primer sequence SEQ ID NO.4 wide affine kind DNA that increases, obtains
s7-nsite labeled fragment has 2 bands, and size is respectively 140bp and 175bp; Rice variety DNA, obtains
s7-isite labeled fragment size is 150bp; Amplification japonica rice variety DNA, obtains
s7-jsite labeled fragment has 2 bands, and size is respectively 140bp and 150bp;
With molecule marker primer W17: left end primer sequence SEQ ID NO.5, the right-hand member primer sequence SEQ ID NO.6 wide affine kind DNA that increases, obtains
s17-nsite labeled fragment size is 200bp; Amplification rice variety DNA, obtains
s17-isite labeled fragment size is 180bp; Amplification japonica rice variety DNA, obtains
s17-jsite labeled fragment has 2 bands, and size is respectively 180bp and 190bp.
2. for a molecule marker combination of primers for the wide affine fertility of indica and japonica subspecies site fragment polymerization, it is characterized in that being comprised of molecule marker primer W5, W7 and W17, wherein, molecule marker primer W5, W7 and W17 are respectively to fertility site
s5site,
s7site and
s17site, the left end primer sequence of molecule marker primer W5 is SEQ ID NO.1, right-hand member primer sequence is SEQ ID NO.2; The left end primer sequence of molecule marker primer W7 is SEQ ID NO.3, and right-hand member primer sequence is SEQ ID NO.4; The left end primer sequence of molecule marker primer W17 is SEQ ID NO.5, and right-hand member primer sequence is SEQ ID NO.6.
3. the application of molecule marker combination of primers claimed in claim 2 in cross-breeding.
4. application according to claim 3, is characterized in that the application of molecule marker combination of primers claimed in claim 2 in the seed selection of the rice varieties of a plurality of wide compatibility genes of polymerization.
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