CN105087768B - A kind of method of the anti-bean weevil kind of molecular marking supplementary breeding mung bean - Google Patents
A kind of method of the anti-bean weevil kind of molecular marking supplementary breeding mung bean Download PDFInfo
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Abstract
It is used to detect primer and its application of the mung bean to bean weevil resistance the invention discloses a kind of.It is provided by the present invention to be used to detect primer pair of the mung bean to bean weevil resistance, for the primer pair being made up of the single stranded DNA shown in sequence in sequence table 1 and the single stranded DNA shown in sequence 2.Test result indicates that the mung bean that 106bp purpose bands (sequence 3) are amplified using primer pair provided by the present invention (sequence 1 and sequence 2) all shows anti-bean weevil；And use primer pair provided by the present invention (sequence 1 and sequence 2) to amplify the mung bean of 120bp purpose bands (sequence 4) and all show to feel bean weevil.It can be seen that, detection mung bean provided by the present invention is reliable, easy, practical to the method for bean weevil resistance, there is important application prospect in the assisted Selection of mungbean germplasm resources evaluation and breeding mark, while reference frame is provided to the mung bean variety of high anti-bean weevil to cultivate.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of method of the anti-bean weevil kind of molecular marking supplementary breeding mung bean,
It is more particularly to a kind of to be used to detect primer pair of the mung bean to bean weevil resistance, and it is in the anti-bean weevil of molecular marking supplementary breeding mung bean
Application in kind.
Background technology
Mung bean (Vigna radiata) is a kind of pulse family, Papillionoideae cowpea platymiscium, and China is the cradle of mung bean
One of, possess the mung bean variety resource of wide variety.The states such as China, Burma are main mung bean exported countrys.Due to its breeding time
Short, wide adaptability, and there is preferable nitrogen fixing capacity, thus be the planting industry rational distribution of resources, rotation of crops crop rotation, intercropping,
The indispensable cereal crops of the mitigation disaster relief and the Important Economic crop of poverty-stricken area farmer richness；Mung bean is rich in albumen simultaneously
Matter, vitamin, several mineral materials and amino acid needed by human, also with clearing heat and detoxicating, hypotensive and cholesterol, prevent artery
Atherosis and other effects, there is higher nutrition and health care and economic value, enjoy favor in the international market.In recent years, it is international
Market increased to the demand of mung bean and the output of whole world mung bean, and the mung bean annual export volume of China is in 20- at present
300000 tons, the general 400-500 dollars of export price.The social economic value of mung bean can not be ignored.
However, bean weevil is the storage pest for endangering mung bean most serious, bean weevil has a very wide distribution, which kind of plant in office and storage food
Callosobruchus chinensis can all occur with the place of beans to endanger.Within a life cycle, because Callosobruchus chinensis endangers General Loss yield 30%-
56%, whole storehouse mung bean can be caused to be destroyed.In China, Callosobruchus chinensis can breed 4-11 generations every year, in addition to mung bean is endangered, can also endanger
Red bean, cowpea, pea, broad bean, Kidney bean, French beans etc..The research to mung bean bean weevil also quite lags both at home and abroad, although both at home and abroad
Anti- bean weevil research for many years is carried out, but the progress in terms of anti-bean weevil related gene is slow.First cause is that mung bean there is no
Whole genome sequence information, SSR molecular marker is deficient, is all marked substantially using RFLP, RAPD and AFLP, and these marks have one
Fixed limitation, it is cumbersome, the cycle is long, cost is high, success rate is low, has radioactive pollution as amount of DNA needed for RFLP marks is big
Etc. drawback.RAPD marks can not differentiate heterozygote and homozygote, the shortcomings of going back existence and stability and poor repeatability.AFLP marks skill
Art difficulty is big, costly.These marks limit molecular studies to the anti-bean weevil of mung bean, this with 10 years before paddy gene heredity
Study much like.But after rice genome sequencing is completed, substantial amounts of SSR is developed, the mark such as STS and SNP, is particularly
The use of SSR marker is greatly promoted the map based cloning research of paddy gene.SSR marker have reproducible, polymorphism is high,
Codominant inheritance, spread all over the advantages that whole gene group so that SSR marker turns into widely used molecular labeling.
