CN1831138A - Molecular marking supplementary breeding method for multi-purpose restoring series of three-series hybrid rice - Google Patents
Molecular marking supplementary breeding method for multi-purpose restoring series of three-series hybrid rice Download PDFInfo
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Abstract
The invention relates to three-class cross breed paddy multifunctional restorer molecule assistant breeding selection method that includes the following steps: selecting wide compatible gene material and taking polymer cross breed, taking molecule mark assistant selection to excellent individual, taking anther culture of the excellent individual containing at least four different targets gene to gain excellent material, and gaining multifunctional restore series after taking test cross, repeating test cross. The invention supplies an actual method for selective cultivating new species.
Description
One, technical field
The present invention relates to a kind of selection of multi-purpose restoring series of three-series hybrid rice, exactly is recovery gene and the wide compatibility gene of using the auxiliary polymerization different sources of molecular marking technique, with the seed selection multi-purpose restoring series of three-series hybrid rice.
Two, background technology
Cytoplasmic male sterility is the basis that crop heterosis utilizes.China utilizes cytoplasmic male sterility to obtain immense success aspect breeding of hybridized rice.Difference according to the tenuigenin source, produce the rice cytoplasmic male sterile of going up utilization at present and can be divided into Yebai, short type, ridge type, D type, Indonesia's paddy field paddy type, K type, red lotus type, bag bench-type, Yunnan one type, the horse association type etc. of losing by cytoplasmic source, then mainly contain 3 kinds of Yebai, red lotus type and bag bench-types by the male sterile extensive guarantor relation of pair cell matter, these 3 kinds of cytoplasmic male sterilities also are to produce to go up the most frequently used type simultaneously.Genetic research shows, wild-abortive cytoplasmic male sterility (WA-CMS) has 2 main fertility restorer gene Rf3, Rf4 of imitating, wherein Rf3 is positioned at paddy rice the 1st karyomit(e), with RFLP mark Y2-33 and SSR mark RM1 close linkage, Rf4 is positioned at paddy rice the 10th karyomit(e), with RFLP mark Y3-8 and SSR mark RM228 close linkage (Runchun Jing et al, 2001, Bot.Bull.Acad.Sin.42:167-171; Yang Cunyi etc., 2002, Agricultural University Of South China's journal, 23 (4): 30-33; Zhang Qunyu etc., 2002, Acta Genetica Sinica, 29 (11): 1001-1004; What brilliance etc., 2002, Acta Genetica Sinica, 27 (4): 304-310).HL cytoplasm male sterile (HL-CMS) also has 2 main fertility restorer gene Rf5, Rf6 of imitating, these 2 are recovered gene and all are located in paddy rice the 10th karyomit(e), linkage analysis proves, the genetic distance of Rf3 and SSR mark RM1108 is 0.9cM only, and Rf6 and RM5373 are divided into from (Liu Xuequn, 2004, Wuhan University's Ph D dissertation).2 main efficient recovery gene Rf1a, Rf1b that closely link to each other of bag bench-type cytoplasmic male sterility (BT-CMS) are by Japan and Chinese scholar clone, also be positioned on paddy rice the 10th karyomit(e), with fL610 mark close linkage (Komori etc., 2003, Euphytica, 129:241-247; Wang Zhonghua, 2004, Agricultural University Of South China's Ph D dissertation).The genetic linkage analysis that Chinese scholar is carried out shows, being located between Rf4, Rf5, Rf6 and Rf1a on paddy rice the 10th karyomit(e), the Rf1b gene is inequipotential (Liu Xuequn, 2004, Wuhan University's Ph D dissertation).
The discovery of paddy rice wide compatibility and the further investigation of wide compatibility gene are for the successful utilization of subspecies indica and japonica hybrid advantage provides theoretical foundation and basic substance.The S5-n that is positioned on paddy rice the 6th karyomit(e) is the wide compatibility gene site of finding the earliest between the paddy rice indica and japonica subspecies, can explain the sterile phenomenon of most typical subspecies indica and japonica hybrids.The S5-n site is between marker gene C (purple bran point) and the wx, and is located between RFLP molecule marker RG138 and the RG2349, apart from RG2349 1.0cM (Zhu Susong, 2005, Agricultural University Of Nanjing's Ph D dissertation) only.
