CN102031301B - Molecular marking method for indica-japonica segment substitution of fertility loci of indica-japonica hybrid - Google Patents

Abstract

The invention provides a molecular marking method for indica-japonica fragment substitution of fertility loci of an indica-japonica hybrid, which belongs to the field of molecular genetics. Molecular marker primers RMz 5, RMz7, RMz8 and RMz9 which are developed and designed by the invention amplify differential fragments at infertile loci of the indica-japonica hybrid, namely S5, S7, S8 and S9. Indica-japonica fragments can be substituted by molecular markers of the fertility gene loci; the required fragments can be precisely and quickly introduced; the selection efficiency of fragment substitution of the indica-japonica fertility loci is greatly improved, and a japonica type indica-compatible light and temperature-sensitive male sterile line 509S can be bred.

Description

Indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method

One, technical field

The invention provides indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method, belong to the molecular genetics field.And utilize this method to select round-grained rice type parent Xian property photo-thermo-sensitive genetic male sterile line 509S.This method is suitable for the seed selection that paddy rice Xian round-grained rice is handed over offspring's fertility site fragment substitution line, can promote the utilization of subspecies indica and japonica hybrid advantage.

Two, background technology

In recent years, along with the continuous increase of China's size of population, and under the cultivated area condition of limited, it is significant to ensureing national food safety to improve rice yield, and the utilization of indica-japonica heterosis is the important channel of improving rice yield.But subspecies indica and japonica hybrid F ₁Usually show partial sterility, thereby limit effective utilization of its powerful hybrid vigour on producing.Being found to be of wide compatibility gene S 5-n overcomes that the fertility obstacle provides approach between indica and japonica subspecies.At present, this gene be widely used in the paddy rice indica-japonica heterosis utilize in (Sci Agric Sin 1992,23:1-6).Along with the expansion of breeding parent scope, investigators find, although the wide affine kind of one of some cross combination parent for carrying S5-n, its hybrid F ₁(Japan J Breed 1993 43:507-516), therefore only depends on wide compatibility gene S 5-n can not solve the hybrid semisterility problem between indica and japonica subspecies fully half sterile phenomenon still to occur.In the practices of breeding, along with hybrid fertile is identified the expansion of scope, identified S8 (Japan J Breed 1993,43:507-516), S9 (Theor Appl Genet; 1996,92:183-190), S15 (Theor Appl Genet, 1996; 92:183-190), S16 (Breeding Science 1995,45:161-170), 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) wait the sterile site of hybrid megagamete.At present, the fertility site that has been found that of what use is made of is applied to paddy rice Xian round-grained rice and hands over molecular marker breeding or blank out.Mingling kind of sterile mechanism based on paddy rice Xian round-grained rice mainly is unit point sporinite-gametophyte cooperating type (IRRI Rice genetics.IRRI; Manila; 1984, pp 119-130), and on some fertility site; Allelotrope in long-grained nonglutinous rice and the japonica rice is respectively SX-i and SX-j, and genotype is that the heterozygote of SX-i/SX-j is owing to the megagamete part abortion of carrying SX-j produces half sterile small ear.This seminar proposes; Under the genetic background that does not change a certain round-grained rice type kind, only on the fertility site, be replaced into the allelotrope of corresponding indica type variety, this kind and rice variety hybridization have affinity; Otherwise; Under indica type background, the allelotrope of the corresponding round-grained rice type of displacement on the fertility site, this kind and the mixing breed of round-grained rice type have affinity.Through this strategy, mingle kind of a sterile problem thereby overcome the Xian round-grained rice, realize the utilization of subspecies indica and japonica hybrid advantage.For this reason; The invention provides indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method; And utilize this method in low segregating population from generation to generation; Through molecular marker assisted selection and the conventional back cross breeding of combination, select round-grained rice type parent Xian property photo-thermo-sensitive genetic male sterile line 509S, realize the utilization of subspecies indica and japonica hybrid advantage.