With the fast development of second generation sequencing technologies, its high flux, it is quick, inexpensive the characteristics of turn into increasing
First choice of the biological study person when solving biological question, especially more shows great potentiality in terms of transcript profile sequencing.
Transcript profile is the inevitable tie for connecting genome hereditary information and biological function, while relative to eucaryote genome sequencing
For, sequence that transcript profile is sequenced to obtain does not contain introne and other non-coding sequences, therefore have can not for transcript profile sequencing
The high performance-price ratio advantage of analogy.It is a kind of simple efficient approach to be sequenced by mung bean transcript profile come developing SSR mark, and these are new
EST-SSR molecular labelings favourable condition is provided to the Molecular and Genetic Study of mung bean.
Since the 1980s, the Food Legume Genetic breeder of the country such as Japan, Thailand, Australia is endeavoured
In the research of anti-bean weevil.Domestic anti-bean weevil resource is more rare, and so far, the high anti-bean weevil mung bean resource of report mainly has
Come from African Madagascar's 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 that bean weevil harm is prevented and treated in production is aluminum phosphide fumigation method, and this not only adds the life of edible beans
The cost of production, and easily cause residues of pesticides, environmental pollution is caused, influences eater's health.Therefore research and develop anti-
Bean weevil gene close linkage marks, and clones anti-bean weevil gene, and it is green to cultivate anti-bean weevil using molecular marker assisted selection breeding technique
Beans new varieties, it is to prevent and treat the method for bean weevil harm the most cost-effective and environmentally friendly, for mitigating bean weevil harm, ensures China's mung bean safety
Production has very important significance.
The content of the invention
The purpose of the present invention is routinely to carry out anti-bean weevil identification after anti-bean weevil breeding method can only rely on harvest for mung bean
A kind of the shortcomings of caused cycle is long, cumbersome and time and effort consuming, there is provided time saving, laborsaving, accurately and fast molecule mark
Remember the selection of assist-breeding mung bean anti-bean weevil kind, and in particular to a kind of to be used to detect primer of the mung bean to object resistance
Pair and its application.
It is provided by the present invention to be used to detect primer pair of the mung bean to bean weevil resistance, specially by the institute of sequence in sequence table 1
The primer pair of single stranded DNA composition shown in the single stranded DNA and sequence 2 shown.
In the primer pair, two single strand dnas both can be packed individually, can also be hybrid packed.It is if mixed
Packaging is closed, the mol ratio of two single strand dnas can be 1:1.
The primer pair falls within protection scope of the present invention in detection mung bean to the application in bean weevil resistance.
It is used to detect kit of the mung bean to bean weevil resistance present invention also offers a kind of.
It is provided by the present invention to be used to detect kit of the mung bean to bean weevil resistance, specifically contain described primer pair,
And PCR reaction buffers, archaeal dna polymerase and 4 kinds of dNTP.
The preparation method of the primer pair and the preparation method of the kit also belong to protection scope of the present invention.
Detection mung bean provided by the present invention specifically may include following steps to the method for bean weevil resistance：
(a1) using the genomic DNA of mung bean to be measured as template, performing PCR amplification is entered with described primer pair, obtains PCR productions
Thing；
(a2) size of PCR primer obtained by detecting step (a1), determines the mung bean to be measured to bean weevil as follows
Resistance：If containing the DNA fragmentation that size is 106bp in the PCR primer, the mung bean to be measured is or candidate is anti-bean weevil
Mung bean；If the DNA fragmentation in the PCR primer containing size for 120bp, the mung bean to be measured is or candidate is that sense bean weevil is green
Beans.
In actual applications, can be non-by the way that PCR primer is carried out when judging whether to contain target DNA fragment in PCR primer
Denaturing polyacrylamide gel electrophoresis (gel strength specifically can be such as 8%), sees on electrophoresis pattern whether contain purpose band：Electrophoresis
Contain purpose band on collection of illustrative plates, then contain target DNA fragment in PCR primer.
