CN105087768A - Molecular marker assisted method for selectively breeding bruchid-resistant mung bean varieties - Google Patents
Molecular marker assisted method for selectively breeding bruchid-resistant mung bean varieties Download PDFInfo
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Abstract
The invention discloses a primer used for detecting resistance, to bruchid, of mung beans and application thereof. A primer pair is composed of single-chain DNA shown in a sequence 1 and single-chain DNA shown in a sequence 2 in a sequence table. Experiment results show that all mung beans, of a stripe (sequence 3) of 106bp mesh, amplified by the primer pair (sequence 1 and sequence 2) are resistant to bruchid; all mung beans, of s tripe (sequence 4) of 120bp mesh, amplified by the primer pair (sequence 1 and sequence 2) are sensitive to bruchid. Consequently, the method is reliable, simple, convenient and practical and has important application prospect in mung bean germ plasma resource evaluation and assisted selection of breeding markers, and a reference basis is provided for cultivating mung bean varieties highly resistant to bruchid.
Description
Technical field
The invention belongs to biological technical field, relate to the method for the anti-bean weevil kind of a kind of molecular marking supplementary breeding mung bean, particularly a kind of for detecting the primer pair of mung bean to bean weevil resistance, and its application in the anti-bean weevil kind of molecular marking supplementary breeding mung bean.
Background technology
Mung bean (Vignaradiata) is a kind of pulse family, Papillionoideae Vigna plant, and China is one of source region of mung bean, has the mung bean variety resource of wide variety.The states such as China, Burma are main mung bean export States.Because its breeding time is short, wide adaptability, and having good nitrogen fixing capacity, is therefore the Important Economic crop of the plant husbandry rational distribution of resources, rotation of crops crop rotation, intercropping, the indispensable food crop of the mitigation disaster relief and poverty-stricken area farmer richness; The amino acid of mung bean rich in proteins, VITAMIN, several mineral materials and needed by human simultaneously, also there is clearing heat and detoxicating, hypotensive and effect such as cholesterol, atherosclerosis, there is higher nutritive health-care and economic worth, enjoy favor in the international market.In recent years, the turnout of world market to the demand of mung bean and whole world mung bean increases all to some extent, and the mung bean annual export volume of China is 20-30 ten thousand tons at present, the general 400-500 dollar of export price.The social economic value of mung bean can not be ignored.
But bean weevil is the most serious storage pest of harm mung bean, and bean weevil has a very wide distribution, in office which kind of to plant and the place of store edible beans Callosobruchus chinensis all can occur endangers.Within a life cycle, because of Callosobruchus chinensis harm General Loss output 30%-56%, whole storehouse mung bean can be caused to be destroyed.In China, Callosobruchus chinensis can breed 4-11 generation every year, except harm mung bean, also can endanger red bean, cowpea, pea, broad bean, Kidney bean, French beans etc.Also quite delayed to the research of mung bean bean weevil both at home and abroad, although carried out anti-bean weevil research for many years both at home and abroad, the progress of anti-bean weevil genes involved aspect is slow.First cause is that mung bean there is no whole genome sequence information, SSR molecular marker is deficient, substantially RFLP, RAPD and AFLP is all used to mark, these marks have some limitations, DNA amount as required in RFLP mark is large, and complex operation, the cycle is long, cost is high, success ratio is low, have the drawbacks such as radiocontamination.RAPD mark can not differentiate heterozygote and homozygote, goes back the shortcoming such as existence and stability and poor repeatability.AFLP labeling technique difficulty is large, costly.These mark the molecular studies limited the anti-bean weevil of mung bean, and this is very similar to the genetic research of paddy gene before 10 years.But after completing paddy rice genome sequencing, develop a large amount of SSR, the marks such as STS and SNP, particularly the use of SSR marker greatly facilitates the map based cloning research of paddy gene.SSR marker have reproducible, polymorphism is high, codominant inheritance, spread all over the advantages such as whole genome, makes SSR marker become widely used molecule marker.
Along with the fast development of s-generation sequencing technologies, its high-throughput, feature that is quick, low cost become the first-selection of increasing biological study person when solving biological question, especially in transcript profile order-checking, more demonstrate great potentiality.Transcript profile is the inevitable tie connecting genome genetic information and biological function, simultaneously relative to eukaryote genome sequencing, transcript profile checks order the sequence that obtains not containing intron and other non-coding sequence, and therefore transcript profile order-checking has unrivaled high performance-price ratio advantage.Carrying out developing SSR mark by the order-checking of mung bean transcript profile is a kind of simple approach efficiently, and these new EST-SSR molecule marker Molecular and Genetic Studies to mung bean provide favourable condition.
