CN103088018A - Intragenic single nucleotide polymorphism (SNP) mark of male sterility restoring gene RF4 of C-type cytoplasm of corn - Google Patents
Intragenic single nucleotide polymorphism (SNP) mark of male sterility restoring gene RF4 of C-type cytoplasm of corn Download PDFInfo
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- CN103088018A CN103088018A CN2012105959735A CN201210595973A CN103088018A CN 103088018 A CN103088018 A CN 103088018A CN 2012105959735 A CN2012105959735 A CN 2012105959735A CN 201210595973 A CN201210595973 A CN 201210595973A CN 103088018 A CN103088018 A CN 103088018A
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Abstract
The invention discloses an intragenic single nucleotide polymorphism (SNP) mark of a male sterility restoring gene RF4 of C-type cytoplasm of corn. The intragenic SNP mark is obtained by the following steps of: constructing a near-isogenic line by using a good inbred line 87-1 of corn as a basic material, and performing DNA (deoxyribonucleic acid) extraction on leaves of a single plant of an offspring of the near-isogenic line; performing molecular marker development on the extracted DNA, and acquiring and processing molecular marker data; screening an 87-1 BAC library by using primers on two sides of a target gene to obtain a clone containing the restoring gene RF4, sequencing the clone, and performing gene prediction according to a target sequencing result; and performing functional analysis according to the result of gene prediction, and obtaining the intragenic SNP mark of the male sterility restoring gene of the C-type cytoplasm. According to the invention, fine positioning of the male sterility restoring gene of the C-type cytoplasm and the intragenic SNP mark of a fertility restoring gene are realized, and the technical problem in molecular detection of the male sterility restoring gene of the C-type cytoplasm of corn is solved.
Description
Technical field
The invention belongs to the gene genetic technical field, relate in particular to the interior SNP mark of gene and the application thereof of corn C type cytoplasmic male sterility fertility restorer gene RF4.
Background technology
Male sterile phenomenon ubiquity in higher plant, male sterile can be divided into again that core is sterile, two types of cytoplasmic sterilities, wherein, cytoplasmic male sterility is to make mutually the sterile type of control by Cytoplasmic male sterile genes (S) and nucleus sterile gene (rfrf).A kind of normal recovery gene that has is arranged in plasmone and cell nucleus gene, and fertility can be restored.The genotype of sterile line is S (rfrf); The genotype of maintenance line is N (rfrf), does male parent and sterile line hybridization with it, and the offspring who obtains can keep maternal sterility; The genotype of restorer is S (RfRf), N (RfRf), does male parent and sterile line hybridization with it, and the offspring who obtains can make maternal sterility be recovered.Just because of this hereditary property of cytoplasmic male sterility, can realize " three series mating " of sterile line, maintenance line, restorer, can be widely used in producing the preparing hybrid kind, be to produce the upper main Types of using.
Utilize sterile line preparation corn hybrid seed, can exempt artificial emasculation, reduce production costs; Avoid improving seed purity because of the not thorough impurity of seeds that causes of artificial emasculation; Save the energy that tassel consumes because growing, increase the output of cross-fertilize seed.According to the special effect principle of the fertility restorer that recovers the gene pairs sterile line, mitochondrial DNA system can be divided into T, S, C three types.The CMS-C type is the sterile type of sporophyte, and the sterile line fertility is highly stable, is the male sterile type of producing widespread use, and therefore clear and definite corn C MS-C Fertility-restoration Mechanism is cloned its fertility restorer gene extremely important to the application of this sterile type.
SNP propose to be used in 1996 by Lander, is take the molecular marking technique of sequencing technologies as the basis, reflection be between different biology individual on genomic level due to the caused DNA sequence polymorphism of the variation of single core thuja acid.Because a base mutation in genome just can form a SNP, thereby its quantity is very abundant.The difference of this single base can show (Collins, 1998) by design specific PCR primer amplification and electrophoresis detection.SNP is as third generation molecule marker, have own unique advantage: SNP no longer with the length polymorphism of DNA fragmentation as detection means, but directly serve as a mark with sequence variations; The detection means of SNP mark is enriched constantly, is not confined to classical gel electrophoresis, replaces DNA microarray and chip technology.Along with the further investigation to SNPs, important breakthrough (Lindblad et al., 2000 have been obtained in pattern animals and plants and important farm crop; Rafalski, 2002). according to Tenaillon and Rafalski report, at corn 3, just there are a SNP difference (Ching et al., 2002 end non-translational region and coding region every 48 bases and 130 bases respectively; Tenaillon et al., 2001; Rafalski, 2002); Nasu etc. (2002) find 2,800 SNPs altogether according to the DNA sequence dna of 3 rice varieties in 250,000 bases, average every 89 bases just have a SNP.
Summary of the invention
The invention provides SNP mark and application thereof in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4, be intended to solve the prior art system and fail to realize the Fine Mapping of corn C type cytoplasmic male sterility fertility restorer gene and the problem of carrying out the gene internal labeling on the DNA sequencing basis.