Utilize wide affinitive material and different Xian, round-grained rice to recover system's hybridization, the offspring select in the system by Continuous Selection and constantly test cross identify that forefathers have cultivated some wide-congenial restoring lines.Ren Guangjun etc. (1999, the rice in China science, 13 (2): 120-122) hybridize with 871028/Lemont, progeny material through how for Continuous Selection, select elite plant strain anther culture, the wide-congenial restoring line of breeding to become the natural setting percentage of 7 sterile lines (Zhenshan 97a, golden 23A, big grain A, Gang No.46A, K17A, II-32A, D62A) of extensive 448 pairs of 5 kinds of source of cytoplasm all to reach more than 80% to F6 generation; Through 4 wide compatibility test kind of evaluations, become extensive 448 to have wide compatibility.Zhu Xudong etc. (2004, China's agronomy circular, 20 (1): 40-42) with samsara 422 for extensively affine, the donor that recovers gene is lost in the open country, with Italian japonica rice Cripto hybridization, in the F2 of 3000 strains colony, select 43 elite seedlings, 122 of F3 test crosses are handed in pairs, F4 selects 7 82 individual plants in the excellent strains system, F5 carries out 53 and hands in pairs, F6 selects 37 individual plants in the excellent strains system, F7 carries out 7 and hands in pairs, and F8 is for all performance is excellent, thereby seed selection becomes to have good general combining ability, it is T461 that the wide affine inclined to one side round-grained rice of wide spectrum recovers, this recovery system can lose extensive open country, short losing, Indonesia paddy field paddy, the bag bench-type, Xian such as Yunnan type, round-grained rice three is a sterile line, and is very approaching with setting percentage indica type and round-grained rice type test kind of hybridization.
Tight chrysanthemum waits (1995 by force, Acta Genetica Sinica, 22 (5): 361-371) with wide affinitive material CPSLO17 and bright extensive 63 hybridization, wide-congenial restoring line TG7, TG8 that F1 cultivates through flower all have 1 pair of wide compatibility gene, and TG7 and TG8 have the fertility restorer gene of 2 couples of WA-CMS and 1 couple of Yunnan one type CMS respectively.Teng Lisheng etc. (1996, Acta Agronomica Sinica, 22 (2): 142-146) with the setting percentage be the fertility index, having studied that japonica rice recovers is that C57 and ZH157 are to BT type, the restorative heredity of WA type CMS, find that a pair of dominance that C57 has BT-CMS simultaneously recovers a pair of Incomplete dominance of gene and WA-CMS by force and recover gene, ZH157 then has a pair of Incomplete dominance recovery gene of BT-CMS simultaneously and a pair of dominance of WA-CMS is recovered gene by force, and the fertility restorer of both strong recovery gene pairs sterile lines plays a leading role.Yellow Qingyang etc. (2000, Scientia Agricultura Sinica, 33 (3): 8-13) when the fertility inheritting of research Cong Guang 41A, Zhenshan 97a and Maxie A, find, though the non-equipotential of the genic male sterile gene of Cong Guang 41A and Zhenshan 97a, Maxie A, extensive guarantor's relation is also obviously different, but when use special blue or green, close positive 23, win excellent No. 2, bright extensive 63,6078, IR661,3037 and IR72 etc. when recovering system with Cong Guang 41A, Zhenshan 97a and Maxie A test cross, F1 all can be normally solid, illustrates that above-mentioned recovery has the recovery gene that recovers these three kinds of sterile lines in being simultaneously.These studies confirm that cultivates the possibility that the paddy rice broad spectrum, multi purpose recovers system.
Studies have shown that in a large number, the paddy rice of different sources is recovered gene and wide compatibility gene polymerization, can cultivate the wide spectrum wide-congenial restoring line, but in the systematic breeding process,, must test through the descendant and could determine whether selected individual plant contains recovery gene and wide compatibility gene owing to do not know the genotype of selected individual plant in advance, in order to reduce blindness, have to select a large amount of individual plants to carry out descendant's test, thereby need the more manpower and materials of cost, prolong the breeding time limit.Therefore yet there are no the report that can all have restorative by force wide spectrum wide-congenial restoring line simultaneously to Yebai, red lotus type, bag bench-type cytoplasmic male sterility.
Along with the biological development of modern rice molecular, people constantly filter out rice cytoplasmic male sterile recovery gene and the closely linked molecule marker of wide compatibility gene with different sources, but reason owing to aspects such as experiment condition and information communications, the actual numerous scientific workers that are engaged in the field breeding are difficult to directly utilize these molecule markers, cause these molecule markers to fail to be used widely in the seed selection of wide spectrum wide-congenial restoring line.