From the eighties in 20th century, the Food Legume Genetic breeder of the country such as Japan, Thailand, Australia is devoted to the research of anti-bean weevil.Domestic anti-bean weevil resource is comparatively rare, and so far, the high resistance bean weevil mung bean resource of report mainly contains and comes from African Madagascar wild species TC1966 and come from Australian wild species ACC41.TC1966 is widely used in mung bean genetic breeding research.
At present, the main method of production preventing and treating bean weevil harm is aluminum phosphide fumigation method, this not only adds edible beans production cost, and easily causes pesticide residue, cause environmental pollution, affect eater healthy.Therefore anti-bean weevil gene close linkage mark is researched and developed, clone anti-bean weevil gene, application molecular marker assisted selection breeding technique cultivates anti-bean weevil New Mung Bean Variety, it is the most economic and method of environmental protection of control bean weevil harm, for alleviating bean weevil harm, ensure that China's mung bean safety in production has very important significance.
Summary of the invention
The shortcomings such as the cycle that anti-bean weevil qualification brings long, complex operation and time and effort consuming are carried out after the object of the invention is can only to rely on results for the conventional anti-bean weevil breeding method of mung bean, there is provided a kind of and save time, selection that is laborsaving, the anti-bean weevil kind of molecular marking supplementary breeding mung bean accurately and fast, be specifically related to a kind of for detecting mung bean to the primer pair of object resistance and application thereof.
Provided by the present invention for detecting the primer pair of mung bean to bean weevil resistance, be specially the primer pair be made up of the single stranded DNA shown in the single stranded DNA shown in sequence in sequence table 1 and sequence 2.
In described primer pair, two single strand dnas both can individually be packed, also can be hybrid packed.If hybrid packed, the mol ratio of two single strand dnas can be 1:1.
Described primer pair also belongs to protection scope of the present invention at detection mung bean to the application in bean weevil resistance.
Present invention also offers a kind of for detecting the test kit of mung bean to bean weevil resistance.
Provided by the present invention for detecting the test kit of mung bean to bean weevil resistance, specifically can contain described primer pair, and PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
The preparation method of described primer pair and the preparation method of described test kit also all belong to protection scope of the present invention.
Detection mung bean provided by the present invention, to the method for bean weevil resistance, specifically can comprise the steps:
(a1) with the genomic dna of mung bean to be measured for template, carry out pcr amplification with described primer pair, obtain PCR primer;
(a2) size of detecting step (a1) gained PCR primer, determine that described mung bean to be measured is to the resistance of bean weevil as follows: if be the DNA fragmentation of 106bp containing size in described PCR primer, then described mung bean to be measured is or candidate is anti-bean weevil mung bean; If be the DNA fragmentation of 120bp containing size in described PCR primer, then described mung bean to be measured be or candidate for feeling bean weevil mung bean.
In actual applications, when judging whether to contain target DNA fragment in PCR primer, by PCR primer being carried out native polyacrylamide gel electrophoresis (gel strength specifically can as 8%), see and whether electrophoretogram contains object band: containing object band on electrophoretogram, then contain target DNA fragment in PCR primer.
In the process, the DNA fragmentation that described size is 106bp is DNA fragmentation shown in sequence in sequence table 3; The DNA fragmentation that described size is 120bp is DNA fragmentation shown in sequence in sequence table 4.
In actual applications, judge whether containing DNA fragmentation shown in sequence 3 or sequence 4 in PCR primer, by carrying out nucleotide sequencing to judge to PCR primer.
In the process, the annealing temperature of carrying out pcr amplification with described primer pair is 50 DEG C.
In one embodiment of the invention, the program of carrying out pcr amplification with described primer pair is specially: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 50 DEG C of annealing 45s, 72 DEG C extend 30s, carry out 33 circulations; Last 72 DEG C extend 5min.
Described primer pair or described test kit or the application of described method in following (I)-(IV) is arbitrary also belong to protection scope of the present invention:
(I) anti-bean weevil mung bean variety is screened;
(II) screening sense bean weevil mung bean variety;
(III) anti-bean weevil mung bean variety is cultivated;
(IV) sense bean weevil mung bean variety is cultivated.