Three, summary of the invention

Technical problem

The objective of the invention is: indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method is provided, has belonged to the molecular genetics field.And utilize this method to select round-grained rice type parent Xian property photo-thermo-sensitive genetic male sterile line 509S.This method is suitable for the seed selection that paddy rice Xian round-grained rice is handed over offspring's fertility site fragment substitution line, can solve the Xian round-grained rice and hand over the sterile problem of inter subspecific hybrid, promotes the utilization of subspecies indica and japonica hybrid advantage.

Subspecies indica and japonica hybrid F ₁Usually the performance partial sterility limits the effective utilization of its powerful hybrid vigour on producing.Being found to be of wide compatibility gene S 5-n overcomes that the fertility obstacle provides approach between indica and japonica subspecies.At present, this gene be widely used in the paddy rice indica-japonica heterosis utilize in (Sci Agric Sin 1992,23:1-6).Along with the expansion of breeding parent scope, investigators find, although the wide affine kind of one of some cross combination parent for carrying S5-n, its hybrid F ₁(Japan J Breed 1993 43:507-516), therefore only depends on wide compatibility gene S 5-n can not solve the hybrid semisterility problem between indica and japonica subspecies fully half sterile phenomenon still to occur.In the practices of breeding, along with hybrid fertile is identified the expansion of scope, identified more than 30 the sterile site of hybrid megagamete (Theor Appl Genet, 2007,114:915-925).At present, the fertility site that has been found that of what use is made of is applied to paddy rice Xian round-grained rice and hands over molecular marker breeding or blank out.Mingling kind of sterile mechanism based on paddy rice Xian round-grained rice mainly is unit point sporinite-gametophyte cooperating type (IRRI Rice genetics.IRRI; Manila; 1984, pp 119-130), and on some fertility site; Allelotrope in long-grained nonglutinous rice and the japonica rice is respectively SX-i and SX-j, and genotype is that the heterozygote of SX-i/SX-j is owing to the megagamete part abortion of carrying SX-j produces half sterile small ear.

Technical scheme

Indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method; It is characterized in that; Respectively molecule marker is carried out in the Xian round-grained rice fragment displacement in S5 site, fertility site, S7 site, S8 site and S9 site with molecule marker primer RMz5, RMz7, RMz8 and RMz9, wherein

Use labeled primer RMz5

Left end primer sequence 5 '-AATGGAGTCGACCGTGTGTTCGTCG-3 '

Right-hand member primer sequence 5 '-CCCCAAACCTGAAATCACCAGTGTT-3 '

The wide affine rice varieties DNA that increases, obtaining S5 site labeled fragment size is 105bp; Amplification rice variety DNA, obtaining S5 site labeled fragment size is 145bp; Amplification japonica rice variety DNA, obtaining S5 site labeled fragment size is 131bp;

Labeled primer RMz7

Left end primer sequence 5 '-GGTAACATGCATATCACGCT-3 '

Right-hand member primer sequence 5 '-GTATGCCGATATGCGTAGCC-3 '

Amplification rice variety DNA, obtaining S7 site labeled fragment size is 200bp; Amplification japonica rice variety DNA, obtaining S7 site labeled fragment size is 215bp;

Labeled primer RMz8

Left end primer sequence 5 '-AACCACCACCACCACGCCTCTG-3 '

Right-hand member primer sequence 5 '-CCTTGGAGAGGAGGAGGCGCGC-3 '

Amplification rice variety DNA, obtaining S8 site labeled fragment size is 131bp; Amplification japonica rice variety DNA, obtaining S8 site labeled fragment size is 145bp;

Labeled primer RMz9

Left end primer sequence 5 '-GGTTGTTGGGAGGGAGAAAGGCT-3 '

Right-hand member primer sequence 5 '-TGGAGGCGACGGCGATGTCCTTG-3 '

Amplification rice variety DNA, obtaining S9 site labeled fragment size is 150bp, amplification japonica rice variety DNA; Obtaining S9 site labeled fragment size is 161bp.