In the process, the DNA fragmentation that the size is 106bp is DNA fragmentation shown in sequence 3 in sequence table；It is described
The DNA fragmentation that size is 120bp is DNA fragmentation shown in sequence 4 in sequence table.
In actual applications, judge whether containing DNA fragmentation shown in sequence 3 or sequence 4 in PCR primer, can be by PCR
Product carries out nucleotide sequencing to judge.
In the process, the annealing temperature of performing PCR amplification is entered as 50 DEG C using the primer pair.
In one embodiment of the invention, with the primer pair enter performing PCR amplification program be specially：95 DEG C of pre-degenerations
5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 45s, 72 DEG C of extension 30s, carry out 33 circulations；Last 72 DEG C of extensions 5min.
The application of described primer pair or the kit or methods described in following (I)-(IV) is any falls within this
The protection domain of 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 for cultivating anti-bean weevil (or sense bean weevil) mung bean variety, including
It is used as parent using anti-bean weevil (or the sense bean weevil) mung bean variety for detecting to obtain by approach described above and carries out breeding
Step.
In the present invention, the anti-bean weevil or the sense bean weevil are embodied in seed percentage of injury this index.Specifically
For, the anti-bean weevil mung bean is mung bean of the seed percentage of injury below 35%；The sense bean weevil mung bean is that seed percentage of injury exists
More than 65% mung bean.
Wherein, the seed percentage of injury is the ratio of aggrieved seed grain number and total grain number of every part of material identification.More have
Body, the assay method of the seed percentage of injury is referring to " journey must the precious anti-bean weevil GENE SOURCES excavation of mung beans and creative utilization research
The text of the Chinese Academy of Agricultural Sciences, Master's thesis in 2006 " one is carried out.
In the present invention, the bean weevil is specially Callosobruchus chinensis.
In one embodiment of the invention, the mung bean to be measured is specially by mung bean parent TC1966 (anti-bean weevil) and green
Beans parent VC1973A (sense bean weevil) F caused by hybridization2F derived from 94 individual plants of segregating population2:3Family.
The present invention utilize second generation high throughput sequencing technologies, it is anti-to mung bean, feel bean weevil near isogene based material VC6089A and
VC1973A carries out transcript profile sequencing, develops the related EST-SSR marks of anti-bean weevil, and filter out anti-bean weevil parent TC1966 and
Feel the polymorphism mark between bean weevil parent material VC1973A, and to caused F294 individual plants of segregating population are verified, are obtained
Anti- bean weevil gene linkage mark (primer pair that sequence 1 and sequence 2 form).It is demonstrated experimentally that the present invention is to by anti-, sense bean weevil parent
F caused by this TC1966 and VC1973A hybridization2Molecular Detection and resistance statistical experiment of 94 individual plants of segregating population to Callosobruchus chinensis
As a result show, the green of 106bp purpose bands (sequence 3) is amplified using primer pair provided by the present invention (sequence 1 and sequence 2)
Beans all show anti-bean weevil；And the bar of 120bp mesh is amplified using primer pair provided by the present invention (sequence 1 and sequence 2)
The mung bean of band (sequence 4) all shows to feel bean weevil.It can be seen that detection mung bean provided by the present invention can to the method for bean weevil resistance
Lean on, be easy, be practical, there is important application prospect in the assisted Selection of mungbean germplasm resources evaluation and breeding mark, be simultaneously
Cultivate and reference frame is provided to the high anti-mung bean variety of bean weevil.
Brief description of the drawings
Fig. 1 is that part SSR repeats primitive type sum amount in embodiment 1.
Fig. 2 is to detect anti-bean weevil parent TC1966 using primer pair A in embodiment 2, feel bean weevil parent VC1973A and it
The F that hybridizes2Segregating population DNA carries out the result of polymorphism checking.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Trizol, RNase H and Superscript IIreversetranscriptase kits are purchased from
Invitrogen companies.DNA polymerase i is purchased from NEB companies.The joint sequence of grappling is purchased from by Illumina in cDNA fragments
Sequencing kit.Quantitative test in following examples, it is respectively provided with and repeats to test three times, results averaged.
