CN102392074B - Method for directly identifying homologous recombination of higher plant DNA - Google Patents
Method for directly identifying homologous recombination of higher plant DNA Download PDFInfo
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- CN102392074B CN102392074B CN 201110363048 CN201110363048A CN102392074B CN 102392074 B CN102392074 B CN 102392074B CN 201110363048 CN201110363048 CN 201110363048 CN 201110363048 A CN201110363048 A CN 201110363048A CN 102392074 B CN102392074 B CN 102392074B
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Abstract
The invention discloses a method for directly identifying homologous recombination of higher plant DNA, which comprises the following steps: inducing chromosome doubling of embryo sac by taking a plant to be identified as a female parent to obtain 2n female gametes; obtaining a hybrid triploid by the hybridization of the 2n female gametes and male gametes of a paternal plant; screening to obtain a codominant molecular marker primer; conducting PCR (Polymerase Chain Reaction) amplification with the screened codominant molecular marker primer; conducting capillary electrophoresis analysis to the amplification product: if only one of codominant molecular markers of the female parent occurs in the amplification product of the triploid, homologous recombination does not occur at relevant locus of the molecular marker, and if all genetic information of the female parent occur in the amplification product of the triploid, homologous recombination occurs to the pistillate parent at the relevant locus of the molecular marker and heteroduplex DNA is formed. The method has important theoretical and application values on plant genetic analysis, discrimination of molecule marker technical method and guidance on triploid breeding and the like.
Description
Technical field
The present invention relates to a kind of analytical procedure of plant genetic proterties, relate in particular to a kind of method of Direct Identification higher plant dna homology restructuring, belong to the plant genetics field.
Background technology
Reduction division is the heterogamous weight break points of syngenesis species, thereby and biological evolution has been accelerated in the hereditary basis variation that makes sexual cell that occurs between homologous chromosomes in the reduction division process to recombinate.Although people have carried out large quantity research for homologous recombination, it remains one of genetics phenomenon of knowing little.Since the sixties in last century, people have carried out systematic research to homologous recombination mechanism in prokaryotic organism and fungal systems, and multiple homologous recombination model (Holliday R.A mechanism for gene conversion in fungi.Genet.Res proposed in succession, 1964,5:282-304; Meselson M S, Radding C M.A general model of genetic recombination.Proc.Natl.Acad.Sci.USA, 1975,72:358-361; Szostak J W, Orr-weaver T L, Rothstein R J, et al., The double-strand break-repair model for recombination.Cell, 1983,33:25-35).The homologous recombination model of extensively being approved at present is the double-strand break model that Szostak etc. (1983) proposes, and it roughly may be summarized to be DNA chain generation double-strand break; The terminal intrusion of single stranded DNA forms the holliday linker; Move the generation heteroduplex DNA by branch; The Holliday intermediate cuts and base mismatch is repaired.And confirm (the Potter H that exists of holliday linker by Electronic Speculum, Dressler D.On the mechanism of genetic recombination:electron microscopic observation intermediates.Proc.Natl.Acad.Sci.USA, 1976,73:3000-3004).The research of present most genetic recombination phenomenons concentrates on (Padmore R in protokaryon and the fungal organism, Cao L, Kleckner N.Temporal comparison of recombination and synaptonemal complex formation during meiosis in S.cerevisiae.Cell, 1991,66:1239-1256; Goldway M, Sherman A, Zenvirth D, Arbel T, Simchen G.A short chromosomal region with major roles in yeast chromosome III meiotic disjunction, recombination and double strand breaks.Genetics, 1993,133:159-169; Jessop L, Allers T, Lichten M.Infrequent co-conversion of markers flanking a meiotic recombination initiation site in Saccharomyces cerevisiae.Genetics, 2005,169:1353-1367).For higher plant, owing to lacking suitable research material and method, there is not yet so far from dna level and directly observe the method for higher plant homologous recombination and judge whether the relevant evidence that heteroduplex DNA forms.