The application of said molecule marking method comprises:

(1) with sterile line samsara 422S and 88 hybridization of japonica rice variety town rice, obtains hybridizing F ₁, with hybrid F ₁Make female parent, make recurrent parent, obtain backcross population BC with town rice 88 ₁F ₁, from BC ₁F ₁Beginning just detected with the mark RMz5 in S5 site and the mark RMz8 in S8 site respectively in seedling stage,

Samsara 422S is wide affine rice variety, when increasing with molecule marker RMz5, can obtain the fragment of wide affine kind S5 site mark 105bp size, when increasing with molecule marker RMz8, can obtain the fragment of S8 site 131bp size;

Town rice 88 is a japonica rice variety, when increasing with molecule marker RMz5, can obtain the fragment of S5 site mark 131bp size; When increasing, can obtain the fragment of S8 site mark 145bp size with molecule marker RMz8;

BC ₁F ₁Detect the segmental strain of long-grained nonglutinous rice system at fertility site S5 and S8 site, just do maternal and background parent hybridization, all use the same method from generation to generation at each, until BC in flowering period ₄F ₂Obtain from generation to generation a stable japonica rice photoperiod-temperature sensitive genie male-sterile line material W0532 and be samsara 422S/ town rice 88//town rice 88/ // town rice 88/ ///press down rice 88;

(2) with have yellow phyllopodium because of 249 yellow and 88 hybridization of japonica rice variety town rice, obtain hybridizing F ₁, with hybrid F ₁Make female parent, make recurrent parent, obtain backcross population BC with town rice 88 ₁F ₁, just detect with the mark RMz7 in S7 site and the mark RMz9 in S9 site respectively in seedling stage from beginning,

249 Huangs are rice variety, when increasing with molecule marker RMz7, can obtain the fragment of S7 site mark 200bp size, when increasing with molecule marker RMz9, can obtain the fragment of S9 site mark 150bp size;

Town rice 88 is a japonica rice variety, when increasing with molecule marker RMz7, can obtain the fragment of S7 site mark 215bp size; When increasing, can obtain the fragment of S9 site mark 161bp size with molecule marker RMz9;

BC ₁F ₁Detect the segmental strain of long-grained nonglutinous rice system at fertility site S7 and S9 site, just do maternal and background parent hybridization, all use the same method from generation to generation at each, until BC in flowering period ₄F ₂Obtain from generation to generation one and stable have yellow phyllopodium because of material W249, i.e. samsara 249 Huangs/town rice 88//town rice 88/ // town rice 88/ ///press down rice 88;

(3) further do maternally, and have yellow phyllopodium and obtain F because of material W249 hybridization with japonica rice photoperiod-temperature sensitive genie male-sterile line material W0532 ₁, selfing obtains separating F ₂Colony, in this segregating population with 4 molecule marker RMz5, RMz7, RMz8 and RMz9 select jointly, simultaneously also carry out Huang Ye and pollen fertility Phenotypic Selection, in following selfing segregating population, all use identical method to select, up to F ₈In generation, when sterile strain system performance neat and consistent, sterility is good; Be sterile plant rate 100%, pollen sterility degree 100%, bagging self-fruitful rate 0; On S5 site, 4 fertility sites, S7 site, S8 site and S9 site, all be replaced into indica type fragment, with the tentative 509S by name of this strain system.

Photo-thermo-sensitive genetic male sterile line 509S will with long-grained nonglutinous rice, japonica rice and breeding on the conventional variety preparing hybrid kind utilized use.

Beneficial effect indica-japonica hybrid fertility provided by the present invention site Xian round-grained rice fragment metathetical molecule marking method has the following advantages:

(1) through the closely linked molecule marker of acquisition of the present invention and indica-japonica hybrid fertility site; Exploitation molecule marker RMz5 on the S5 site; Exploitation molecule marker RMz7 on the S7 site, exploitation molecule marker RMz8 on the S8 site, exploitation molecule marker RMz9 on the S9 site.

