CN103416297B - New method for mapping corn S-type cytoplasm male sterility fertility restoring gene Rf3 - Google Patents
New method for mapping corn S-type cytoplasm male sterility fertility restoring gene Rf3 Download PDFInfo
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Abstract
The invention belongs to the technical field of corn gene mapping, in particular to a new method for mapping a corn S-type cytoplasm male sterility fertility restoring gene Rf3. According to a conventional method for mapping a qualitative character gene, a backcross or selfing population is constructed, the gene type of an individual is inferred through phenotype identification, molecular marker detection is performed, and the position of the gene is determined through two-point and three-point tests. When the Rf3 gene is mapped with the conventional method, the phenotype identification is affected by human and environmental factors easily, the workload is large and the size of the population to be mapped is limited. The mapping method aims to overcome the defects of the conventional method for mapping the Rf3 gene and does not need the phenotype identification. The new method is characterized in that due to the fact that the gene type of the utilized population to be mapped is already known, the phenotype does not need to be identified, a large population can be constructed, and enough recombined individuals can be found in one generation to fine map the Rf3 gene. The method for mapping the Rf3 gene is effective and promotes three-line sterility seed production by the utilization of the Rf3 gene in production.
Description
Technical field
The invention belongs to corn gene field of locating technology, be specifically related to the novel method of a kind of mapping maize S type cytoplasmic male sterilty fertility restorer gene Rf3.
Background technology
One of world's Three major grain crops is not only by corn, or important feed and insutrial crop.From future development, corn will be the crop that demand growth is the fastest in the world, and the production potential excavating corn are the keys meeting this demand.The raising of corn yield is mainly realized by plantation cross-fertilize seed, and therefore, preparation has high yield potential, highly purified cross-fertilize seed is the key link ensureing corn with high yield.Produce in the process of cross-fertilize seed and need to carry out maternal emasculation, in the process of intensive manufacture cenospecies, maternal emasculation workload is large, and seed costs is high, and often because emasculation is not thorough, produces selfed seed and cause purity of hybrid to reduce.The new way of the cross-fertilize seed sterilization production of hybrid seeds has been opened up in the excavation of corn male sterility germ plasm resource and research.Utilize nuclear male sterility (Genic Male Sterility, GMS) SPT method for seed producing and utilize the three-line indica hybrid riace of cytoplasmic male sterilty (Cytoplasmic Male Sterility, CMS) to be two kinds of main method of the sterilization production of hybrid seeds.At present, SPT method for seed producing has received the patent protection of Pioneer Electronic Corp. of the U.S..In T, C and S three groups of CMS sterile lines, the sterile line of T and CI subgroup and cross-fertilize seed subject to infecting of southern corn leaf blight T and C microspecies, and S group sterile line is to T, C microspecies performance immunity.Therefore, S type CMS sterile line has very important practice significance to China's Maize Production.
The expression of CMS by plasmone and cell nucleus gene co-controlling, plasmone be sterile type and in nucleus without Restore gene time, it shows as male sterile; Under normal cell plasmagene or nucleus Restore gene existent condition, its fertility restorer is normal.The Sterile cytoplasm classification of S type CMS is S, and its male sterile Restore gene is a dominant gene Rf3, and be positioned on corn No. 2 chromosome long arm, belong to gametophyte male sterile, Fertility reaction is controlled by gametophytic genotype.At F
1in generation, the pollen of 50% is with Rf3, and showing as loose powder can educate; The pollen of 50%, with rf3 gene, shows as not loose powder.
In production, the most kytoplasm of corn inbred line of use is for can educate type, and without Restore gene.Therefore, in the three-line indica hybrid riace utilizing CMS, need the maternal kytoplasm transformation of cross-fertilize seed to be sterile type and by Restore gene transformation in male parent.Wherein, because kytoplasm is mainly undertaken transmitting by megagamete and little to the Phenotype of plant, therefore the transformation of kytoplasm is fairly simple.The transformation of Restore gene then needs the mode by backcrossing to carry out.
In crop breeding, the backcross transformation of gene has two kinds of modes: based on the conventional breeding methods of Phenotypic Selection and the breeding method of molecular marker assisted selection.The former efficiency is low, the cycle long, blindness is large, can not meet the demand of modern breeding; The blindness that the latter can improve breeding efficiency, shorten breeding cycle, reduces breeding, becomes the trend of modern breeding gradually.Want to carry out the auxiliary backcross transformation of molecule marker to gene, its prerequisite to know the accurate location of gene, namely gene location.