The object of the present invention is to provide the interior SNP mark of gene of a kind of corn C type cytoplasmic male sterility fertility restorer gene RF4, the method comprises the following steps:
Step 1, build the near isogenic line that comprises restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) take maize elite inbred line 87-1 as base mateiral, get single-strain blade near isogenic line offspring's the litmus mouth phase and carry out DNA extraction;
Step 3 with the primer screening 87-1BAC library of target gene both sides, obtains comprising the clone who recovers gene Rf4, and this clone is checked order, and carries out predictive genes according to the target sequencing result;
Further, in step 1, build when comprising the near isogenic line of restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) the target gene Rf4 source self-mating system A619 near isogenic line take maize elite inbred line 87-1 as base mateiral.
Further, near isogenic line offspring's the male loose powder phase of taking out, individual plant is carried out the fertility investigation, Fertility rating adopts 6 grade standards of Li Jing hero to reach, and 6 grade standards of Li Jingxiong are:
When flower pesticide exposes 0%, it is 0 grade; When flower pesticide exposes 0-5%, it is 1 grade; When flower pesticide exposes 6-25%, it is 2 grades; When flower pesticide exposes 26-50%, it is 3 grades; When flower pesticide exposes 51-75%, it is 4 grades; When flower pesticide exposes 76-100%, it is 5 grades;
The reclassify standard of Khey-Pour is: when flower pesticide does not ftracture fully, be the I level; When flower pesticide partly ftractures, be the II level; During the flower pesticide normal crack, be the III level;
Be 4 grades in 6 grade standards at Li Jingxiong, and when being the III level in the reclassify standard of Khey-Pour, this individual plant is fertile plant;
Be 5 grades in 6 grade standards at Li Jingxiong, and when being the III level in the reclassify standard of Khey-Pour, this individual plant is fertile plant;
Be 0 grade in 6 grade standards at Li Jingxiong, and when being the I level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 1 grade in 6 grade standards at Li Jingxiong, and when being the I level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 2 grades in 6 grade standards at Li Jingxiong, and when being the II level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 3 grades in 6 grade standards at Li Jingxiong, and when being the II level in the reclassify standard of Khey-Pour, this individual plant is sterile strain.
Further, getting near isogenic line offspring's the litmus mouth phase implementation method that single-strain blade carries out DNA extraction is:
Get the fresh corn male sterility mutant blade grind into powder in liquid nitrogen of 5g, in the centrifuge tube of the 50mL that packs into;
Add the SDS extracting solution 15mL that is preheated to 65 ℃ in centrifuge tube, add the beta-mercaptoethanol mixing of 30-50 μ L, 65 ℃ of water-bath 40-60min;
Take out centrifuge tube, add 5M KAC5mL, ice-water bath 10min;
Add the mixed solution of the chloroform of 15mL and primary isoamyl alcohol and abundant mixing, both hands slowly shook 1 hour, and solution is blackish green at the bottom of the centrifuge tube, places 10min under room temperature, and chloroform and primary isoamyl alcohol allocation ratio are 24: 1;
At 4 ℃ of temperature, with 12000r/min, the centrifugal 15min of rotating speed;
Slowly shift in supernatant liquor to a clean 50mL centrifuge tube with the 1mL liquid-transfering gun that cuts off head, and add wherein the Virahol of 15mL precooling, rotate gently mixing, be placed in 4 ℃ of refrigerators, DNA cotton-shaped the separating out that be white in color becomes bulk, and wherein Virahol needs place under-20 ℃ to spend the night;
With glass needle, the careful hook of DNA is gone out, be placed in dry free of contamination 1.5mL centrifuge tube, 75% washing with alcohol 2 times, then add absolute ethanol washing once, again add at last dehydrated alcohol that DNA is preserved under 4 ℃ of conditions;
Used time is outwelled ethanol, and seasoning DNA adds TE or ultrapure water dissolving DNA.
Further, in step 2, the implementation method of the DNA that extracts from single-strain blade being carried out molecular markers development is:
Search the DNA sequence dna of the BACs in 8.00,8.01 districts on the maizeGDB website, and at http://linux1.softberry.com/berry.phtml? topic=fgenesh﹠amp; Group=programs﹠amp; Subgroup=gfind predicted gene structure;
According to the prediction of gene structure result, find the corresponding sequence of intron, exon on DNA sequence dna;
Intron sequences is compared at http://blast.ncbi.nlm.nih.gov/ together with the exon sequence of each 100bp of both sides, finds out the single-copy sequence of target section;
The single-copy sequence that obtains is designed primer on Primer Primer5.0.
Further, when design intron mark, to the upstream and downstream primers of intron, on the exon of intron both sides, amplified production length is at 200bp respectively for two chains of primer, and beginning and end position are no less than 30bp apart from 5 ' and 3 ' end respectively; Primer length 18-25bp; Annealing temperature is between 56 ℃-65 ℃; And the Tm value of upstream and downstream primer is more or less the same in 2 ℃; The content of G+C is between 40%-60%.