(2) can carry out the displacement of Xian round-grained rice fragment to S5, S7, S8, S9 fertility site through the present invention and carry out molecule marker, the fragment locality specific of selection, it is convenient to identify.Through detecting the molecule marker in fertility site, promptly measurable Xian round-grained rice mingles the size of kind of fertility restorer, and then is used for the breeding of hybrid rice the offspring that the Xian round-grained rice hands over rapid screening in the segregating population to go out Gao Yu.This method is easy to detect fast, and is not affected by environment;

(3) the assistant breeding select target is clear and definite, practices thrift cost.Hand in the traditional breeding way at the Xian round-grained rice, at first will plant bigger secondary segregating population, need to drop into great amount of manpower, 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 through the molecule marker in early stage fertility site; Can in less segregating population, hand over the molecule marker in fertility site, just identify the segmental individual plant of displacement in seedling stage through detecting the Xian round-grained rice; Eliminate other plant, not only save production cost but also improve efficiency of selection greatly.Through this method, photo-thermo-sensitive genetic male sterile line 509S only used for 6 years just selected, and conventional breeding method is 2 years in advance.Successfully be used for and the combination of long-grained nonglutinous rice preparing hybrid, the offspring shows powerful Xian round-grained rice and mingles kind of an advantage, in the Nanjing, all shows as good quality and high output during continuous 2 years district, Nanchang tries like cross combination 509S/W102.

Description of drawings

Molecule marker RMz5 selects individual plant to Fig. 1 in the S5 site

M is Marker, and 1,7,8,9,10,11,12 swimming lanes are represented background parent town rice 88 segmental individual plants; 3,6 for having rice variety 249 yellow segmental individual plants; 2,4,5 swimming lanes are for having wide affine kind samsara 422 segmental individual plants.

Molecule marker RMz7 detects long-grained nonglutinous rice, japonica rice to Fig. 2 in the S7 site

M is Marker, and 1,9,11,12 for having town's rice 88 segmental individual plants; 4,5,6,7,10, swimming lane is for having the segmental individual plant of rice variety; 2,3,8 is heterozygosis individual plant fragment.

Molecule marker RMz8 surveys long-grained nonglutinous rice, japonica rice to Fig. 3 in the S8 site

M is Marker, and 1,7,8,11,12 for having the segmental individual plant of rice variety; 2,3,4,5, swimming lane is for having the segmental individual plant of japonica rice variety; 6,9,10 is the heterozygosis individual plant.

Molecule marker RMz9 detects long-grained nonglutinous rice, japonica rice to Fig. 4 in the S9 site

M is Marker, and 1,7,8,11,12 for having the segmental individual plant of rice variety; 2,4,5,6 swimming lanes are for having the segmental individual plant of japonica rice variety; 3,9,10 is the heterozygosis individual plant.

Fig. 5 utilizes the SSR mark to comprising 24 kind cluster analyses of 509S.

In the drawings, photo-thermo-sensitive genetic male sterile line 509S by cluster japonica rice variety this type, wherein with japonica rice variety more light have higher similarity.

The biomaterial preservation

The indica type photo-thermo-sensitive genetic male sterile line 509S of round-grained rice type parent; Belong to paddy rice (Oryza sativa); Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on 08 23rd, 2010; No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, Institute of Microorganism, Academia Sinica, preserving number: CGMCCNO.4105.

Embodiment

Result of study shows that the Xian round-grained rice mingles kind of sterile mechanism and belongs to unit point sporinite-gametophyte cooperating type; And on some fertility site; Allelotrope in long-grained nonglutinous rice and the japonica rice is respectively SX-i and SX-j, and genotype is that the heterozygote of SX-i/SX-j is owing to the megagamete part abortion of carrying SX-j produces half sterile small ear.Only on the fertility site, be replaced into the allelotrope of corresponding another subspecies, might overcome the Xian round-grained rice like this and mingle kind sterile.Wherein, this experiment is to S5, S7, and Fine Mapping has been carried out in S8 and S9 fertility site, and corresponding molecule marker is also developed.Like the RMz5 that is labeled as in S5 site, the S7 site be labeled as RMz7, the S8 site be labeled as RMz8, the S9 site be labeled as RMz9 (seeing table 1).