Mitochondrial DNA belongs to qualitative character.Qualitative character refers to the proterties showing as discontinuous change.This proterties mostly is Dominant gene, less by E&H background influence, and therefore location is got up relatively easy.The ultimate principle of the assignment of genes gene mapping is 2 tests and three point test, and it primarily works is assembly target group.According to the difference of the colony of assembly, qualitative trait gene location mainly can be divided into following two kinds of methods:
Method one, utilizes the qualitative trait gene of backcross population (BC colony) to locate
BC colony is by first-filial generation F
1backcross with recessive parent and obtain.Judge individual genotype by phenotypic evaluation, detect its molecular marker gene type, utilize test at 2 and three point test calculates genetic distance between gene and each molecule marker, by the assignment of genes gene mapping between two molecule markers.
Method two, utilizes inbreeding population (F
2colony) qualitative trait gene location
F
2colony is by first-filial generation F
1selfing obtains.From colony, filter out phenotype is recessive individuality, detects its molecular marker gene type, utilizes test at 2 and three point test calculates genetic distance between gene and each molecule marker, by the assignment of genes gene mapping between two molecule markers.
Above two kinds of its core concepts of method are the same, namely infer the genotype of segregating population by phenotype and carry out Markers for Detection to it, and the genetic distance utilizing 2 tests and three point test to calculate between gene and molecule marker carrys out gene location.Difference is that the individuality utilized in BC colony has two types: heterozygote and recessiveness individuality, and F
2in colony, due to dominant homozygote and heterozygote cannot be distinguished, so generally only utilize recessive individuality to carry out the location of gene.
That the Rf3 assignment of genes gene mapping mainly utilizes is BC
1f
1colony, this colony is to F by common material (N (rf3/rf3))
1(S (Rf3/rf3)) pollination obtains.In flowering period, phenotypic evaluation is carried out to each individual plant, judge that it is fertile plant or sterile strain according to its loose powder situation.From colony, the molecule marker of random choose part individual plant (n >=200) on corn No. 2 chromosome long arm detects, found and the closely linked molecule marker of Rf3 gene by 2 tests, to be positioned between two molecule markers at the beginning of Rf3 gene by three point test, then find in first positioning area and exploitation molecule marker, and detect whole target group with it, find the individual plant that restructuring occurs in first positioning area, according to the position Fine Mapping Rf3 gene that restructuring occurs.
This localization method relates generally to two aspects: the detection of molecule marker and the qualification of phenotype.At present, molecular marking technique reaches its maturity, and can not become the restraining factors of the assignment of genes gene mapping, so the result of phenotypic evaluation just seems particularly important, once occur that error directly will affect the result of location.The loose powder of corn is very large by the impact of weather, and there are differences due to the growth in colony between individuality, and the loose powder time is also inconsistent, can not once just the phenotype of whole colony have been investigated, the investigation that needs carry out repeatedly.So by the loose powder situation of visual inspection individual plant judge its phenotype often result there is error and workload is large.The method of this phenotypic evaluation have impact on the reliability of positioning result on the one hand, also limit the size of Rf3 assignment of genes gene mapping colony on the other hand, often can not find enough restructuring individual plants carry out Fine Mapping to it a generation.
Summary of the invention
The present invention is directed to that existing corn Rf3 assignment of genes gene mapping phenotypic evaluation is subject to artificially, shortcoming that such environmental effects and the workload day of one's doom formulate position group size, a kind of Rf3 gene positioning method not needing to carry out phenotypic evaluation is proposed.Feature of the present invention is that the target group's genotype utilized is known, and phenotype, without the need to qualification, can build large group, sufficient restructuring individuality just can be found to carry out Fine Mapping to Rf3 gene a generation.The present invention proposes a kind of effective means for location Rf3 gene, promotes that it is the sterilization production of hybrid seeds that production utilizes Rf3 gene to carry out three.