Further, in step 2, molecular marker data is collected when processing, the banding pattern of parent's restorer Cms-ES87-1 (Rf4Rf4) is designated as 1, the banding pattern of parent's sterile line Cms-ES87-1 (rf4rf4) is designated as 2;
Banding pattern recording method near isogenic line offspring BC1F1 colony is: the isozygoty banding pattern consistent with restorer is designated as 1, and the isozygoty banding pattern consistent with sterile line is designated as 2, and the banding pattern of heterozygosis is designated as 3, and disappearance is designated as 0.
Further, in step 3, with the primer screening 87-1BAC library of target gene both sides, obtain comprising the clone who recovers gene Rf4, and this clone is checked order, carry out predictive genes according to the target sequencing result on FGENESH.
further, in step 4, carry out functional analysis according to the predictive genes result, and when obtaining in the gene of C type cytoplasmic male sterility fertility restorer gene the SNP mark, according to predicting the outcome, carry out functional analysis on GO, by the sequencing result analysis as can be known, from the about 45000Nt of nearest two the mark X-21-1 of target gene and the physical distance between X-33, predict by gene function, have 9 possible gene regions between two marks, wherein 2 have corresponding gene on the corn inbred line B73 that has checked order, these two gene regions are carried out gene sequencing in different restorers and sterile line, the sequence of restorer and the sequence of sterile line are found respectively the difference of 4 SNP.
SNP mark and application thereof in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 provided by the invention, build the near isogenic line that comprises restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) take maize elite inbred line 87-1 as base mateiral, get single-strain blade near isogenic line offspring's the litmus mouth phase and carry out DNA extraction; The DNA that extracts from single-strain blade is carried out molecular markers development, and molecular marker data is collected and processed; With the primer screening 87-1BAC library of target gene both sides, obtain comprising the clone who recovers gene Rf4, and this clone is checked order, carry out predictive genes according to the target sequencing result; Carry out functional analysis according to the predictive genes result, and obtain the interior SNP mark of gene of C type cytoplasmic male sterility fertility restorer gene; The method has realized the Fine Mapping of C type cytoplasmic male sterility fertility restorer gene and the interior SNP mark of gene of fertility restorer gene, utilize this SNP mark, solved the molecular detection technology problem of corn C type cytoplasmic male sterility fertility restorer gene, practical, have stronger propagation and employment and be worth.
Description of drawings
Fig. 1 is the realization flow figure of SNP mark and application thereof in the gene of the corn C type cytoplasmic male sterility fertility restorer gene RF4 that provides of the embodiment of the present invention;
Fig. 2 is the growth schematic diagram of sterile individual plant in the near isogenic line offspring that the embodiment of the present invention provides;
Fig. 3 is the growth schematic diagram that can educate individual plant in the near isogenic line offspring that the embodiment of the present invention provides;
Fig. 4 is the schematic diagram that molecule marker that the embodiment of the present invention provides carries out the BC1 population analysis;
Fig. 5 is the schematic diagram to the Fine Mapping of recovering gene Rf4 that the embodiment of the present invention provides;
Fig. 6 is BAC clone and the 87-1 positive control qualification result schematic diagram that the embodiment of the present invention provides;
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is described in further detail.Should be appreciated that specific embodiment described herein only in order to explaining the present invention, and be not used in and limit invention.
Fig. 1 shows the realization flow of SNP mark in the gene of the corn C type cytoplasmic male sterility fertility restorer gene RF4 that the embodiment of the present invention provides and application thereof.
The method comprises the following steps:
Step S101, build the near isogenic line that comprises restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-1 (rf4rf4) take maize elite inbred line 87-1 as base mateiral, get single-strain blade near isogenic line offspring's the litmus mouth phase and carry out DNA extraction;
Step S102 carries out molecular markers development to the DNA that extracts from single-strain blade, and molecular marker data is collected and processed;
Step S103 with the primer screening 87-1BAC library of target gene both sides, obtains comprising the clone who recovers gene Rf4, and this clone is checked order, and carries out predictive genes according to the target sequencing result;
Step S104 carries out functional analysis according to the predictive genes result, and obtains the interior SNP mark of gene of C type cytoplasmic male sterility fertility restorer gene.
In embodiments of the present invention, in step S101, build when comprising the near isogenic line of restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) the target gene Rf4 source self-mating system A619 near isogenic line take maize elite inbred line 87-1 as base mateiral.