Anti- bean weevil mung bean TC1966 and VC6089A, and sense bean weevil mung bean VC1973A：It is recorded in " Sun Lei, Cheng Xuzhen, king
Plain China, Wang Lixia, Liu Changyou, Mei Li, Xu Nin cultivate the anti-bean weevil characteristic hereditaries of green V2709 and gene Primary Location Chinese agricultures
Science, 2008,41 (5):In 1291-1296 ", the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Callosobruchus chinensis：Sense bean weevil mung bean seed is placed on temperature as 27 DEG C or so, humidity 70%-80% for green No. 1 as in, and
Keep that Callosobruchus chinensis is bred and raised in the insectary of dark.Particular reference：Journey must be precious, Wang Suhua, Jin Dasheng, Wang Panlong,
Poplar and the anti-bean weevil breeding lines overall merit plant genetic resources journals of enlightening mung beans, 2003,4 (2):110-113.
Embodiment 1, for identifying acquisition of the mung bean to the SSR primers of bean weevil resistance
First, RNA-seq analyses and the design of SSR primers
(1) acquisition of transcript profile data
For trying mung bean：Mung bean is anti-, sense bean weevil NIL VC6089A and VC1973A.
Using mung bean children's green bean RNA of 16 days after Trizol reagents extraction florescence, (the anti-bean weevil gene of empirical tests was being bloomed
Start to express in the young green bean of 16 days after phase), with Oligo (dT) enrichment with magnetic bead mRNA.Add fragmentation
MRNA is broken into short-movie section by buffer, and using mRNA as template, first cDNA chain, Ran Houjia are synthesized with hexabasic base random primer
Enter buffer solution, dNTPs, RNase H and DNA polymerase I synthesis Article 2 cDNA chains, tried by QiaQuick PCR
Agent box purifies and adds the elution of EB buffer solutions to do end reparation afterwards, adds A and connects sequence measuring joints, then uses agarose gel electrophoresis
Clip size selection is carried out, finally enters performing PCR amplification, the sequencing library built is surveyed with IlluminaHiseq 2000
Sequence.Reverse transcription and synthetic double chain cDNA, cDNA is purified, carry out end reparation, add A and connect sequence measuring joints, then use agarose
Gel electrophoresis carries out clip size selection, finally enters performing PCR amplification.Specific method is as follows：
1st, mung bean Total RNA extraction
Extracted using the Trizol methods of routine, purifying, DNA enzymatic processing, obtain concentration >=50ng/ μ l, μ g of total amount >=3,
The Total RNA samples that OD260/280 is 1.8-2.2 (electrophoresis detection and NanoDrop initial surveies, further preferably select sample progress
Qubit is quantitative and Agilent2100 is detected).
2nd, mRNA separation and interrupt at random
Go out the mRNA with polyA with the Beads enrichment with oligo-dT, then interrupted at random using ultrasonic wave, reclaimed
200-700bp fragment.
3rd, the synthesis of the chains of cDNA first and the second chain
The synthesis of the chains of cDNA first is with random 6 polymers and Superscript II reverse transcriptase examinations
Agent box is carried out.The chains of cDNA second are completed with RNase H and DNA polymerase i.
4th, in cDNA fragments grappling joint sequence.PCR amplifications carry out 15 with the primer in above-mentioned joint sequence and followed
The PCR amplifications of ring.
5th, library construction and detection are using the sequence obtained in above-mentioned steps, according to Illumina companies sample prep
Kit carries out library construction and detection.
6th, RNA-seq sequencing
The library built up is added to the corresponding of Illumina sequenators (Genome Analyzer II) with 5-7pM concentration
On passage, 36 circulations are run.