Three, summary of the invention
The present invention is the innovation on a kind of method, be with the hybridization in the molecular marking technique of modern molecular biology, anther culture technique and the traditional Crop Genetic Breeding, backcross, reestablish diplomatic relations, test cross and multiple choices technology organically combine, for crop breeding of new variety, novel material initiative provide quick, accurately, can predict and practical method.Specifically this supplementary breeding method comprises choosing and contains the wide compatibility gene material and recover system with Yebai, red lotus type and bag bench-type respectively and carry out convergent cross, elite seedling in its large group segregating generation is aided with molecular marking technique to be selected, the elite seedling of selecting that contains a plurality of goal gene is carried out anther culture, until the stable elite clone that all isozygotys that obtains to contain 4 kinds of the above object genes, obtain stable multi-purpose restoring through test cross, the selection of reestablishing diplomatic relations again.Described molecular marking technique be exactly extraction separation from generation to generation in the DNA of elite seedling be template, carry out PCR with two primers of certain nucleotide sequence (5 '-3 ') and react and the SSR polymorphism analysis, to determine to contain a plurality of goal gene.On electrophorogram, can know and demonstrate different goal gene.
Particular content is as follows:
1, choose have wide compatibility gene material (as CPSLO17, zero wheel 11, CB-1, train short by 64,02428, HP121 etc.) respectively with Yebai (bright extensive 63,057, bright extensive 86, another name for Sichuan Province extensive 527, Zhenjiang 084, Zhejiang extensive 7954,9019 etc.), red lotus type (is raised rice No. 6, green rice 24 etc.), bag bench-type (C57, C418, R18, extensive No. 9 of Anhui, C fort etc.) etc. recovery system hybridization, and according to breeding objective, carry out the convergent cross of different modes: is seed selection based on the many of Yebai with recovering---Yebai recovery system ///the wide affine system of round-grained rice/wide affine system of red lotus type recovery system // Xian/bag bench-type recovery system, is seed selection based on the many of red lotus type with recovering---red lotus type recover system ///the wide affine system of round-grained rice/Yebai recovers the wide affine system of system // Xian/bag bench-type and recovers system, is seed selection based on the many of bag bench-type with recovering---the bag bench-type recover system ///round-grained rice wide affine be 1/ Yebai recovery system // round-grained rice extensively affine be that the recovery of 2/ red lotus type is.
2, in the segregating generation of large group, select elite seedling according to the leaf attitude of strain, growing way appearance etc. earlier, with whether containing goal gene in the molecular markers for identification primary election individual plant, mark contains the selected individual plant of at least 4 kinds of various objectives genes simultaneously again.
3, to the selected individual plant that contains at least 4 kinds of various objectives genes of mark, get part children fringe at heading stage and carry out anther culture, isozygoty when quickening a plurality of goal gene; Determine selected individual plant according to the comprehensive agronomy proterties again when ripe and press individual plant results capacity seed.Following season plant into large group again and carry out the molecular marker assisted selection second time, and get young fringe and carry out anther culture, until obtaining to contain more than 4 kinds the various objectives gene stable elite clone that all isozygotys at least.
4, plant above-mentioned elite clone, be carried out to test cross with dissimilar sterile lines, following season will be selected the high elite clone of combining ability, repetition measurement again; According to the repetition measurement result, select the excellent person of stablizing of performance to be shaped into multi-purpose restoring.
Four, description of drawings
Accompanying drawing is depicted as the part electrophorogram, knows among the figure to demonstrate different goal gene.
Fig. 1, Rf3 detect the paddy rice Yebai and recover gene
Fig. 1, Rf4 detect the paddy rice red lotus type and recover gene
Fig. 3, Rf1 detect paddy rice BT type and recover gene
Five, embodiment
Be example based on the many of Yebai with recovering now, be described below with seed selection.