In embodiments of the present invention, near isogenic line offspring's the male loose powder phase of taking out, individual plant is carried out the fertility investigation, Fertility rating adopts 6 grade standards of Li Jing hero to reach, and 6 grade standards of Li Jingxiong are:
When flower pesticide exposes 0%, it is 0 grade; When flower pesticide exposes 0-5%, it is 1 grade; When flower pesticide exposes 6-25%, it is 2 grades; When flower pesticide exposes 26-50%, it is 3 grades; When flower pesticide exposes 51-75%, it is 4 grades; When flower pesticide exposes 76-100%, it is 5 grades;
The reclassify standard of Khey-Pour is: when flower pesticide does not ftracture fully, be the I level; When flower pesticide partly ftractures, be the II level; During the flower pesticide normal crack, be the III level;
Be 4 grades in 6 grade standards at Li Jingxiong, and when being the III level in the reclassify standard of Khey-Pour, this individual plant is fertile plant;
Be 5 grades in 6 grade standards at Li Jingxiong, and when being the III level in the reclassify standard of Khey-Pour, this individual plant is fertile plant;
Be 0 grade in 6 grade standards at Li Jingxiong, and when being the I level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 1 grade in 6 grade standards at Li Jingxiong, and when being the I level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 2 grades in 6 grade standards at Li Jingxiong, and when being the II level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 3 grades in 6 grade standards at Li Jingxiong, and when being the II level in the reclassify standard of Khey-Pour, this individual plant is sterile strain.
In embodiments of the present invention, getting near isogenic line offspring's the litmus mouth phase implementation method that single-strain blade carries out DNA extraction is:
Get the fresh corn male sterility mutant blade grind into powder in liquid nitrogen of 5g, in the centrifuge tube of the 50mL that packs into;
Add the SDS extracting solution 15mL that is preheated to 65 ℃ in centrifuge tube, add the beta-mercaptoethanol mixing of 30-50 μ L, 65 ℃ of water-bath 40-60min;
Take out centrifuge tube, add 5M KAC5mL, ice-water bath 10min;
Add the mixed solution of the chloroform of 15mL and primary isoamyl alcohol and abundant mixing, both hands slowly shook 1 hour, and solution is blackish green at the bottom of the centrifuge tube, places 10min under room temperature, and chloroform and primary isoamyl alcohol allocation ratio are 24: 1;
At 4 ℃ of temperature, with 12000r/min, the centrifugal 15min of rotating speed;
Slowly shift in supernatant liquor to a clean 50mL centrifuge tube with the 1mL liquid-transfering gun that cuts off head, and add wherein the Virahol of 15mL precooling, rotate gently mixing, be placed in 4 ℃ of refrigerators, DNA cotton-shaped the separating out that be white in color becomes bulk, and wherein Virahol needs place under-20 ℃ to spend the night;
With glass needle, the careful hook of DNA is gone out, be placed in dry free of contamination 1.5mL centrifuge tube, 75% washing with alcohol 2 times, then add absolute ethanol washing once, again add at last dehydrated alcohol that DNA is preserved under 4 ℃ of conditions;
Used time is outwelled ethanol, and seasoning DNA adds TE or ultrapure water dissolving DNA.
In embodiments of the present invention, in step S102, the implementation method of the DNA that extracts from single-strain blade being carried out molecular markers development is:
Search the DNA sequence dna of the BACs in 8.00,8.01 districts on ma izeGDB website, and at http://linux1.softberry.com/berry.phtml? topic=fgenesh﹠amp; Group=programs﹠amp; Subgroup=gfind predicted gene structure;
According to the prediction of gene structure result, find the corresponding sequence of intron, exon on DNA sequence dna;
Intron sequences is compared at http://blast.ncbi.nlm.nih.gov/ together with the exon sequence of each 100bp of both sides, finds out the single-copy sequence of target section;
The single-copy sequence that obtains is designed primer on Primer Primer5.0.
In embodiments of the present invention, when design intron mark, to the upstream and downstream primers of intron, two chains of primer are respectively on the exon of intron both sides, amplified production length is at 200bp, and beginning and end position are no less than 30bp apart from 5 ' and 3 ' end respectively; Primer length 18-25bp; Annealing temperature is between 56 ℃-65 ℃; And the Tm value of upstream and downstream primer is more or less the same in 2 ℃; The content of G+C is between 40%-60%.
In embodiments of the present invention, in step S102, molecular marker data is collected when processing, the banding pattern of parent's restorer Cms-ES87-1 (Rf4Rf4) is designated as 1, the banding pattern of parent's sterile line Cms-ES87-1 (rf4rf4) is designated as 2;
Banding pattern recording method near isogenic line offspring BC1F1 colony is: the isozygoty banding pattern consistent with restorer is designated as 1, and the isozygoty banding pattern consistent with sterile line is designated as 2, and the banding pattern of heterozygosis is designated as 3, and disappearance is designated as 0.