Another object of the present invention is to provide a kind of method of cultivating anti-bean weevil (or sense bean weevil) mung bean variety, comprises adopting detecting by the above method described anti-bean weevil (or feeling bean weevil) mung bean variety obtained and carrying out the step of breeding as parent.
In the present invention, described anti-bean weevil or described sense bean weevil are embodied in this index of seed percentage of injury.Specifically, described anti-bean weevil mung bean is the mung bean of seed percentage of injury below 35%; Described sense bean weevil mung bean is the mung bean of seed percentage of injury more than 65%.
Wherein, described seed percentage of injury is the ratio of total number that seed grain number and every part of material identify of being injured.More concrete, the measuring method of described seed percentage of injury see " Cheng Xuzhen. the anti-bean weevil GENE SOURCES of mung bean is excavated and is studied with creative utilization. the Chinese Academy of Agricultural Sciences, Master's thesis in 2006 " literary composition carries out.
In the present invention, described bean weevil is specially Callosobruchus chinensis.
In one embodiment of the invention, described mung bean to be measured is specially the F produced by mung bean parent TC1966 (anti-bean weevil) and mung bean parent VC1973A (sense bean weevil) hybridization
2the F that segregating population 94 individual plants are derivative
2:3family.
The present invention utilizes s-generation high throughput sequencing technologies,, sense bean weevil near isogene based material VC6089A and VC1973A anti-to mung bean carries out transcript profile order-checking, develop the EST-SSR mark that anti-bean weevil is relevant, and the polymorphism mark filtered out between anti-bean weevil parent TC1966 and sense bean weevil parent material VC1973A, and to the F produced
2segregating population 94 individual plants are verified, obtain anti-bean weevil gene linkage mark (primer pair that sequence 1 and sequence 2 form).Experiment proves, the present invention hybridizes to by anti-, sense bean weevil parent TC1966 and VC1973A the F produced
2segregating population 94 individual plants show the Molecular Detection of Callosobruchus chinensis and resistance statistical experiment result, and the mung bean adopting primer pair provided by the present invention (sequence 1 and sequence 2) to amplify 106bp object band (sequence 3) all shows anti-bean weevil; And the mung bean adopting primer pair provided by the present invention (sequence 1 and sequence 2) to amplify 120bp object band (sequence 4) all shows sense bean weevil.Visible, the method of detection mung bean provided by the present invention to bean weevil resistance is reliable, easy, practical, in the assisted Selection that mungbean germplasm resources evaluation and breeding mark, there is important application prospect, for cultivating, reference frame being provided to the mung bean variety of bean weevil high resistance simultaneously.
Accompanying drawing explanation
Fig. 1 is part SSR repeating group element type and quantity in embodiment 1.
Fig. 2 adopts primer pair A to detect anti-bean weevil parent TC1966 in embodiment 2, the F of sense bean weevil parent VC1973A and their hybridization
2segregating population DNA carries out the result of polymorphism checking.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Trizol, RNaseH and SuperscriptIIreversetranscriptase test kit is all purchased from Invitrogen company.DNA polymerase i is purchased from NEB company.In cDNA fragment, the joint sequence of grappling is purchased from by Illumina sequencing kit.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Anti-bean weevil mung bean TC1966 and VC6089A, and sense bean weevil mung bean VC1973A: be recorded in " Sun Lei; Cheng Xuzhen, Wang Suhua, Wang Lixia; Liu Changyou; Mei Li, Xu Nin. cultivate the anti-bean weevil characteristic hereditary of green V2709 and gene Primary Location. Scientia Agricultura Sinica, 2008; in 41 (5): 1291-1296 ", the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Callosobruchus chinensis: be about 27 DEG C as No. 1, medium green is placed on temperature sense bean weevil mung bean seed, humidity is 70%-80%, and keeps breeding in dark insectary and raising Callosobruchus chinensis.Concrete reference: Cheng Xuzhen, Wang Suhua, Jin Dasheng, Wang Panlong, Yang Youdi. mung bean anti-bean weevil breeding lines comprehensive evaluation. plant genetic resources journal, 2003,4 (2): 110-113.
Embodiment 1, for the identification of the acquisition of mung bean to the SSR primer of bean weevil resistance
One, the design of RNA-seq analysis and SSR primer
(1) acquisition of transcript profile data
For examination mung bean: mung bean is anti-, sense bean weevil near isogenic line VC6089A and VC1973A.