With regard to plant, after the reduction division of plant sporocyte is finished, its megaspore and microspore cytological development all need could form ovum or sperm by once above mitotic division, and mitotic division has the effect that makes heteroduplex DNA " isozygotying ", so that existence that can't the direct-detection heteroduplex DNA.This mainly is because in the megaspore and microspore cytological development process, form the sister chromosome of two DNA chains " isozygotying " through mitotic dna replication dna by the heteroduplex DNA of Holliday junction structure generation, and through chromosome segregation mitosis anaphase (separating behind the subtrahend), two sister chromosomees enter respectively different gametes.Because the existence of homologous recombination, these two sister chromosome hereditary basiss are not identical, no matter but after this through how many times mitotic division, but each Haploid gamete only has in the on all four sister chromosome of DNA chain.Like this, in the normal diploid hybrid generation, can only judge whether to occur genetic recombination by two or more chain sites or character analysis, and only for a pair of proterties or a site, then whether impalpable homologous recombination occurs, and whether more can't directly differentiate the formation of heteroduplex DNA.
Summary of the invention
Technical problem to be solved by this invention is that the detection method for existing plant homologous recombination is difficult to differentiate the problem whether homologous recombination occurs for a pair of proterties or a site, the method that provides a kind of new Direct Identification higher plant dna homology to recombinate, thus whether the method can have heteroduplex DNA formation to differentiate the size whether this site homologous recombination and recombination fraction occur by detecting.
Technical problem to be solved by this invention is achieved through the following technical solutions:
A kind of method of Direct Identification higher plant dna homology restructuring may further comprise the steps:
(1) take plant to be identified as maternal, applies the physics and chemistry processing and induce embryo sac chromosome doubling to select to obtain the 2n megagamete that two sister chromosomees coexist as same gamete; (2) the microgamete hybridization with 2n megagamete and paternal plant obtains the Triploid induction material; (2) screening obtains the codominant marker primer of the variant and maternal heterozygosis of Parent; (3) DNA that extracts female parent and triploid material is amplification template, as the pcr amplification primer, carries out respectively pcr amplification with the codominant marker primer of screening; (4) amplified production is carried out respectively capillary electrophoresis analysis: if only occur one of them of maternal codominant marker in the amplified production of Triploid induction material, show at this molecule marker related locus homologous recombination does not occur; If occurred maternal whole genetic information in the amplified production of Triploid induction material, illustrated that then pistillate parent to be identified homologous recombination has occured and formed heteroduplex DNA at this molecule marker related locus.
Only carry out the physics and chemistry processing at suitable megaspore developmental stage and could obtain the 2n megagamete that two sister chromosomees coexist as same gamete.The present invention is by further test discovery, only in the mitotic division first time of Embryo Sac Development period (being that the monokaryon blastular is examined between the Embryo Sac Developments to two period) blastular is applied the physics and chemistry processing and could obtain the 2n megagamete that two sister chromosomees coexist as same gamete, thereby separate behind the heteroduplex DNA subtrahend that stops homologous recombination to form, and by with a paternal hybrid relevant information being fixed in the triploid, be the basis that the inventive method is successfully detected the dna homology restructuring.
Publication number is the method that the Chinese invention patent of CN 101147465A discloses a kind of inducing plant embryo sac chromosome doubling to select and breed triploid, this application technical problem to be solved is triploid plantlets induction starting time and efficiency, and the duration of response that this application has disclosed inducing plant embryo sac chromosome doubling to select was four nuclear blastular periods.The present invention is by further test discovery, period it is applied at four nuclear blastulars and to separate after physics and chemistry is processed the heteroduplex DNA subtrahend that can not stop homologous recombination to form, only apply the physics and chemistry processing and induce the blastular mitotic chromosome first time to double when the monokaryon blastular is examined Embryo Sac Development to two, guarantee makes heteroduplex DNA coexist as in the same 2n gamete through two sister chromosomees that copy formation.
It can be the various conventional meanses of inducing plant polyploid that described physics and chemistry is processed, and for example can carry out immersion treatment with colchicine solution, and the concentration of colchicine solution can be 0.3-0.5%, and the time of processing can be 6-48 hour etc.; In addition, also can adopt high temperature that blastular is processed, described high temperature can be 38-44 ℃ etc., and the described treatment time can be 1-8 hour etc.; In a word, the various conventional meanses of these inducing plant polyploids are those skilled in the art and understand thoroughly.
