CN101717819A

CN101717819A - Method for screening specific molecular markers of rice fertility restorer genes

Info

Publication number: CN101717819A
Application number: CN200910155608A
Authority: CN
Inventors: 黄得润; 庄杰云; 元芳丽; 樊叶杨
Original assignee: China National Rice Research Institute
Current assignee: China National Rice Research Institute
Priority date: 2009-12-17
Filing date: 2009-12-17
Publication date: 2010-06-02

Abstract

The invention provides a method for screening specific molecular markers of rice fertility restorer genes, which is characterized by applying rice germ plasm resources carrying and not carrying fertility restorer genes and screening the molecular markers, the sterile lines and (or) the restorer lines of which are in specific banding patterns, from the molecular markers closely linked with the rice fertility restorer genes Rf3 and Rf4. With the markers adopted to carry out auxiliary selection on the molecular markers of rice fertility restorer genes, the breeding efficiency of the sterile lines and the restorer lines of indica type rice can be improved.

Description

The screening method of specific molecular markers of rice fertility restorer genes

Technical field

The present invention relates to a kind of method of utilizing rice germplasm resource screening specific molecular marker, particularly utilize the method for rice germplasm resource Screening of Rice fertility restorer gene specific molecular marker.

Background technology

Rice cytoplasmic male sterile (CMS) and fertility restorer thereof are the bases of realizing the paddy rice three series mating.In many CMS, Yebai is to use the earliest, use area sterile cytoplasm type the most widely in China's ternary hybrid rice, and other kytoplasm types (K type, short lose type, D type, ridge type and Indonesia paddy field paddy type) more to producing upward application have similar extensive guarantor's relation.The fertility restorer of this class kytoplasm is controlled by two major genes mainly, is respectively Rf3 and the long-armed Rf4 of the 10th karyomit(e) that is positioned at the 1st the short arm of a chromosome.

In the practices of breeding, recover the screening of gene and normally determined by a large amount of offspring's test crosses, its shortcoming be the cycle long, workload is big, the while phenotypic evaluation is subjected to the influence of envrionment conditions to a certain extent, seed selection efficient is low.In recent years, effectively used at breeding practice gradually based on the molecular marker assisted selection of genotype identification.On the localized basis of fertility restorer gene, can screen and recover the closely linked molecule marker of gene, but often run into the combination specificity problem of mark polymorphism, promptly between specific parent, be polymorphic mark, between the parent of breeding population, may present singlet, need screen polymorphism mark again at the parent of concrete breeding population, both increase workload, improve the difficulty of breeding work again.If can the bigger germ plasm resource of number of applications, filter out the specific marker relevant with fertility restorer gene, just can provide great facility for carrying out of rice fertility restorer genes molecular marker assisted selection.

Summary of the invention

The purpose of this invention is to provide a kind of method of screening the fertility restorer gene specific molecular marker, thereby the molecule marker that obtains to can be used for the assisting sifting indica cms line and recover system quickens the rice breeding process, improves efficiency of selection, saves cost.

The present invention is achieved by the following technical solution: with 43 parts of indica restorer lines that carry paddy rice CMS fertility restorer gene Rf3 and Rf4 and 23 parts of indica cms lines that do not carry these two genes is material, select and Rf3 and the closely linked PCR of Rf4 gene (polymerase chain reaction) mark, use corresponding primer and carry out pcr amplification, amplified production is electrophoresis detection on 6% non-denaturing polyacrylamide gel.According to 66 parts of germ plasm resource Molecular Detection results, (detected band does not detect in recovering system in the sterile line if one is marked at sterile line and recovers to present between the system specificity difference, and detected band does not detect in sterile line yet in the recovery system), or (particular bands only detects in partial sterility system to detect specific band at sterile line, and in any recovery is, do not detect), or (particular bands is only detecting in the part recovery is to detect specific band in recovery system, and detect in what sterile line not in office), then with the specific molecular marker of this marker for judgment for its linked gene of evaluation.

6 linked markers through using the Rf3 gene and 5 linked markers of Rf4 gene detect 66 parts of rice materials, microsatellite marker RM10338 and RM10353 are defined as identifying the specific molecular marker of Rf3 gene, RM6100 and RM1108 are defined as identifying the specific molecular marker of Rf4 gene; Wherein, RM10338 presents the specificity difference between sterile line and recovery system, and other 3 are marked at recovery system and detect specific band.

Description of drawings

Figure 1A is that 23 parts of Yebai male sterile lines and 43 parts of dna marker detected results of recovering system are the detected result of molecule marker RM10338;

Figure 1B is that 23 parts of Yebai male sterile lines and 43 parts of dna marker detected results of recovering system are the detected result of molecule marker RM6100.

Specific embodiments

Shown in Figure 1A, Figure 1B, M:Marker; 1:II-32B; The excellent 62B of 2:D; 3:V20B; 4: rich B; 5: the fragrant 29A in river; 6: phenanthrene changes B; 7: Feng Yuan A; 8: ridge 46B; 9: golden 23B; 10: Long Tepu B; 11: interior fragrant 2A; 12: interior fragrant 85A; 13: day rich A; 14: the blue or green B early of association; 15: seal 32B; 16: excellent IB; 17: Zhenshan 97B; 18: middle 100A; 19: middle 1A; 20: middle 2B; 21: middle 3B; 22: middle 9B; 23: middle Zhejiang A; 24:207; 25:253; 26:926; 27:CDR22; 28:R402; 29:To974; 30:ZDZ057; 31: survey 64; 32: No. 1, polyphyly; 33: grace extensive 58; 34: spoke extensive 718; 35: wide extensive 128; 36: osmanthus 99; 37: river extensive 151; 38: Lu extensive 17; 39: Milyang 46; 40: continuous extensive 501; 41: continuous extensive 725; 42: bright extensive 63; 43: bright extensive 70; 44: bright extensive 77; 45: bright extensive 86; 46: another name for Sichuan Province extensive 162; 47: another name for Sichuan Province extensive 527; 48: salt extensive 559; 49: suitable extensive 1577; 50: press down extensive 084; 51:CH238; 52:CH59; 53: in extensive 218; 54: in extensive 8006; 55: in extensive 111; 56: in extensive 1176; 57: in extensive 333; 58: in extensive 161; 59: special blue or green; 60:IR24; 61: in extensive 8012; 62: in extensive 8015; 63: in extensive 465; 64: in extensive 7492; 65:R600; 66: extensive 66.