1, preparation convergent cross
Another name for Sichuan Province extensive 527/ // 02428/ raises rice No. 6 // train short 64/C418.This polymerization is handed among the used parent, and another name for Sichuan Province extensive 527 is that the Yebai long-grained nonglutinous rice recovers system, have restorer strong (containing Rf3, Rf4 gene), combining ability height, product are of fine quality, the leaf attitude of strain is good, than characteristics such as blast resistings; The 02428th, the leaf attitude of strain is good, has the wide affinitive material of japonica rice of S5-n wide compatibility gene; Raising rice is for No. 6 that the red lotus type long-grained nonglutinous rice recovers system, has restorer strong (containing Rf5, Rf6 gene), combining ability height, characteristics such as product are of fine quality, the leaf attitude of strain is good, strong stress resistance; Training short 64 is that the leaf attitude of strain is good, has the wide affinitive material of long-grained nonglutinous rice of S5-n wide compatibility gene; C418 is that bag bench-type japonica rice recovers system, and characteristics such as have restorer strong (containing Rf1a, Rf1b gene), combining ability height, product are of fine quality, the leaf attitude of strain is good have certain wide compatibility simultaneously.
2, the molecular marker assisted selection of segregating generation
Because Rf4, Rf5, Rf6, Rf1 gene coexist on paddy rice the 10th karyomit(e), and be not allelotrope, the reorganization that therefore will obtain 4 beneficial genes just needs bigger segregating population.Early generations such as F2, F3 are handed in polymerization at the large group of strain more than 4000, earlier according to about 400 individual plants of primary election such as the leaf attitudes of strain, again with closely linked molecule markers of gene such as Rf3, Rf4, Rf5, Rf6, Rf1 and S5-n the genomic dna of primary election individual plant being carried out label screening respectively, eliminate the individual plant that does not contain these genes or only contain the minority gene, and the individual plant of as far as possible selecting said gene all to isozygoty, obtain the selected individual plant that the 40-50 strain contains 4 kinds of various objectives genes at least.
2.1, the extraction of genomic dna
About water intaking rice young leaflet tablet 0.5g, place the 2.0ml centrifuge tube, adding liquid nitrogen is ground to Powdered, add 700ul DNA extraction damping fluid and (contain 100mmol/L Tris-HCl, 20mmol/L EDTA, 500mmol/L NaCl, 1.5%SDS), vibration water-bath 1 hour under 60 ℃ of conditions adds isopyknic chloroform when taking out the back to room temperature: primary isoamyl alcohol (24: 1), left standstill 30 minutes after putting upside down abundant mixing back and forth, centrifugal 15 minutes of 10000rpm, supernatant liquor is moved in the 1.5ml centrifuge tube, add the Virahol of isopyknic precooling, mixing, left standstill under the room temperature 30 minutes, centrifugal 10 minutes of 10000rpm abandons supernatant, precipitation 400ul 70% washing with alcohol, centrifugal 10 minutes of 10000rpm, abandon supernatant, repeated washing once is deposited under the room temperature and is dissolved in after the drying in the 50ul redistilled water.
2.2, the two primer sequences of mark, it is synthetic to give birth to the worker by Shanghai.
| Gene-mark | Primer sequence | Clip size | Reference |
| Rf3-RM1 | F：5′-GCGAAAACACAATGCAAAAA-3′ R：5′-GCGTTGGTTGGACCTGAC-3′ | 114bp | What brilliance etc., 2002, Acta Genetica Sinica, 29 (9): 798～802 |
| Rf4-RM228 | F：5′-CTGGCCATTAGTCCTTGG-3′ R：5′-GCTTGCGGCTCTGCTTAC-3′ | 154bp | Runchun Jing et al， 2001，Bot.Bull. Acad.Sin.42： 167-171 |
| Rf5-RM1108 | F：5′-GCTCGCGAATCAATCCAC-3′ R：5′-CTGGATCCTGGACAGACGAG-3′ | 124bp | Liu Xuequn, 2004, Wuhan University's Ph D dissertation |
| Rf6-RM5373 | F：5′-GGAGATGCTATAGCAGCAGTG-3′ R：5′-ATTGCTCCTTACCACCTTGC-3′ | 110bp | Liu Xuequn, 2004, Wuhan University's Ph D dissertation |
| Rf1-fL610 | F：5′-TTGACGCCTTCGTCCTCT-3′ R：5′-AGACGTAACAAGATGATCG-3′ | 590bp | Ichikawa N et al.， 1997，Molecular Breeding，3：195-202 |
| Sn-5-RM50 | F：5′-ACTGTACCGGTCGAAGACG-3′ R：5′-AAATTCCACGTCAGCCTCC-3′ | 210bp | Yan Changjie etc., 2000, Acta Genetica Sinica, 27 (5): 409-417 |
2.3, the SSR polymorphism analysis
In the 10ul reaction system, contain 2ul 5 * SSR Buffer, 1ul 2mM dNTPs, 0.5ul 50mM MgCl
2, the upstream and downstream primer of each 0.8ul 2.5uM, 0.5U Taq archaeal dna polymerase, oryza sativa genomic dna template 1ul, the sterilization distilled water complements to 10ul.