7th, data analysis
Impurity data is rejected, the result after being assembled to RNA-seq is integrated.What before the step of obtained is original number
According to wherein containing the joint sequence added, referred to as Clean reads after removing it, it is possible to spliced and assembled.Specifically
Method is using the Clean reads that will be obtained, using the Trinity (versions spliced for transcript profile：v2012-10-05；Ginseng
Number is set：Min_kmer_cov is 2, and other parameters are default parameters) software spliced.Sequencing sequence is spelled with Trinity
A transcript profile is connected into, in this, as the reference sequences of subsequent analysis.Take transcript conduct most long in every gene
Unigene。
8th, bioinformatic analysis
Unigene sequences obtained above and albumen database nr, Swiss-Prot, KEGG and KOG are subjected to blastx
Compare (evalue ＜ 0.00001), the albumen for taking comparison result best determines Unigene sequence direction.If different storehouses it
Between comparison result it is contradictory, then Unigene sequence direction is determined by nr, Swiss-Prot, KEGG and KOG priority,
The Unigene being all less than with above-mentioned 4 storehouses, predict its code area with software ESTScan and determine the direction of sequence.For energy
The Unigene in sequence direction is determined, provides the sequence in its from 5 ' to 3 ' direction；Unigene for sequence direction can not be determined,
Provide the sequence that composite software obtains.These genes have been carried out with functional annotation, including KOG classification and GO annotations.
(2) identification of SSR primers
Perl language is installed, from http://pgrc.1pk-gatersleben.de/misa/ downloads est_trimmer.pl
And run, remove and be less than the too short sequences of 100bp and the sequence long more than 2000bp in transcript profile sequence, operation order 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).From http://
CD_HIT softwares are downloaded in www.bioinformatics.org/cd-hit, redundant sequence is removed using it：
A.fasta.results is copied in cd_hit files and RNTO B.fasta, runs cd_hit.exe, Perl environment
It is lower operation order be:C:\perl\bin\cd_hit>Cd_hit.exe-1B.fasta-oC.fasta-cl.00-n5-M2000,
Three files are exported, wherein C.fsata files are used to handle (A, B and C are short title) in next step.From http://
Pgrc.1pk-gatersleben.de/misa/ downloads misa.pi programs to identify and the SSR in positioning sequence；Parameter setting
It is as follows：Mononucleotide, dinucleotides, trinucleotide, tetranucleotide, the number of repetition of pentanucleotide and Hexanucleotide are at least
10、6、5、4、3、3.By C.fsata file copies to C disks perl under bin catalogues, order is run under Perl environment:C:\perl\
bin>Perlmisa.plC.fas, two files of C.fasta.misa and C.fasta.statistics are produced after operation, wherein
C.fasta.misa is used for follow-up design of primers.
(3) design of SSR primers
Use primer3 modules Batch Design SSR primers under Perl environment:Design of primers parameter is Tm55-65 DEG C, primer
Length is 18-22bp.P3_out.pi is run, running order under Perl environment is:C:\perl\bin>perlp3_
In.plC.fasta.misa, generate entitled C.fasta.p3in primer3 input file；Replicate again
C.fasta.p3in files to C disks perl bin primer3 under bin roots, operation primer3_core.exe is realized batch
The design of primers of amount, under Perl environment operation order be：C:\perl\bin\primer3\bin>primer3_core.exe<
C.fasta.p3in>C.fasta.p3out, produce entitled C.fasta.p3out file；Finally will
C.fasta.p3out files be copied to C disks perl under bin catalogues, run p3_out.pi, its order is：C:\perl\bin>
Perl p3_out.pl C.fasta.p3out C.fasta.misa, obtain C.fasta.results files after operation, be
Designed primer.
2nd, the excavation in the anti-bean weevil correlation SSR sites of mung bean high flux
Entered using the above method using anti-, sense bean weevil NIL VC6089A and VC1973A young green bean as material
Row high-flux sequence, the excavation in high flux SSR sites is carried out to mung bean transcript profile sequence using Perl language, obtains 48693
unigenes.SSR Density Distribution frequency of occurrences highests are single base microsatellites, and proportion highest is A/T, next to that four
Nucleotides (Fig. 1).