(1) label screening:

(1) on the one hand, with sterile line samsara 422S and 88 hybridization of japonica rice variety town rice, obtain hybridizing F ₁, with hybrid F ₁Make female parent, make recurrent parent, obtain backcross population BC with town rice 88 ₁F ₁, from BC ₁F ₁Beginning just detected with the mark RMz5 in S5 site and the mark RMz8 in S8 site respectively in seedling stage.Samsara 422S is wide affine kind, when increasing with molecule marker RMz5, can obtain the fragment of 105bp size, when increasing with molecule marker RMz8, can obtain the fragment (seeing table 2) of 131bp size.Town rice 88 is a japonica rice variety, when increasing with molecule marker RMz5, can obtain the fragment of 131bp size; When increasing, can obtain the fragment of 145bp size with molecule marker RMz8.Detect the segmental strain of the long-grained nonglutinous rice that needs system in the fertility site, just do female parent and background parent hybridization, all use the same method from generation to generation at each, until BC in flowering period ₄F ₂Obtain from generation to generation a stable japonica rice photoperiod-temperature sensitive genie male-sterile line material W0532 (samsara 422S/ town rice 88//town rice 88/ // town rice 88/ ///press down rice 88).

(2) on the other hand, with have yellow phyllopodium because of 249 yellow and 88 hybridization of japonica rice variety town rice, obtain hybridizing F ₁, with hybrid F ₁Make female parent, make recurrent parent, obtain backcross population BC with town rice 88 ₁F ₁, from BC ₁F ₁Beginning just detected with the mark RMz7 in S7 site and the mark RMz9 in S9 site respectively in seedling stage.249 Huangs are rice variety, when increasing with molecule marker RMz7, can obtain the fragment of 200bp size, when increasing with molecule marker RMz9, can obtain the fragment of 150bp size.Town rice 88 is a japonica rice variety, when increasing with molecule marker RMz7, can obtain the fragment of 215bp size; When increasing, can obtain the fragment of 161bp size with molecule marker RMz9.Detect the segmental strain of the long-grained nonglutinous rice that needs system in the fertility site, just do female parent and background parent hybridization, all use the same method from generation to generation at each, until BC in flowering period ₄F ₂Obtain from generation to generation one and stable have yellow phyllopodium because of material W249 (samsara 249 Huangs/town rice 88//town rice 88/ // town rice 88/ ///press down rice 88).

(3) further japonica rice photoperiod-temperature sensitive genie male-sterile line material W0532 is done maternally, and have yellow phyllopodium and obtain F because of material W249 hybridization ₁, selfing obtains separating F ₂Colony, (RMz8 RMz9) carries out the selection of mark for RMz5, RMz7, also carries out Phenotypic Selection (Huang Ye and pollen fertility selection) simultaneously with 4 molecule markers in this segregating population.In following selfing segregating population, all use identical method to select, until F ₈In generation, when sterile strain system performance neat and consistent, sterility is good, on 4 fertility sites, all is replaced into indica type fragment, and can numerous property when cutting stubble regeneration good, is this tentative 509S by name.

(4) after photo-thermo-sensitive genetic male sterile line 509S breeds, make female parent with it, extensively hybridize (seeing table 3) with the conventional variety of the excellence of utilizing in the current production.The result can know from table, and the small ear fertility of sterile line 509S and japonica rice test kind of Barilla, autumn light hybridization hybrid shows as partly sterile, and acts normally with the small ear fertility of long-grained nonglutinous rice test kind of Nanjing 11, IR36 hybridization hybrid.Though this result hints the japonica rice genetic background of sterile line 509S 92% for town rice 88,100% all has been replaced into the long-grained nonglutinous rice fragment on sterile site, therefore, acts normally with the indica hybrid hybrid fertile.Simultaneously, in the cross combination of sterile line and long-grained nonglutinous rice preparation, show powerful Xian round-grained rice and mingle kind of an advantage, in the Nanjing, all show as good quality and high output during continuous 2 years district, Nanchang tries like cross combination 509S/W102 (kind power CNA20060245.4).Present method is handed over heterotic breeding practice utilization for the Xian round-grained rice a kind of new feasible way is provided.