The tender pod RNA of mung bean children that utilizes Trizol reagent to extract after flowering period 16 days (empirical tests anti-bean weevil gene starts to express in the tender pod of children of 16 days after flowering period), with Oligo (dT) enrichment with magnetic bead mRNA.Add fragmentationbuffer and mRNA is broken into short-movie section, take mRNA as template, with hexabasic base random primer synthesis Article 1 cDNA chain, then add damping fluid, dNTPs, RNaseH and DNApolymeraseI synthesize Article 2 cDNA chain, end reparation is being done after adding EB buffer solution elution through QiaQuickPCR kits, add A and connect sequence measuring joints, then clip size selection is carried out with agarose gel electrophoresis, finally carry out pcr amplification, the sequencing library IlluminaHiseq2000 built checks order.Reverse transcription synthetic double chain cDNA, purifying cDNA, carries out end reparation, adds A and connects sequence measuring joints, then carries out clip size selection with agarose gel electrophoresis, finally carries out pcr amplification.Concrete grammar is as follows:
1, the extraction of mung bean TotalRNA
Conventional Trizol method is adopted to extract, purifying, DNA enzymatic process, obtain TotalRNA sample that concentration >=50ng/ μ l, total amount >=3 μ g, OD260/280 are 1.8-2.2 (electrophoresis detection and NanoDrop initial survey, more preferably select sample carry out Qubit quantitatively and Agilent2100 detection).
2, mRNA separation and interrupt at random
Go out the mRNA with polyA with the Beads enrichment with oligo-dT, then utilize ultrasonic wave to interrupt at random, reclaim the fragment of 200-700bp.
3, the synthesis of cDNA first chain and the second chain
The synthesis of cDNA first chain carries out with random 6 polymers and SuperscriptIIreversetranscriptase test kit.CDNA second chain completes with RNaseH and DNA polymerase i.
4, the joint sequence of grappling in cDNA fragment.The primer of pcr amplification in above-mentioned joint sequence carries out the pcr amplification of 15 circulations.
5, library construction and detection utilize the sequence obtained in above-mentioned steps, carry out library construction and detection according to Illumina company sampleprepkit.
6, the order-checking of RNA-seq
The library of building up is added to the concentration of 5-7pM in the respective channel of Illumina sequenator (GenomeAnalyzerII), runs 36 circulations.
7, data analysis
Reject impurity data, the result after RNA-seq assembling is integrated.What step before obtained is raw data, wherein containing the joint sequence added, is called Cleanreads after being removed, and just can carry out splicing and assemble.Concrete grammar utilizes the Cleanreads that will obtain, and adopts the Trinity (version: v2012-10-05 for transcript profile splicing; Optimum configurations: min_kmer_cov is 2, and other parameter is default parameters) software splices.Sequencing sequence is spliced into a transcript profile, in this, as the reference sequences of subsequent analysis with Trinity.Get transcript the longest in every bar gene as Unigene.
8, bioinformatic analysis
Unigene sequence obtained above and albumen database nr, Swiss-Prot, KEGG and KOG are carried out blastx comparison (evalue < 0.00001), get the sequence direction that the best albumen of comparison result determines Unigene.If the comparison result between different sink is contradictory, the sequence direction of Unigene is then determined by the priority of nr, Swiss-Prot, KEGG and KOG, catch up with state 4 storehouses all less than Unigene, predict its coding region with software ESTScan and determine the direction of sequence.For the Unigene that can determine sequence direction, provide the sequence in its from 5 ' to 3 ' direction; For the Unigene that cannot determine sequence direction, provide the sequence that composite software obtains.Functional annotation is carried out to these genes, has comprised KOG classification and GO annotation.