Molecule marker is take the genetic marker of dna molecular polymorphism as the basis, comprising detecting the codominant marker of plant because of the heteroduplex of homologous recombination formation, such as the SSR molecule marker.Whether adopt codominant marker (such as SSR) to analyze the triploid offspring's in embryo sac chromosome doubling to select source genetic information, a situation arises just can to have heteroduplex DNA formation to detect homologous recombination by analysis.According to the maternal sample that will detect, the suitable male parent of selection and hybridization of female parent is screened the codominant marker primer that just can obtain and maternal heterozygosis variant for Parent by Relational database in conjunction with capillary electrophoresis again.
Draw No. 3 poplars (Populus pseudo-simonii * P.nigra ' Zheyin3# ') take pistillate parent to be identified as the wise man and be example, the selected Beijing of the present invention poplar ' (P. * beijingensis) is for drawing the male parent of No. 3 Yang Jinhang hybridization with the wise man.The present invention is by willow SSR data (http://www.ornl.gov/sci/ipgc/ssr_resource.htm) and obtain three pairs of SSR primers of and maternal heterozygosis variant for Parent in conjunction with the capillary electrophoresis repeated screening, and concrete sequence sees Table 1.
Utilization of the present invention comes from the special triploid colony of embryo sac chromosome doubling to select acquisition and the codominant marker that filters out, whether exist heteroduplex DNA to differentiate relevant DNA section by analysis and whether have genetic recombination, show the plant number of these all genetic information of plant in the Triploid induction colony that the statistics embryo sac chromosome doubling to select obtains, thereby calculate comparatively easily the homologous recombination rate of the actual generation of a certain gene locus in certain cross combination.The inventive method is differentiated for plant genetic analysis, molecular marking technique method and is instructed triploid Breeding etc. to have important theory and using value.
Description of drawings
The triploid strain in Fig. 1 primer GCPM_2570 amplification embryo sac chromosome doubling to select source and the capillary electrophoresis collection of illustrative plates behind the parent thereof.
The triploid strain in Fig. 2 primer PMGC_93 amplification embryo sac chromosome doubling to select source and parent's capillary electrophoresis collection of illustrative plates thereof.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can make amendment or replace the details of technical solution of the present invention and form, but these modifications and replacing all fall within the scope of protection of the present invention.
Embodiment 1 detects ' wise man draws poplar No. 3 ' homologous recombination situation test in sporocyte reduction division process
One, test materials:
Maternal material to be detected: ' wise man draws poplar No. 3 ' (P.pseudo-simonii * P.nigra ' Zheyin3# ');
The male parent material: ' Beijing poplar ' (P. * beijingensis).
Two, test method and detected result
When the Embryo Sac Development stage of ' wise man draws poplar No. 3 ' is in and monokaryon blastular period processes (colchicine solution with 0.3-0.5% soaked female inflorescence 6-48 hour) between the two nuclear Embryo Sac Developments blastular being applied physics and chemistry, obtain the 2n megagamete that two sister chromosomees coexist as same gamete; (microgamete of P. * beijingensis) is hybridized the acquisition triploid with 2n megagamete and ' Beijing poplar ';
According to willow SSR database (http://www.ornl.gov/sci/ipgc/ssr_resource.htm), screening is variant for ' wise man draws poplar No. 3 ' and ' Beijing poplar ', and ' wise man draws poplar No. 3 ' is three pairs of codominance SSR primers of heterozygosis, is respectively GCPM_2570, PMGC_93 and WPMS_16 (table 1).
Table 1 is suitable for detecting the SSR primer of the maternal homologous recombination of ' wise man draws poplar No. 3 ' * ' Beijing poplar '
Adopt respectively the polymorphism primers such as GCPM_2570 and PMGC_93 that parent and triploid are carried out pcr amplification and capillary electrophoresis analysis, wherein: 1) utilize primer GCPM_2570 amplification maternal ' wise man draws poplar No. 3 ', at 221bp and 232bp place two peak spectrums are arranged, naming it is the ab type; Male parent ' Beijing poplar ' has two peak spectrums at 217bp and 232bp place, and naming it is cb ' type; The position at peak spectrum place represents the size of the purpose fragment that this primer amplification goes out, and the height of peak spectrum represents the relative dna content of this purpose fragment.The information of combination line spectrum can be divided into 6 types with the Triploid colony plant that embryo sac chromosome doubling to select obtains, and is followed successively by abc; Abb '; Aab '; Aac; Bbc and bbb ' type.The triploid (abc and abb ') that wherein comprises its maternal all genetic information has experienced the dna homology restructuring and has formed heteroduplex in this site.2) utilize primer PMGC_93 amplification maternal ' wise man draws poplar No. 3 ', at 367bp and 375bp place two peak spectrums are arranged, naming it is the ab type; Male parent ' Beijing poplar ' has two peak spectrums at 355bp and 375bp place, and naming it is cb ' type; The Triploid plant that embryo sac chromosome doubling to select obtains can be divided into 6 types equally, be followed successively by abc; Abb '; Aab '; Aac; Bbc and bbb ' type.The triploid (abc and abb ') that wherein comprises its maternal all genetic information has experienced the dna homology restructuring and has formed heteroduplex in this site.