It is right to design acquisition SSR primers 3000 altogether using primer3.0 Batch Designs software, utilizes Catalase DNA to carry out
Design of primers success rate detects, the results showed that, share 651 pairs of SSR primers and detect clear band in 100-300bp, show to draw
Thing design success rate is higher.
Wherein, 6 pairs are shared in anti-, polymorphic between sense bean weevil NIL VC6089A and VC1973A primer, using this 6
F is verified to SSR primers2Segregating population, obtain 1 pair of polymorphism mark and with the primer pair of anti-bean weevil gene close linkage
(being designated as primer pair A), sequence is as follows：
Sense primer：5 '-AATTTCAAACAGCCAAGC-3 ' (sequence 1)；
Anti-sense primer：5 '-TATGTTTATAACTTACAA-3 ' (sequence 2).
Embodiment 2, foundation and application of the mung bean to the method for bean weevil resistance are detected using primer pair A
First, foundation of the mung bean to the resistance method of bean weevil is detected using primer pair A
(1) PCR is expanded
Mung bean genomic DNA to be measured is extracted, after DNA concentration is diluted into 50ng/ μ L, using it as template, using primer pair A
(primer pair that artificial synthesized two single stranded DNAs shown in sequence in sequence table 1 and sequence 2 form) enters performing PCR.If the base
Because group DNA does not have to that temporarily -20 DEG C of preservations can be placed in.
Reaction system (10 μ l)：Genomic DNA 40ng, 1 × Taq enzyme buffer solution (10mmolL-1Tris-HCl, pH8.8；
10mmol L-1KCl；10mmol L-1(NH4)2SO4；1.5mmol L-1 MgCl2；0.1%Triton X-100), 1mmol L- 1DNTPs, upstream and downstream primer to final concentration are 0.25 μm of ol L-1, 1U Taq archaeal dna polymerases, ddH2O complements to 10 μ l.
Response procedures are：95 DEG C of pre-degeneration 5min；95 DEG C of denaturation 30s, 50 DEG C of annealing 45s, 72 DEG C of extension 30s, carry out 33
Individual circulation；Last 72 DEG C of extensions 5min.
After reaction terminates, product adds 2 μ L sample loading buffers, using 100bp DNA ladder as DNA molecular amount standard, adopts
Electrophoresis is carried out with 8% non-denaturing polyacrylamide gel, and electrophoretic buffer is 0.5 × TBE, 200V voltage stabilizing electrophoresis 2-2.5h, extremely
Electrophoresis terminates when sample loading buffer moves on to gel bottom.After electrophoresis terminates, dyed using argentation, gel is finally placed in gel
Taken pictures in imaging system.
(2) sequencing and analysis of target stripe
(3) resistance according to result judgement mung bean to be measured to bean weevil
If containing the DNA fragmentation (sequence 3) that size is 106bp in the PCR primer, the mung bean to be measured is anti-bean weevil
Mung bean；It is if green for sense bean weevil containing the DNA fragmentation (sequence 4) that size is 120bp, the mung bean to be measured in the PCR primer
Beans.
2nd, primer pair A is used to detect effective checking of the mung bean to bean weevil resistance
For trying mung bean：The F as caused by anti-bean weevil mung bean variety TC1966 and the VC1973A hybridization of sense bean weevil mung bean variety2Point
Peel off F derived from 94 individual plants of body2:3Family, and anti-bean weevil mung bean variety TC1966, VC6089A and sense beans as control
As mung bean variety VC1973A.
On the one hand, using primer pair A to carrying out bean weevil Resistance detecting for examination mung bean (referring specifically to above step two).