(2) identification of SSR primer
Perl language is installed, download est_trimmer.pl from http://pgrc.1pk-gatersleben.de/misa/ and run, remove in transcript profile sequence and be less than the too short sequence of 100bp and be greater than the long sequence of 2000bp, action command is: C: perl bin>perlest_trimmer, piA.fasta-amb=2,50-tr5=T, 5,50-tr3=A, 5,50-cut=100,2000.Export two file A.fasta.log and A.fasta.results (A is short title).CD_HIT software is downloaded from http://www.bioinformatics.org/cd-hit, it is utilized to remove redundant sequence: A.fasta.results to be copied in cd_hit folder and RNTO B.fasta, run cd_hit.exe, under Perl environment, action command is: C: perl bin cd_hit>cd_hit.exe-1B.fasta-oC.fasta-cl.00-n5-M2000, export three files, wherein C.fsata file is used for next step process (A, B and C are short title).Misa.pi program is downloaded with the SSR identified and positioning sequence from http://pgrc.1pk-gatersleben.de/misa/; Optimum configurations is as follows: the multiplicity of mononucleotide, dinucleotides, trinucleotide, tetranucleotide, pentanucleotide and Hexanucleotide is at least 10,6,5,4,3,3.By C.fsata file copy to C dish perl under bin catalogue, action command under Perl environment: C: perl bin>perlmisa.plC.fas, produce C.fasta.misa and C.fasta.statistics two files after running, wherein C.fasta.misa is used for follow-up design of primers.
(3) design of SSR primer
Primer3 module Batch Design SSR primer under use Perl environment: design of primers parameter is Tm55-65 DEG C, and primer length is 18-22bp.Under running p3_out.pi, Perl environment, action command is: C: perl bin>perlp3_in.plC.fasta.misa, create the input file of the primer3 of a C.fasta.p3in by name; Copy again C.fasta.p3in file to C dish perl bin primer3 under bin root directory, run the design of primers that primer3_core.exe realizes batch, under Perl environment, action command is: C: perl bin primer3 bin>primer3_core.exe<C.fasta. p3in>C.fasta.p3out, produce the file of a C.fasta.p3out by name; Finally by C.fasta.p3out file copy to C dish perl under bin catalogue, run p3_out.pi, its order is: C: perl bin>perlp3_out.plC.fasta.p3outC.fasta.misa, obtain C.fasta.results file after operation, be the primer designed.
Two, the anti-bean weevil of mung bean high-throughput is correlated with the excavation in SSR site
Application aforesaid method uses the tender pod of children that is anti-, sense bean weevil near isogenic line VC6089A and VC1973A to carry out high-flux sequence as material, utilizes Perl language to carry out the excavation in high-throughput SSR site to mung bean transcript profile sequence, obtains 48693 unigenes.What the SSR density distribution frequency of occurrences was the highest is the micro-satellite of single base, and that proportion is the highest is A/T, is secondly tetranucleotide (Fig. 1).
Primer3.0 Batch Design software is used to design acquisition SSR primer 3000 altogether right, Catalase DNA is utilized to carry out the detection of design of primers success ratio, result shows, has 651 pairs of SSR primers and clear band detected at 100-300bp, show that design of primers success ratio is higher.
Wherein, between anti-, sense bean weevil near isogenic line VC6089A and VC1973A, polymorphic primer has 6 right, utilizes these 6 pairs of SSR primers to verify F
2segregating population, obtain 1 pair of polymorphism mark and with the closely linked primer pair of anti-bean weevil gene (being designated as primer pair A), sequence is as follows:
Upstream primer: 5 '-AATTTCAAACAGCCAAGC-3 ' (sequence 1);
Downstream primer: 5 '-TATGTTTATAACTTACAA-3 ' (sequence 2).
Embodiment 2, utilize primer pair A detect mung bean to the foundation of the method for bean weevil resistance and application
One, primer pair A is utilized to detect mung bean to the foundation of the resistance method of bean weevil
(1) pcr amplification
Extract mung bean genomic dna to be measured, after DNA concentration dilution to 50ng/ μ L, with it for template, adopt primer pair A (primer pair be made up of sequence in sequence table 1 and shown two single stranded DNAs of sequence 2 of synthetic) to carry out PCR.If described genomic dna temporarily need not be placed in-20 DEG C of preservations.
Reaction system (10 μ l): genomic dna 40ng, 1 × Taq enzyme damping fluid (10mmolL
-1tris-HCl, pH8.8; 10mmolL
-1kCl; 10mmolL
-1(NH
4)
2sO
4; 1.5mmolL
-1mgCl
2; 0.1%TritonX-100), 1mmolL
-1dNTPs, upstream and downstream primer to final concentration is 0.25 μm of olL
-1, 1UTaqDNA polysaccharase, ddH
2o complements to 10 μ l.
Response procedures is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 50 DEG C of annealing 45s, 72 DEG C extend 30s, carry out 33 circulations; Last 72 DEG C extend 5min.