The sequence of table 1 labeled primer and amplified fragments size

Table 2 molecule marker is the amplification PCR products size in different types of varieties

(2) materials and methods:

Rice variety: Nanjing 11 (the Shanghai Agricultural journal, 2005,21:13-18), IR64 (Science Bulletin, 2005,50:32-37); IR24 (Science Bulletin, 2005,50:32-37), 9311 (Scientia Agricultura Sinica, 2004,37:788-794); Kasalath (Agricultural University Of Nanjing's journal, 2006,29:123-126), bright extensive 63 (Acta Genetica Sinica, 2003,30:804-810); Osmanthus towards No. 2 (Science Bulletin, 2003,48:1864-1867), special blue or green (rice in China science, 2003; 17:129-133), and IR36 (Acta Agronomica Sinica, 2003,29:574-580), totally 9 kinds;

Japonica rice variety: autumn light (Acta Agronomica Sinica, 2003,29:574-580), Barilla (Acta Agronomica Sinica, 2003; 29:574-580), Japanese fine (Theor Appl Genet, 2007,114:939-946), force is educated No. 3 (Scientia Agricultura Sinicas of round-grained rice; 2009,42:11-12), Asominori (Science Bulletin, 2005,50:32-37); More light (the rice in China science, 2003,17:73-76), 509S, totally 7 kinds;

Wide affine kind: 02428 (Science Bulletin, 2005,50:32-37), heat is ground (Theor Appl Genet, 1996 No. 2; 45:183-190), and Dular (the heredity circular, 2001,28:540-549), Cpslo17 (Guangxi agro-ecology science; 2002,21:199-203), N22 (Guangxi agro-ecology science, 2002,21:199-203); PA64 (the Hunan agricultural sciences, 2005,3:11-12), K.N (Plant Breed, 2005; 124:440-445), and C418 (the Hunan agricultural sciences, 2005,3:11-12), totally 8 parts;

At clip rice leaf in tillering phase, and frozen in-70 ℃ of refrigerators.Utilize the SDS method (Plant Mol Bioi Rep, 1983,1:19-21) extract genomic DNA.The DNA that extracts is dissolved in (10mM Trisbase, 0.1mM EDTA) in the 200ulTE damping fluid.Detect the quality of sample DNA with MBA 2000UV/VIS Spectrometer.Be diluted to the working fluid of 20ng/ul then with distilled water, be stored in 4 ℃ of refrigerators, be used as template during analysis.The PCR reaction is with reference to (Theor Appl Genet, 1997, method 95:553-567).95 ℃ of sex change 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 1min, carry out 35 circulations altogether, and last 72 ℃ are extended 7min, 10 ℃ of preservations.PCR is reflected in the MJ Reseach PTC-225 thermal cycler and carries out.Amplified production is electrophoresis on 8% non-denaturing polyacrylamide gel, then according to (Biotechniques 1994, and method 17:915-919) is analyzed behind cma staining and reading.

Reaction system comprises: the about 10ng of dna profiling, 10 * LA buffer, 1 μ l, LA Taq (5u/ μ l) 0.1 μ l, MgCl ₂(25mM) 1 μ l, dNTPs (2.5mM) 1 μ l, justice, each 1.5 μ l of antisense primer (2mM) add ddH then ₂O makes volume arrive 10 μ l, and the PCR program is: 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min.

Data analysis

Each detects a site to the SSR primer, and each bar polymorphum band is an allelotrope; Each band that is observed is regarded as a proterties, and assignment was " 1 " when this band was arranged, and assignment is not " 0 " when having this band; The hereditary difference of per two parts of materials is according to (Columbia University Press; NY, 1979, method pp:106-107) is asked genetic distance:

GD＝1-[2N/(Ni+Nj)]

N is the total bands of two parts of materials in the formula, and Ni and Nj are respectively total band number of i and two parts of materials of j.According to the genetic distance of gained, carry out cluster analysis with UPGMA (unweighted pair-group method with arithmetic mean, the non-weighting combo of arithmetical mean method), and draw dendrogram.