In embodiments of the present invention, in step S103, with the primer screening 87-1BAC library of target gene both sides, obtain comprising the clone who recovers gene Rf4, and this clone is checked order, carry out predictive genes according to the target sequencing result on FGENESH.
in embodiments of the present invention, in step S104, carry out functional analysis according to the predictive genes result, and when obtaining in the gene of C type cytoplasmic male sterility fertility restorer gene the SNP mark, according to predicting the outcome, carry out functional analysis on GO, by the sequencing result analysis as can be known, from the about 45000Nt of nearest two the mark X-21-1 of target gene and the physical distance between X-33, predict by gene function, have 9 possible gene regions between two marks, wherein 2 have corresponding gene on the corn inbred line B73 that has checked order, these two gene regions are carried out gene sequencing in different restorers and sterile line, find the difference of 4 SNP, wherein, the sequence of restorer is:
GAATAGATGATCTGATGA?TTATCGCAG?C?AGCATTACATTTTTTGCATGAAATCGGAT
GGCTTCTTCAAAGGAGCCATGTGCGAGCTACGTCTGAGCAGCGACAATATTGTCCTGA
CGCTTCCCTGTTGCAAGATTTAGATGGCTGCTATCCTTTGCAGTTGATCAGGAATGGT
GTGCTGTTCTAAGGAAGCTTCTGAACACCATGTTCCAGGGTGATATTGATGTATTGTCA
The sequence of sterile line is:
GAATAGATGATCTGATGA?TTATCGCAG?C?AGCATTACATTTTTTGCATGAAATCGGAT
GGCTTCTTCAAAGGAGCCATGTGCGAGCTACGTCTGAGCAGCGACAATATTGTCCTGA
CGCTTCCCTGTTGCAAGATTTAGATGGCTGCTATCCTTTGCAGTTGATCAGGAATGGT
GTGCTGTTCTAAGGAAGCTTCTGAACACCATGTTCCAGGGTGATATTGATGTATTGTCA。
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
1, the Fine Mapping of C type cytoplasmic male sterility fertility restorer gene
1.1 target group builds
Built the near isogenic line that comprises single-gene Rf4Rf4, rf4rf4 (NIL) take maize elite inbred line 87-1 as base mateiral, respectively restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4), target gene Rf4 source self-mating system A619 in the 87-1 near isogenic line, this material is verified contains single recovery gene Rf4 (Hu et al., 2006), and built the approximately BC of 45000 strains
1F
1Segregating population is used for Fine Mapping.
Plantation 45000 strains of BC1F1 colony, individual plant is listed, and litmus mouth phase individual plant is got blade and is kept in-70 ℃ of refrigerators, is used for the extraction of DNA.Individual plant is carried out fertility investigation taking out the male loose powder phase.Fertility rating adopts 6 grade standards of Li Jing hero and the reclassify standard of Khey-Pour.
Wherein: 4/III, 5/III are fertile plant, and 0/I, 1/I are sterile strain, and 2/II, 3/II are the partial sterility strain.
1.2DNA extract
(1) get the fresh corn male sterility mutant blade grind into powder in liquid nitrogen of 5g, in the centrifuge tube of the 50mL that packs into;
(2) add the SDS extracting solution 15mL that is preheated to 65 ℃ in centrifuge tube, then add the beta-mercaptoethanol mixing of 30-50 μ L, 65 ℃ of water-bath 40-60min;
(3) take out centrifuge tube, add 5M KAC5mL, ice-water bath 10min;
(4) add the 15mL chloroform: primary isoamyl alcohol (24: 1) is mixing fully, and both hands shake slowly that solution is blackish green at the bottom of the centrifuge tube, approximately 1 hour, place 10min under room temperature;
(5) use chloroform: after primary isoamyl alcohol (24: 1) balance, at 4 ℃ of temperature, with 12000r/min, the centrifugal 15min of rotating speed;
(6) slowly shift in supernatant liquor to a clean 50mL centrifuge tube with the 1mL liquid-transfering gun that cuts off head, and add wherein the Virahol of approximately 15mL precooling (20 ℃ of placements are spent the night), and rotate gently mixing, be placed in 4 ℃ of refrigerators, DNA cotton-shaped the separating out that be white in color, agglomerating;
(7) with glass needle, the careful hook of DNA is gone out, be placed in dry free of contamination 1.5mL centrifuge tube, 75% washing with alcohol 2 times, then add absolute ethanol washing and once again add at last dehydrated alcohol that DNA is preserved (4 ℃);
(8) used time is outwelled ethanol, and then seasoning DNA adds TE or ultrapure water dissolving DNA.
1.3 molecular markers development
The sequence of searching the BACs in 8.00,8.01 districts on the maizeGDB website, with microsatellite sequence research tool SSRHUNTER one by one BACs seek the SSR site, BLAST on NCBI, seek the SSR site of single copy, utilize primer-design software Primer5.0 to SSR site flank conserved sequence design upstream and downstream primer.The design of primers principle is: implementation sequence length is greater than 100bp, the beginning of SSR sequence and end position are no less than 25bp apart from 5 ' and 3 ' end respectively, primer length 18-25bp, annealing temperature is between 55 ℃-65 ℃, and the Tm value of upstream and downstream primer is more or less the same in 2 ℃, (G+C) content 40%-60%, pcr amplification product length 100-300bp.