After reaction terminates, product adds 2 μ L sample loading buffers, take 100bpDNAladder as DNA molecular amount standard, 8% non-denaturing polyacrylamide gel is adopted to carry out electrophoresis, electrophoretic buffer is 0.5 × TBE, 200V voltage stabilizing electrophoresis 2-2.5h, and when moving on to bottom gel to sample loading buffer, electrophoresis terminates.After electrophoresis terminates, adopt argentation dyeing, finally gel is placed on gel imaging system and takes pictures.
(2) cloning and characterization of target stripe
(3) resistance of mung bean to be measured to bean weevil is judged according to result
If be the DNA fragmentation (sequence 3) of 106bp containing size in described PCR primer, then described mung bean to be measured is anti-bean weevil mung bean; If in described PCR primer containing size be the DNA fragmentation (sequence 4) of 120bp, then described mung bean to be measured is sense bean weevil mung bean.
Two, primer pair A is for detecting the effective checking of mung bean to bean weevil resistance
For examination mung bean: hybridize by anti-bean weevil mung bean variety TC1966 and sense bean weevil mung bean variety VC1973A the F produced
2the F that segregating population 94 individual plants are derivative
2:3family, and anti-bean weevil mung bean variety TC1966, VC6089A in contrast and sense bean weevil mung bean variety VC1973A.
On the one hand, primer pair A is adopted to carry out bean weevil Resistance detecting (specifically see above step 2) to for examination mung bean.
On the other hand, the anti-bean weevil qualification adopting the present inventor place team to set up and stage division, adopt indoors artificial connect worm method to carry out for examination mung bean bean weevil Resistance Identification (reference: Cheng Xuzhen. the anti-bean weevil GENE SOURCES of mung bean is excavated and is studied with creative utilization. the Chinese Academy of Agricultural Sciences, Master's thesis in 2006), specific as follows: to get and hybridize by anti-bean weevil mung bean variety TC1966 and sense bean weevil mung bean variety VC1973A the F produced
2the F that segregating population 94 individual plants are derivative
2:3family seed, and the seed of the wild mung bean TC1966 of anti-bean weevil in contrast and sense bean weevil cultivation mung bean VC1973A, every part of material random selecting 30 healthy seeds, are placed in anti-bean weevil authentication room plastic box, temperature remains between 25 DEG C ~ 29 DEG C, and humid control is between 70% ~ 80%.Put into 400 ~ 500 Callosobruchus chinensis adults just sprouted wings 1 ~ 3 day in each plastic box, allow it lay eggs at random, when every seed fall ovum amount more than 5 time, removing adult.40 ~ 45 days killed seed grain numbers of " Invest, Then Investigate " and resistance class.Repetitive identified is carried out to above material, if 3 times are repeated.Wherein, seed percentage of injury is killed seed number (have worm channel with seed coat surface or be broken for seed during finger extruding the standard of being injured) and the ratio supplying to plant experimentally subnumber.Seed percentage of injury is anti-bean weevil mung bean below 35%; Seed percentage of injury is sense bean weevil mung bean more than 65%, and anti-bean weevil qualification grade scale is in table 1.
Table 1 mung bean anti-bean weevil characteristic grading evaluation criteria
The F produced is hybridized by anti-bean weevil mung bean variety TC1966 and sense bean weevil mung bean variety VC1973A
2the F that segregating population 94 individual plants are derivative
2:3family seed resistance grade detected result is as shown in table 2, finds to be numbered 1,5,11,14,17,20, No. 56 totally 7 individual plant sense bean weevil, and its seed percentage of injury is all higher than 85%, and the seed percentage of injury of all the other individual plants is all below 15%, shows as anti-bean weevil.The result (Fig. 2) adopting primer pair A to carry out Molecular Detection just conforms to it, only be numbered 1,5,11,14,17,20, No. 56 totally 7 individual plants obtain when adopting primer pair A to carry out pcr amplification the object band that size is 120bp, and all the other individual plants obtain when adopting primer pair A to carry out pcr amplification the object band that size is 106bp.Further, the object band sequencing result being 106bp to size confirms that it is being just sequence 3; The object band sequencing result that size is 120bp is confirmed that it is being just sequence 4.