The present invention carry out the Rf3 assignment of genes gene mapping build colony time with F
1(S (Rf3/fr3)) is male parent, and Zheng 58 (N (rf3/rf3)) pollinates for female parent.Because S type CMS belongs to gametophyte male sterile, thus only with Rf3 pollen can loose powder and participate in fertilization, the colony's genotype obtained thus is all Rf3/rf3.Because this colony's genotype is known, so do not need target group's seed to seed into the ground, inferring genotype in flowering period by observing loose powder situation, directly can propose to seed the qualification that DNA carries out molecule marker.
First from colony, random choose some individuals (n >=200) carries out molecule marker on corn No. 2 chromosome long arm and Rf3 gene 2 and tests.Assuming that molecule marker to be detected genotype in restorer is A/A, the genotype in conventional corn is a/a.The first situation, when itself and Rf3 gene wide apart (genetic distance is greater than 50cM), F
1can produce the gamete of 4 types: Rf3A, rf3a, Rf3a and rf3A, ratio is 1: 1: 1: 1, F
1when pollinating to Zheng 58, rf3a and rf3A type pollen not loose powder, so can only produce two kinds of genotypic offspring: Rf3A/rf3a and Rf3a/rf3a, ratio is 1: 1 (Figure 1A).The second situation, when itself and the complete linkage of Rf3 gene, F
1only can produce the gamete of 2 types: Rf3A and rf3a, ratio is 1: 1, F
1when pollinating to Zheng 58, rf3a type pollen not loose powder, so can only produce a kind of genotypic offspring: Rf3A/rf3a (Figure 1B).The third situation, when itself and the incomplete sex-linkage of Rf3 gene, if the genetic distance between them is xcM (0<x<50), F
1the gamete of 4 types can be produced: Rf3A, rf3a, Rf3a and rf3A, ratio is 1-x%: 1-x%: x%: x%, F
1when pollinating to Zheng 58, rf3a and rf3A type pollen not loose powder, so can only produce two kinds of genotypic offspring: Rf3A/rf3a and Rf3a/rf3a, ratio is 1-x%: x% (>1: 1) (Fig. 1 C).By 2 tests find with the closely linked molecule marker of Rf3 gene after, to be positioned between two molecule markers at the beginning of Rf3 gene by three point test again, then find in first positioning area and exploitation molecule marker, and detect whole target group with it, find the individual plant that restructuring occurs in first positioning area, according to the position Fine Mapping Rf3 gene that restructuring occurs.Assuming that will be positioned between molecule marker A1 and A2 at the beginning of Rf3 gene, have found again two molecule marker B1 and B2 between A1 and A2, the genotype of restorer is A1B1B2A2/A1B1B2A2, and the genotype of common material is a1b1b2a2/a1b1b2a2, F
1if recombinate between B1 and B2 when producing gamete, the genotype of restructuring gamete is A1B1b2a2 and a1b1B2A2, F
1after pollinating to Zheng 58, if detect the individuality that genotype is A1B1b2a2/a1b1b2a2, illustrate that Rf3 gene is between A1 and B2, if detect the individuality that genotype is a1b1B2A2/a1b1b2a2, then Rf3 gene between B1 and A2 (Fig. 2) is described.
Compare conventional localization method, the advantage utilizing the present invention to carry out the corn Rf3 assignment of genes gene mapping is, because target group's genotype is known, so do not need target group's seed to seed into the ground to carry out phenotypic evaluation, directly can propose to seed the detection that DNA carries out molecule marker, first avoid phenotypic evaluation result and forbidden caused deviations, secondly do not limit by group size, save man power and material, improve location efficiency, again locate not by the restriction in corn planting season, save time.
Accompanying drawing explanation
Fig. 1: utilize localization method of the present invention, molecule marker and Rf3 gene are carried out to the schematic diagram of 2 tests, A figure represents the genotype individual in (genetic distance is greater than 50cM) target group when molecule marker and Rf3 gene wide apart and ratio thereof, B figure represents the genotype individual in target group when molecule marker and the complete linkage of Rf3 gene and ratio thereof, the genotype of individuality and ratio thereof in target group when the genetic distance of C figure expression between molecule marker and Rf3 gene is xcM (0<x<50).
Fig. 2: will be positioned at the beginning of Rf3 gene after between molecule marker A1 and A2, utilizes the inner individuality that restructuring occurs between first positioning area Rf3 gene to be carried out to the schematic diagram of Fine Mapping.
Fig. 3: the Technology Roadmap of the present invention of setting forth for example with the position fixing process of Rf3 gene.