(3) result and analysis:

2.1 photo-thermo-sensitive genetic male sterile line 509S fertility site metathetical molecular selection

At first, sterile line samsara 422S and 88 hybridization of japonica rice variety town rice obtain hybridizing F ₁, with hybrid F ₁Make female parent, make recurrent parent, obtain backcross population BC with town rice 88 ₁F ₁, from BC ₁F ₁Beginning just detected with molecule marker in seedling stage, detected the segmental strain of the long-grained nonglutinous rice that needs system in the fertility site, just did female parent and background parent hybridization in flowering period, all used the same method from generation to generation at each, until BC ₄F ₂Obtain from generation to generation a stable japonica rice photoperiod-temperature sensitive genie male-sterile line material W0532 (samsara 422S/ town rice 88//town rice 88).Simultaneously, do female parent with 249 Huangs, rice 88 backs, hybridization town utilize identical method, until BC backcrossing town rice 88 once in backcross population ₄F ₂Obtain from generation to generation one stable have yellow phyllopodium because of japonica rice material W249 (249 Huangs/town rice 88//town rice 88).Utilize these two intermediate materials to hybridize once more and obtain F ₁, selfing obtains F ₂Colony is numbered S530, in the self progeny, utilizes molecule marker and Phenotypic Selection (pollen fertility and Huang Ye Phenotypic Selection); Positive season plantation F8 strain system and corresponding cross-fertilize seed in 2005, sterile strain is the performance neat and consistent, hands in the segregating population; All use identical method to select, until F ₈In generation, when sterile strain system performance neat and consistent, sterility is good (under high temperature of positive season of Nanjing (>=24 ℃) condition; Sterile plant rate 100%, pollen sterility degree 100%, bagging self-fruitful rate 0); On S5 site, 4 fertility sites, S7 site, S8 site and S9 site, all be replaced into indica type fragment; And cut when regeneration stubble can numerous property good (ear setting percentage more than 30% the middle ten days and the last ten days in September in Nanjing, control plant new variety specificity, consistence and stability test guide, B.68.4.3 regulation can numerous property among paddy rice standard GB/T 19557.7-2004; Reach one-level), with the tentative 509S by name of this strain system.

2.2 photo-thermo-sensitive genetic male sterile line 509S and conventional variety hybridization F ₁Pollen fertility and setting percentage

After breeding photo-thermo-sensitive genetic male sterile line 509S, with the conventional variety preparing hybrid combination (seeing table 3) that is utilizing in sterile line and typical long-grained nonglutinous rice, japonica rice and the breeding.The result can know from table, and the pollen fertility of sterile line 509S and japonica rice test kind of Barilla, autumn light hybridization hybrid is normal, is respectively 97.62 ± 0.93 and 88.18 ± 4.31; But the small ear fertility shows as partly sterile, has only 55.64 ± 2.42 and 62.01 ± 3.13 respectively.The pollen fertility of sterile line 509S and long-grained nonglutinous rice test kind of Nanjing 11, IR36 hybridization hybrid is normal, is respectively 99.49 ± 0.77 and 91.98 ± 0.27; The small ear fertility is also acted normally, and reaches 92.77 ± 0.61 and 85.35 ± 0.77 respectively.Simultaneously, sterile line 509S is normal with most of wide affine mixing breed hybrid fertile.This result hints that sterile line 509S major part on sterile site all has been replaced into the long-grained nonglutinous rice fragment, therefore, acts normally with the indica hybrid hybrid fertile.