Table 2 is respectively for seed percentage of injury and the resistance rank measurement result of examination mung bean
Embodiment 3, VC1973A and VC6089A hybridize the molecular marker assisted selection of backcross progeny
(1) not anti-bean weevil main breed and anti-bean weevil kind TC1966 are hybridized and backcross: select full seed, and sense bean weevil recurrent parent VC1973A and anti-bean weevil donor parents TC1966 is sowed at land for growing field crops; Treat that Post flowering is hybridized, collect seed after pod maturation, be F
1; Next year is by F
1be sowed at land for growing field crops with recurrent parent seed, treat that Post flowering is hybridized, collect seed after pod maturation, be BC
1f
1; 3rd year by BC
1f
1be sowed at land for growing field crops with recurrent parent seed, treat that Post flowering is hybridized, after pod maturation, collect seed by strain;
(2) seedling stage Marker-assisted selection: the progeny seed that step (1) is obtained, a point individual plant is seeded in land for growing field crops; When after seed germination, first pair of ternately compound leaf is open and flat, each individual plant gets blade sample after listing, grind into powder in liquid nitrogen, DNA is extracted with CTAB, pcr amplification is carried out according to the method for embodiment 2, the individual plant that namely individual plant that can amplify 106bp size fragment has anti-bean weevil gene is retained, and other is eliminated;
(3) anti-bean weevil qualification is carried out after harvesting time: to the plant normal harvest be selected in through step (2) molecular marker screening, and carry out anti-bean weevil qualification, anti-bean weevil qualification grade scale method as described in Example 2, therefrom select anti-bean weevil individual plant selfing, be the new lines that anti-bean weevil genotype is comparatively isozygotied;
(4) results and analysis: by comparing the result that Markers for Detection anti-bean weevil plant and anti-bean weevil are identified, finds that the accuracy rate of Markers for Detection is more than 90%.
Claims (10)
1., for detecting the primer pair of mung bean to bean weevil resistance, be the primer pair be made up of the single stranded DNA shown in the single stranded DNA shown in sequence in sequence table 1 and sequence 2.
2. for detecting the test kit of mung bean to bean weevil resistance, containing primer pair according to claim 1, and PCR reaction buffer, archaeal dna polymerase and 4 kinds of dNTP.
3. primer pair according to claim 1 or test kit according to claim 2 are detecting mung bean to the application in bean weevil resistance.
4. detect the method for mung bean to bean weevil resistance, comprise the steps:
(a1) with the genomic dna of mung bean to be measured for template, carry out pcr amplification with primer pair according to claim 1, obtain PCR primer;
(a2) size of detecting step (a1) gained PCR primer, determine that described mung bean to be measured is to the resistance of bean weevil as follows: if be the DNA fragmentation of 106bp containing size in described PCR primer, then described mung bean to be measured is or candidate is anti-bean weevil mung bean; If be the DNA fragmentation of 120bp containing size in described PCR primer, then described mung bean to be measured be or candidate for feeling bean weevil mung bean.
5. method according to claim 4, is characterized in that: the DNA fragmentation that described size is 106bp is DNA fragmentation shown in sequence in sequence table 3; The DNA fragmentation that described size is 120bp is DNA fragmentation shown in sequence in sequence table 4.
6. the method according to claim 4 or 5, is characterized in that: the annealing temperature of carrying out pcr amplification with described primer pair is 50 DEG C.
7. arbitrary described primer pair or test kit or the application of method in following (I)-(IV) is arbitrary in claim 1-6:
(I) anti-bean weevil mung bean variety is screened;
(II) screening sense bean weevil mung bean variety;
(III) anti-bean weevil mung bean variety is cultivated;
(IV) sense bean weevil mung bean variety is cultivated.
8. cultivate the method for anti-bean weevil mung bean variety, comprise adopting and detect by described method arbitrary in claim 4-6 the described anti-bean weevil mung bean variety that obtains to carry out breeding step as parent.
9. cultivate the method for sense bean weevil mung bean variety, comprise the described sense bean weevil mung bean variety adopting and obtained by described method detection arbitrary in claim 4-6 carries out breeding step as parent.
10., according to described primer pair arbitrary in claim 1-9 or test kit or application or method, it is characterized in that: described bean weevil is Callosobruchus chinensis.
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| CN111961127B (en) * | 2020-08-24 | 2021-10-12 | 安徽省农业科学院作物研究所 | Molecular marker closely linked with green bean caulicle color and application thereof |
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| WO2023070937A1 (en) * | 2021-10-29 | 2023-05-04 | 海南大学 | Ssr marker for detecting bruchus rufimanus boheman-resistant variety of vicia faba l. and use thereof |
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