Genome sequence according to the corn inbred line B73 that announces on the www2.genome.arizona.edu/genomes/maize website, find 8.00, the 8.01 corresponding base sequences in district, compare on http://blast.ncbi.nlm.nih.gov/, seek the DNA sequence dna (removing the single copy and the intron sequences list copy that comprise the SSR site) of single copy, the single-copy sequence of acquisition utilizes Primer5.0 to carry out design of primers.The design of primers principle: primer length is arranged on 18～22bp, and forward and reverse primer length difference is less than 3bp; The Tm value of primer is controlled at 56～62 ℃, and forward and reverse primer Tm value difference is different should be less than 3 ℃; PCR product length is controlled at 400bp and is advisable with interior.
The intron zone of the gene exon of comparing, conservative property is relatively poor, easily produces insertion and deletion, thereby it is larger to produce the possibility of polymorphism.The detailed process of exploitation intron mark is: (1) at first finds corresponding DNA sequence dna; (2) exist
Http:// linux1.softberry.com/berry.phtml? topic=fgenesh﹠amp; Group=programs﹠amp; Subgroup
=gfindThe structure of predicted gene; (3) according to predicting the outcome, find the corresponding sequence of intron, exon on DNA sequence dna; (4) intron sequences is compared at http://blast.ncbi.nlm.nih.gov/ together with the exon sequence of each 100bp of both sides, finds out the single-copy sequence of target section; (5) single-copy sequence that obtains is designed primer on Primer Primer5.0.Design intron mark precaution: to the upstream and downstream primers of intron, on the exon of intron both sides, amplified production length is in the 200bp left and right respectively for two chains of primer; Beginning and end position are no less than 30bp apart from 5 ' and 3 ' end respectively; Primer length 18-25bp; Annealing temperature is between 56 ℃-65 ℃; And the Tm value of upstream and downstream primer is more or less the same in 2 ℃; (G+C) content 40%-60%.
1.4 molecular marker data is collected and is processed
The banding pattern of parent's restorer Cms-ES87-1 (Rf4Rf4) is designated as 1, the banding pattern of parent's sterile line Cms-ES87-1 (rf4rf4) is designated as 2, the banding pattern recording method of near isogenic line offspring BC1F1 colony: the isozygoty banding pattern consistent with restorer is designated as 1, the isozygoty banding pattern consistent with sterile line is designated as 2, the banding pattern of heterozygosis is designated as 3, and disappearance is designated as 0.
2, the prediction of BAC sequenced genes and snp analysis
With the primer screening 87-1BAC library of target gene both sides, obtain comprising the clone who recovers gene Rf4, and this clone is checked order.According to sequencing result, carry out predictive genes and functional analysis on FGENESH (http://linux1.softberry.com/) and GO (www.geneontology.org/).
The 87-1BAC library (Fig. 6) that has built with the molecular marker screening of target gene both sides is the cloning and sequencing that comprises goal gene that screens.
3, the target gene order-checking obtains SNP
According to predicting the outcome, GO (
Www.geneontology.org/) on carry out functional analysis.By the sequencing result analysis as can be known, from the about 45000Nt of nearest two the mark X-21-1 of target gene and the physical distance between X-33, predict by gene function, have 9 possible gene regions between two marks.Wherein, 2 have corresponding gene on the corn inbred line B73 that has checked order.These two gene regions are carried out gene sequencing in different restorers and sterile line, find the difference of 4 SNP.Wherein, in restorer, its sequence is as shown in sequence table, and in sterile line, its sequence is as shown in sequence table.
The present invention is directed to SNP mark in the gene that the corn C multiple genes is not arranged now, utilize this mark can solve the fact of the gene internal labeling of corn C type cellular type cytoplasmic male sterility fertility restorer gene, propose the molecular detection technology problem of the extensive matter male sterile of this fertility fertility restorer gene.
The method of SNP mark in the gene of the corn C type cytoplasmic male sterility fertility restorer gene RF4 that the embodiment of the present invention provides, build the near isogenic line that comprises restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) take maize elite inbred line 87-1 as base mateiral, get single-strain blade near isogenic line offspring's the litmus mouth phase and carry out DNA extraction; The DNA that extracts from single-strain blade is carried out molecular markers development, and molecular marker data is collected and processed; With the primer screening 87-1BAC library of target gene both sides, obtain comprising the clone who recovers gene Rf4, and this clone is checked order, carry out predictive genes according to the target sequencing result; Carry out functional analysis according to the predictive genes result, and obtain the interior SNP mark of gene of C type cytoplasmic male sterility fertility restorer gene; The method has realized the Fine Mapping of C type cytoplasmic male sterility fertility restorer gene and the interior SNP mark of gene of fertility restorer gene, utilize this SNP mark, solved the molecular detection technology problem of corn C type cytoplasmic male sterility fertility restorer gene, practical, have stronger propagation and employment and be worth.
The above is only preferred embodiment of the present invention, not in order to limiting the present invention, all any modifications of doing within the spirit and principles in the present invention, is equal to and replaces and improvement etc., within all should being included in protection scope of the present invention.