The PCR response procedures is 95 ℃ of pre-sex change 5 minutes, connect 94 ℃ 40 seconds, 57 ℃ 45 seconds, 72 ℃ 60 seconds, totally 30 circulations, connect 72 ℃ 7 minutes, 4 ℃ of preservations then.Reaction product is horizontal strip electrophoresis on 3% sepharose, and voltage stabilizing 180V write down the result after electrophoresis 1.5-2 hour in gel imaging system; Or on 6%PAGE glue, walk vertical slab electrophoresis, and voltage stabilizing is between 220V-300V, and electrophoresis time was generally 1.5-2 hour.Electrophoresis finishes back silver and dyes colour developing, record mark band or photographic recording on the film scenograph.
3, anther culture
Get young fringe and carry out anther culture from containing more than 4 kinds the selected individual plant of various objectives gene, quicken isozygotying of goal gene.
4, test cross primary election
At the early generation of seed selection, with representational sterile line selected individual plant is carried out preliminary survey and identify.To auxiliary 40-50 the selected individual plant of molecule marker, use Yebai (Zhenshan 97a), red lotus type (the safe A in Guangdong) and bag bench-type (6,000 hot A) sterile line to be carried out to respectively to handing over preliminary survey.According to the performance of test cross, selection is 10-15 to the strain that 3 kinds of sterile lines all show combining ability height, setting percentage good (75%).From this 10 several strains system, select about 100 of the excellent individual plants of performance, and confirm about 10 of the individual plant that target gene all isozygotys with molecule marker.
5, repetition measurement final election
In the middle generation of seed selection, the individual plant of selecting each target gene all to isozygoty carries out repetition measurement with the sterile line of number of different types and identifies.With each individual plant that selects in the preliminary survey, all plant in next season the 300-500 strain than large group, therefrom selecting elite seedling is that sterile line Zhenshan 97a, Xieqingzao A, golden 23A, Gang No.46A, II-32A, D62A, K17A, Guangdong safe A, green three A, 6,000 hot A, 80-4A, 9201A, 23A and two-line sterile line are trained short 64S, X07S, 7001S etc. and carried out extensive repetition measurement with dissimilar three respectively.Select to show in the repetition measurement evaluation and recover to compose individual plant wide, that setting percentage is high, combining ability is strong, the comprehensive agronomy proterties is stablized excellence or strain system.
6, final election expands numerous
During repetition measurement identified the individual plant of choosing or
Strain is an expanding propagation, the further increasing selected to press, and the sterile line that is used in performance excellence in the repetition measurement repeatedly repeats testcross and screening targetedly, identifies wide compatibility on the one hand, identify hybrid vigour on the other hand, thereby cultivate wide spectrum, wide affine multi-purpose restoring.
Claims (1)
1, a kind of molecular marking supplementary breeding method of multi-purpose restoring series of three-series hybrid rice, comprise choosing and contain the wide compatibility gene material and recover system with Yebai, red lotus type and bag bench-type respectively and carry out convergent cross and its segregating generation is carried out molecular marker assisted selection and anther culture, test cross and repetition measurement, it is characterized in that: described molecular marker assisted selection be exactly extraction separation from generation to generation in the DNA of elite seedling be template, two primers with certain nucleotide sequence (5`-3`) carry out PCR reaction and SSR polymorphism analysis, and described pair of primer is as follows:
F：5’-GCGAAAACACAATGCAAAAA-3’
R：5’-GCGTTGGTTGGACCTGAC-3’
F：5’-CTGGCCATTAGTCCTTGG-3’
R：5’-GCTTGCGGCTCTGCTTAC-3’
F：5’-GCTCGCGAATCAATCCAC-3’
R：5’-CTGGATCCTGGACAGACGAG-3’
F：5’-GGAGATGCTATAGCAGCAGTG-3’
R：5’-ATTGCTCCTTACCACCTTGC-3’
F：5’-TTGACGCCTTCGTCCTCT-3’
R：5’-AGACGTAACAAGATGATCG-3’
F：5’-ACTGTACCGGTCGAAGACG-3’
R：5’-AAATTCCACGTCAGCCTCC-3’。
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