The positioning result of Fig. 4: Rf3 gene, A figure represents just positioning result, and B figure represents Fine Mapping result.
Embodiment
By the position fixing process of Rf3 gene, the present invention is described in detail:
In (1) 2012 year summer, Changping County, Beijing farm, to be maternal with the corn inbred line SD13 (S (Rf3/Rf3)) of Restore gene, Zheng 58 (N (rf3/rf3)) is male parent, assembly F
1(S (Rf3/rf3)).
In (2) 2012 year winter, Nan Fan base, Lingshui, Hainan, uses F
1pollinate to Zheng 58, gather in the crops 20000 seeds (N (Rf3/rf3)).
(3) 20000 seed simple grains extract DNA.
(4) screening and exploitation have polymorphic and on corn No. 2 chromosome long arm that band is special molecule marker between SD13 and Zheng 58.
(5) in above-mentioned 20000 parts of DNA, random choose 2000 parts detects with molecule marker, utilizes 2 methods of testting to have found some molecule markers closely linked with Rf3 gene: bnlg1520, A7, A105, UMC1525, A81, A121, A165, CG2, A134, A70, p22 and UMC1736 and the molecule marker with the complete linkage of Rf3 gene: 1-17,1-62,2-43, SEQ58, CAP33.Utilize the method for three point test to depict the genetic map of these marks and Rf3 gene, be positioned between A165 and CG2 by the beginning of Rf3 gene, the physical distance on B73 reference sequences is about 1Mb (Fig. 4 A).
(6) detect above-mentioned 20000 parts of DNA with molecule marker A165 and CG2, find the individual plant occurring to recombinate between A165 and CG2.
(7) with all molecule markers between A165 and CG2, restructuring individual plant is detected, the position occurred according to recombinating is by Rf3 gene Fine Mapping between molecule marker 2-43 and 4-1, and the physical distance on B73 reference sequences is about 600kb (Fig. 4 B).
Claims (1)
1. a method of mapping maize S type cytoplasmic male sterilty fertility restorer gene Rf3, is characterized in that:
(1) with the corn inbred line SD13 with Restore gene for female parent: S, genotype are for Rf3/Rf3, and Zheng 58 is male parent: N, genotype is rf3/rf3, and assembly F1:S, genotype are Rf3/rf3;
(2) take F1 as male parent, Zheng 58 is maternal, assembly target group: N, Rf3/rf3;
(3) target group's single seed extracts DNA;
(4) random choose some individuals, n >=200, detect with the molecule marker on corn No. 2 chromosome long arm, the method utilizing test at 2 is found and the closely linked molecule marker of Rf3 gene, utilize the method for three point test to draw these marks and the genetic map of Rf3 gene, will be positioned between certain two molecule marker at the beginning of Rf3 gene;
(5) find in first positioning area and develop molecule marker, and detecting whole target group with it, finding the inner individuality that restructuring occurs between first positioning area, according to the position Fine Mapping Rf3 gene of restructuring generation.
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| UY35928A (en) * | 2013-12-31 | 2015-07-31 | Dow Agrosciences Llc | ? GEN Rf3 CYTOPLASMATIC ANDROESTERILITY RESTORER (CMS) TYPE S ?. |
| UY35927A (en) * | 2013-12-31 | 2015-07-31 | Dow Agrosciences Llc | ? GENE RESTORATOR RF3 TYPE S OF MALE CYLOPLASM MATIC STERILITY (CMS), MOLECULAR MARKERS AND ITS USES ?. |
| CN103947538A (en) * | 2014-04-28 | 2014-07-30 | 中国农业科学院烟草研究所 | Method for positioning two non-allelic genes for controlling same character of plant |
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| CN101760539A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Development and class of genetic marker |
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| CN101760539A (en) * | 2008-12-19 | 2010-06-30 | 李祥 | Development and class of genetic marker |
Non-Patent Citations (2)
| Title |
|---|
| 玉米Rf3近等基因系的分子标记辅助回交选育与效益分析;夏军红等;《作物学报》;20020531;第28卷(第3期);339-344 * |
| 运用近等基因系(NIL)、AFLP、RFLP和SCAR标记对玉米S组育性恢复基因(Rf3)的研究;王泽立等;《遗传学报》;20011231;第28卷(第3期);465-470 * |
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