Claims (9)
1. SNP mark in the gene of a corn C type cytoplasmic male sterility fertility restorer gene RF4, is characterized in that, the method comprises the following steps:
Step 1, build the near isogenic line that comprises restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) take maize elite inbred line 87-1 as base mateiral, get single-strain blade near isogenic line offspring's the litmus mouth phase and carry out DNA extraction;
Step 2 is carried out molecular markers development to the DNA that extracts from single-strain blade, and molecular marker data is collected and processed;
Step 3 with the primer screening 87-1BAC library of target gene both sides, obtains comprising the clone who recovers gene Rf4, and this clone is checked order, and carries out predictive genes according to the target sequencing result;
Step 4 is carried out functional analysis according to the predictive genes result, and obtains the interior SNP mark of gene of C type cytoplasmic male sterility fertility restorer gene.
2. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, it is characterized in that, in step 1, build when comprising the near isogenic line of restorer Cms-ES87-1 (Rf4Rf4) and sterile line Cms-ES87-l (rf4rf4) the target gene Rf4 source self-mating system A619 near isogenic line take maize elite inbred line 87-1 as base mateiral.
3. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, it is characterized in that, the male loose powder phase of taking out the near isogenic line offspring is carried out the fertility investigation to individual plant, and Fertility rating adopts 6 grade standards of Li Jing hero to reach, and 6 grade standards of Li Jingxiong are:
When flower pesticide exposes 0%, it is 0 grade; When flower pesticide exposes 0-5%, it is 1 grade; When flower pesticide exposes 6-25%, it is 2 grades; When flower pesticide exposes 26-50%, it is 3 grades; When flower pesticide exposes 51-75%, it is 4 grades; When flower pesticide exposes 76-100%, it is 5 grades;
The reclassify standard of Khey-Pour is: when flower pesticide does not ftracture fully, be the I level; When flower pesticide partly ftractures, be the II level; During the flower pesticide normal crack, be the III level;
Be 4 grades in 6 grade standards at Li Jingxiong, and when being the III level in the reclassify standard of Khey-Pour, this individual plant is fertile plant;
Be 5 grades in 6 grade standards at Li Jingxiong, and when being the III level in the reclassify standard of Khey-Pour, this individual plant is fertile plant;
Be 0 grade in 6 grade standards at Li Jingxiong, and when being the I level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 1 grade in 6 grade standards at Li Jingxiong, and when being the I level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 2 grades in 6 grade standards at Li Jingxiong, and when being the II level in the reclassify standard of Khey-Pour, this individual plant is sterile strain;
Be 3 grades in 6 grade standards at Li Jingxiong, and when being the II level in the reclassify standard of Khey-Pour, this individual plant is sterile strain.
4. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, is characterized in that, gets near isogenic line offspring's the litmus mouth phase implementation method that single-strain blade carries out DNA extraction to be:
Get the fresh corn male sterility mutant blade grind into powder in liquid nitrogen of 5g, in the centrifuge tube of the 50mL that packs into;
Add the SDS extracting solution 15mL that is preheated to 65 ℃ in centrifuge tube, add the beta-mercaptoethanol mixing of 30-50 μ L, 65 ℃ of water-bath 40-60min;
Take out centrifuge tube, add 5MKAC5mL, ice-water bath 10min;
Add the mixed solution of the chloroform of 15mL and primary isoamyl alcohol and abundant mixing, both hands slowly shook 1 hour, and solution is blackish green at the bottom of the centrifuge tube, places 10min under room temperature, and chloroform and primary isoamyl alcohol allocation ratio are 24: 1;
At 4 ℃ of temperature, with 12000r/min, the centrifugal 15min of rotating speed;
Slowly shift in supernatant liquor to a clean 50mL centrifuge tube with the 1mL liquid-transfering gun that cuts off head, and add wherein the Virahol of 15mL precooling, rotate gently mixing, be placed in 4 ℃ of refrigerators, DNA cotton-shaped the separating out that be white in color becomes bulk, and wherein Virahol needs place under-20 ℃ to spend the night;
With glass needle, the careful hook of DNA is gone out, be placed in dry free of contamination 1.5mL centrifuge tube, 75% washing with alcohol 2 times, then add absolute ethanol washing once, again add at last dehydrated alcohol that DNA is preserved under 4 ℃ of conditions;
Used time is outwelled ethanol, and seasoning DNA adds TE or ultrapure water dissolving DNA.
5. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, is characterized in that, in step 2, the implementation method of the DNA that extracts from single-strain blade being carried out molecular markers development is:
Search the DNA sequence dna of the BACs in 8.00,8.01 districts on ma izeGDB website, and at http://linux1.softberry.com/berry.phtml? topic=fgenesh﹠amp; Group=programs﹠amp; Subgroup=gfind predicted gene structure;
According to the prediction of gene structure result, find the corresponding sequence of intron, exon on DNA sequence dna;
Intron sequences is compared at http://blast.ncbi.nlm.nih.gov/ together with the exon sequence of each 100bp of both sides, finds out the single-copy sequence of target section;
The single-copy sequence that obtains is designed primer on Primer Primer5.0.
6. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 5, it is characterized in that, when design intron mark, upstream and downstream primers to intron, two chains of primer are respectively on the exon of intron both sides, amplified production length is at 200bp, and beginning and end position are no less than 30bp apart from 5 ' and 3 ' end respectively; Primer length 18-25bp; Annealing temperature is between 56 ℃-65 ℃; And the Tm value of upstream and downstream primer is more or less the same in 2 ℃; The content of G+C is between 40%-60%.
7. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, it is characterized in that, in step 2, molecular marker data is collected when processing, the banding pattern of parent's restorer Cms-ES87-1 (Rf4Rf4) is designated as 1, and the banding pattern of parent's sterile line Cms-ES87-1 (rf4rf4) is designated as 2;
Banding pattern recording method near isogenic line offspring BC1F1 colony is: the isozygoty banding pattern consistent with restorer is designated as 1, and the isozygoty banding pattern consistent with sterile line is designated as 2, and the banding pattern of heterozygosis is designated as 3, and disappearance is designated as 0.
8. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, it is characterized in that, in step 3, primer screening 87-1BAC library with the target gene both sides, obtain comprising the clone who recovers gene Rf4, and this clone is checked order, carry out predictive genes on FGENESH according to the target sequencing result.
9. SNP mark in the gene of corn C type cytoplasmic male sterility fertility restorer gene RF4 as claimed in claim 1, it is characterized in that, in step 4, carry out functional analysis according to the predictive genes result, and when obtaining in the gene of C type cytoplasmic male sterility fertility restorer gene the SNP mark, according to predicting the outcome, carry out functional analysis on GO, by the sequencing result analysis as can be known, from the about 45000Nt of nearest two the mark X-21-1 of target gene and the physical distance between X-33, predict by gene function, have 9 possible gene regions between two marks, wherein 2 have corresponding gene on the corn inbred line B73 that has checked order, these two gene regions are carried out gene sequencing in different restorers and sterile line, the sequence of restorer and the sequence of sterile line are found respectively the difference of 4 SNP.
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| CN106455508A (en) * | 2013-12-31 | 2017-02-22 | 美国陶氏益农公司 | Maize cytoplasmic male sterility (cms) s-type restorer rf3 gene, molecular markers and their use |
| CN107058525A (en) * | 2017-03-21 | 2017-08-18 | 济南大学 | A kind of method that corn unknown gene function is predicted based on gene expression amount and character dynamic correlation |
| CN108441572A (en) * | 2018-02-06 | 2018-08-24 | 北京市农林科学院 | The identification method of DCIPThe chloroplast of maize cytoplasm type based on KASP technologies |
| CN108486264A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | Differentiate corn male sterility c-type cytoplasm type using chloroplastic marker |
| CN108486265A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | The identification method of corn male sterility cytoplasm type based on KASP technologies |
| CN108486266A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | The molecular labeling of DCIPThe chloroplast of maize genome and the application in cultivar identification |
| CN108531635A (en) * | 2018-03-06 | 2018-09-14 | 河南农业大学 | The sites corn C type cytoplasmic male sterility restoring gene Rf4 compact linkage molecule label and its application |
| CN113846179A (en) * | 2021-10-25 | 2021-12-28 | 四川农业大学 | Corn C-type sterile cytoplasm restoring gene Rf12 and application of close linkage marker thereof |
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| CN108441572A (en) * | 2018-02-06 | 2018-08-24 | 北京市农林科学院 | The identification method of DCIPThe chloroplast of maize cytoplasm type based on KASP technologies |
| CN108486264A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | Differentiate corn male sterility c-type cytoplasm type using chloroplastic marker |
| CN108486265A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | The identification method of corn male sterility cytoplasm type based on KASP technologies |
| CN108486266A (en) * | 2018-02-06 | 2018-09-04 | 北京市农林科学院 | The molecular labeling of DCIPThe chloroplast of maize genome and the application in cultivar identification |
| CN108486266B (en) * | 2018-02-06 | 2022-06-24 | 北京市农林科学院 | Molecular marker of corn chloroplast genome and application of molecular marker in variety identification |
| CN108486265B (en) * | 2018-02-06 | 2022-06-24 | 北京市农林科学院 | Method for identifying type of male sterile cytoplasm of corn based on KASP technology |
| CN108531635A (en) * | 2018-03-06 | 2018-09-14 | 河南农业大学 | The sites corn C type cytoplasmic male sterility restoring gene Rf4 compact linkage molecule label and its application |
| CN113846179B (en) * | 2021-10-25 | 2022-04-15 | 四川农业大学 | Corn C-type sterile cytoplasm restoring gene Rf12 and application of close linkage marker thereof |
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