2.3 cluster analysis

Can know that from sterile line 509S seed selection process the photoperiod-temperature sensitive genie male-sterile line gene of 509S is from parent's samsara 422S, its genetic background overwhelming majority is from town rice 88.Through the assisted Selection of mark, on main indica-japonica hybrid hybrid dysgenesis site, 509S has been replaced into the long-grained nonglutinous rice fragment.In order to confirm that from molecular level the genetic background of 509S is japonica rice, we have carried out cluster analysis.Select 184 pairs of SSR marks that are distributed on 12 karyomit(e)s of paddy rice for use, 24 kinds are carried out cluster analysis.Common property is given birth to 785 polymorphic sites, and every pair of mark produces 2～8 of polymorphic site scopes, 3.4 of average out to; Parent's genetic distance range of variation is 0.65～0.96.Cluster analysis result is (Fig. 5) as follows, and 24 rice varieties are divided into 2 big types in long-grained nonglutinous rice and japonica rice, and 509S is gathered in this big type in japonica rice, with japonica rice variety more light have higher similarity.

The invention provides indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method, belong to the molecular genetics field.And utilize this method to select round-grained rice type parent Xian property photo-thermo-sensitive genetic male sterile line 509S.The molecule marker primer RMz5 of development and Design, RMz7, RMz8 and RMz9 can be respectively at S5, S7, these 4 Xian round-grained rice of S8 and S9 hand over sterile site to amplify differential fragment.Molecule marker through the fertile gene site carries out the segmental displacement of Xian round-grained rice, can accurately and fast import needed fragment, has improved Xian round-grained rice fertility site fragment metathetical efficiency of selection greatly.

Material involved in the present invention is public material: samsara 422S (hybrid rice, 1995,6:7-9; Acta Agronomica Sinica, 1996,22:495-499), town rice 88 (Scientia Agricultura Sinica, 2009,42 (1): 103-109), 249 Huangs (PlantPhysiology, 2007,145:29-40).

Table 3 photo-thermo-sensitive genetic male sterile line 509S makes pollen fertility and small ear fertility maternal and long-grained nonglutinous rice, japonica rice and wide affine mixing breed hybrid F1.

Claims (1)

1. indica-japonica hybrid fertility site Xian round-grained rice fragment metathetical molecule marking method; It is characterized in that; Respectively molecule marker is carried out in the Xian round-grained rice fragment displacement in S5 site, fertility site, S7 site, S8 site and S9 site with molecule marker primer RMz5, RMz7, RMz8 and RMz9, wherein

Use labeled primer RMz5

Left end primer sequence 5 '-AATGGAGTCGACCGTGTGTTCGTCG-3 '

Right-hand member primer sequence 5 '-CCCCAAACCTGAAATCACCAGTGTT-3 '

The wide affine rice varieties DNA that increases, obtaining S5 site labeled fragment size is 105bp; Amplification rice variety DNA, obtaining S5 site labeled fragment size is 145bp; Amplification japonica rice variety DNA, obtaining S5 site labeled fragment size is 131bp;

Labeled primer RMz7

Left end primer sequence 5 '-GGTAACATGCATATCACGCT-3 '

Right-hand member primer sequence 5 '-GTATGCCGATATGCGTAGCC-3 '

Amplification rice variety DNA, obtaining S7 site labeled fragment size is 200bp; Amplification japonica rice variety DNA, obtaining S7 site labeled fragment size is 215bp;

Labeled primer RMz8

Left end primer sequence 5 '-AACCACCACCACCACGCCTCTG-3 '

Right-hand member primer sequence 5 '-CCTTGGAGAGGAGGAGGCGCGC-3 '

Amplification rice variety DNA, obtaining S8 site labeled fragment size is 131bp; Amplification japonica rice variety DNA, obtaining S8 site labeled fragment size is 145bp;

Labeled primer RMz9

Left end primer sequence 5 '-GGTTGTTGGGAGGGAGAAAGGCT-3 '

Right-hand member primer sequence 5 '-TGGAGGCGACGGCGATGTCCTTG-3 '

Amplification rice variety DNA, obtaining S9 site labeled fragment size is 150bp, amplification japonica rice variety DNA; Obtaining S9 site labeled fragment size is 161bp.

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