Concrete screening method:

1. the screening of fertility restorer gene specific molecular marker

66 parts of rice materials comprise 23 Yebai male sterile lines or maintenance line (II-32B, the excellent 62B of D, V20B, rich B, the fragrant 29A in river, phenanthrene changes B, Feng Yuan A, ridge 46B, gold 23B, Long Tepu B, interior fragrant 2A, interior fragrant 85A, it rich A, the blue or green B early of association, seal 32B, excellent IB, Zhenshan 97B, middle 100A, middle 1A, middle 2B, middle 3B, middle 9B, in Zhejiang A) and 43 recover to be (207,253,926, CDR22, R402, To974, ZDZ057, survey 64, No. 1, polyphyly, grace extensive 58, spoke extensive 718, wide extensive 128, osmanthus 99, river extensive 151, Lu extensive 17, Milyang 46, continuous extensive 501, continuous extensive 725, bright extensive 63, bright extensive 70, bright extensive 77, bright extensive 86, another name for Sichuan Province extensive 162, another name for Sichuan Province extensive 527, salt extensive 559, suitable extensive 1577, press down extensive 084, CH238, CH59, in extensive 218, in extensive 8006, in extensive 111, in extensive 1176, in extensive 333, in extensive 161, special blue or green, IR24, in extensive 8012, in extensive 8015, in extensive 465, in extensive 7492, R600, extensive 66).

11 of the PCR marks that is applied to screen, comprise with the chain microsatellite marker of Rf3 6 (RM10338, RM10340, RM10353, RM6289, RM3746, RM10376), with chain mark microsatellite marker 3 (RM6100, RM3510, RM1108) and 2 of the preface mark marks (0860,0159) of Rf4.Adopt general experimental technique to carry out DNA extraction, pcr amplification, amplified production is electrophoresis detection on 6% non-denaturing polyacrylamide gel, and Silver Nitrate silver dyes colour developing.

Shown in Figure 1A, in the detected result of Rf3 linked marker RM10338, the banding pattern of 23 parts of sterile line material and 43 parts of banding patterns that recover system are all inequality, and promptly RM10338 can be picked as the specific marker of differentiating the Rf3 different genotype; Shown in Figure 1B, in the detected result of Rf4 lock mark RM6100,43 are recovered to have in the system 38 to detect all undetected band of all sterile lines (long fragment), and promptly RM6100 can be picked as and identify that Rf4 recovers the specific molecular marker of gene.

2. segregating population checking

Be the reliability of verifying that above-mentioned mark is selected, be chosen in isolating 112 RILs on Rf3 and the Rf4 gene, using RM10338, RM10353, RM6100 and RM1108 identifies, 20 parts of candidates of screening acquisition recover is and 27 parts of candidate's maintenance lines, respectively with middle 9A and Xieqingzao A configuration cross combination, the natural setting percentage of examination F1.It is to be respectively 60.3% and 63.1% with the average setting percentage of the F1 of middle 9A and Xieqingzao A that the candidate recovers, and the average setting percentage of F1 of candidate's maintenance line and middle 9A and Xieqingzao A is respectively 18.8% and 14.2%.These results show that it is effective utilizing RM10338, RM10353, RM6100 and RM1108 to carry out the genotypic selection of paddy rice CMS fertility restorer gene.

Claims

1. the screening method of a specific molecular markers of rice fertility restorer genes is characterized in that being applied in and carries not homoallelic rice germplasm resource on the target gene seat, filters out the molecule marker that is specificity difference on isoallele not; Promptly use the rice germplasm resource carry and not carry fertility restorer gene, from rice fertility restorer genes Rf3 and the closely linked molecule marker of Rf4, filter out sterile line and (or) recover the molecule marker that system is special banding pattern.

2. the screening method of a kind of specific molecular markers of rice fertility restorer genes as claimed in claim 1, it is characterized in that selecting for use 23 Yebai cytoplasmic male sterile lines and 43 recoveries is the long-grained nonglutinous rice material, from with the closely linked molecule marker of Rf3, filter out sterile line and (or) recover molecule marker RM10338 and RM10353 that system is special banding pattern.

3. the screening method of a kind of specific molecular markers of rice fertility restorer genes as claimed in claim 2, it is characterized in that selecting for use 23 Yebai cytoplasmic male sterile lines and 43 recoveries is the long-grained nonglutinous rice material, from with the closely linked molecule marker of Rf4, filter out and recovering molecule marker RM6100 and the RM1108 that system is special banding pattern.

4. as the screening method of claim 2 and 3 described a kind of specific molecular markers of rice fertility restorer genes, it is characterized in that identifying the genotype of each material of segregating population on Rf3 and Rf4 seat, obtain to recover system and sterile line material, identify its effect of checking by fertility.