Position by the place, peak and the relative size of peak value are determined the genotype of each plant.The A1 sample is maternal ' wise man draws poplar No. 3 ' in Fig. 1, and defining its genotype is ab; The A2 sample is male parent ' Beijing poplar ', and defining its genotype is cb '.A3-A8 derives from triploid 6 kinds of genotype that embryo sac chromosome doubling to select obtains (abc successively, abb ', aab ', aac, bbc, bbb '), wherein, in this SSR site, B3 and B4 Triploid induction plant contain all genetic information (abc and abb ') of its female parent, show to have experienced the dna homology restructuring in this site and formed heteroduplex.
The B1 sample is ab for ' wise man draws poplar No. 3 ' defines its genotype in Fig. 2; The B2 sample is cb ' for ' Beijing poplar ' defines its genotype; B3-B8 is that six kinds of genotype that derive from the triploid plantlets of embryo sac chromosome doubling to select acquisition (are followed successively by abc, abb ', aab ', aac, bbc, bbb '), wherein at this gene locus place, the plant of B3 and B4 (abc and abb ') representative contains all genetic information of its female parent, shows to have experienced the dna homology restructuring in this site and formed heteroduplex.
Claims (6)
1. the method for Direct Identification poplar dna homology restructuring may further comprise the steps:
(1) take poplar to be identified as maternal, applies the physics and chemistry processing and induce embryo sac chromosome doubling to select to obtain the 2n megagamete that two sister chromosomees coexist as same gamete; (2) the microgamete hybridization with 2n megagamete and paternal plant obtains the Triploid induction material; (2) screening obtains the codominant marker primer of the variant and maternal heterozygosis of Parent; (3) DNA that extracts female parent and Triploid induction material is amplification template, as the pcr amplification primer, carries out respectively pcr amplification with the codominant marker primer of screening; (4) amplified production is carried out respectively capillary electrophoresis analysis: if only occur one of them of maternal codominant marker in the amplified production of Triploid induction material, show at this molecule marker related locus homologous recombination does not occur; If occurred maternal whole genetic information in the amplified production of Triploid induction material, illustrate that then pistillate parent to be identified homologous recombination has occured and formed heteroduplex DNA at this molecule marker related locus, wherein, being in the monokaryon blastular in maternal megaspore growth course examines between the Embryo Sac Developments to two period, be for the first time mitotic division of blastular period, apply described physics and chemistry and process.
2. it is characterized in that in accordance with the method for claim 1: described female parent is ' wise man draws poplar No. 3 ' (P.pseudo-simonii * P.nigra ' Zheyin3# '); Described male parent is ' Beijing poplar ' (P. * beijingensis).
3. according to claim 1 or 2 described methods, it is characterized in that, described physics and chemistry is processed and is comprised: carry out immersion treatment or induce with high temperature with colchicine solution.
4. in accordance with the method for claim 3, it is characterized in that: the concentration of described colchicine solution is 0.3-0.5%, and the described treatment time is 6-48 hour; Described high-temperature temperature is 38-44 ℃, and the described treatment time is 1-8 hour.
5. it is characterized in that in accordance with the method for claim 1: described codominant marker primer is the SSR primer.
6. according to claim 1 or 5 described methods, it is characterized in that: described codominant marker primer is selected from any a group in following 3 groups of primers: the primer pair that (1) is comprised of SEQ ID No.1 and SEQ ID No.2; (2) primer pair that is formed by SEQ ID No.3 and SEQ ID No.4; (3) primer pair that is formed by SEQ ID No.5 and SEQ ID No.6.
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