On the other hand, the anti-bean weevil identification established using team where the present inventor and stage division, are used
Indoors artificial connects worm method to carrying out bean weevil Resistance Identification (bibliography for examination mung bean：Journey must the precious anti-bean weevil GENE SOURCES hair of mung beans
Pick and the creative utilization research Chinese Academy of Agricultural Sciences, Master's thesis in 2006), it is specific as follows：Take by anti-bean weevil mung bean variety
TC1966 and F caused by the VC1973A hybridization of sense bean weevil mung bean variety2F derived from 94 individual plants of segregating population2:3Family seed, with
And the wild mung bean TC1966 of anti-bean weevil as control and sense bean weevil cultivation mung bean VC1973A seed, every part of material select at random
30 healthy seeds are taken, are placed in anti-bean weevil authentication room plastic box, temperature is maintained between 25 DEG C~29 DEG C, and humid control exists
Between 70%~80%.400~500 Callosobruchus chinensis adults just to have sprouted wings 1~3 day are put into each plastic box, allow it to produce at random
Ovum, when every seed falls ovum amount more than 5, remove adult.Killed seed grain number and resistance class are investigated after 40~45 days.
Repetitive identified is carried out to above material, if 3 repetitions.Wherein, seed percentage of injury is that killed seed number (has worm channel with seed coat surface
Or seed is broken for aggrieved standard when being extruded with finger) and supply to plant experimentally the ratio of subnumber.Seed percentage of injury is anti-below 35%
Bean weevil mung bean；Seed percentage of injury is shown in Table 1 more than 65% for sense bean weevil mung bean, anti-bean weevil identification grade scale.
The anti-bean weevil characteristic grading evaluation criteria of the mung bean of table 1
The F as caused by anti-bean weevil mung bean variety TC1966 and the VC1973A hybridization of sense bean weevil mung bean variety2Segregating population 94
F derived from individual plant2:3Family seed resistance grade testing result is as shown in table 2, it is found that numbering is 1,5,11,14,17,20, No. 56
Totally 7 individual plant sense bean weevils, its seed percentage of injury are above 85%, and the seed percentage of injury of remaining individual plant is below 15%, table
It is now anti-bean weevil.The result (Fig. 2) that Molecular Detection is carried out using primer pair A is just being consistent with it, only numbering be 1,5,11,14,
17th, 20, No. 56 totally 7 individual plants using primer pair A to obtain purpose band of the size for 120bp when entering performing PCR amplification, and its
Remaining individual plant use primer pair A enter performing PCR amplification when obtained purpose band of the size for 106bp.Further, it is to size
106bp purpose band sequencing result confirms that it is just being sequence 3；It is confirmed to the purpose band sequencing result that size is 120bp
Just it is being sequence 4.
Seed percentage of injury of the table 2 respectively for examination mung bean is resistance rank measurement result
The molecular marker assisted selection of embodiment 3, VC1973A and VC6089A hybridization backcross progenies
(1) not anti-bean weevil main breed hybridizes and is returned with anti-bean weevil kind TC1966：Select full seed, sense bean weevil wheel
Return parent VC1973A and anti-bean weevil donor parents TC1966 and be sowed at crop field；Treat that Post flowering is hybridized, kind is collected after pod maturation
Son, as F1；Next year is by F1Crop field is sowed at recurrent parent seed, treats that Post flowering is hybridized, seed is collected after pod maturation, i.e.,
For BC1F1；3rd year by BC1F1Crop field is sowed at recurrent parent seed, treats that Post flowering is hybridized, is received after pod maturation by strain
Take seed；
(2) seedling stage Marker-assisted selection：The progeny seed that step (1) is obtained, divides individual plant to be seeded in crop field；Treat that seed is sent out
When first pair of trifoliolate leaf is open and flat after bud, blade sample is taken after each individual plant is listed, grind into powder in liquid nitrogen, extracted with CTAB
DNA, enter performing PCR amplification according to the method for embodiment 2, the individual plant that can amplify 106bp size fragments has anti-bean weevil gene
Individual plant retained, it is other to be eliminated；
(3) anti-bean weevil identification is carried out after harvest time：To through step (2) molecular marker screening be selected in plant normal harvest,
And anti-bean weevil identification is carried out, anti-bean weevil identifies grade scale method as described in Example 2, therefrom selects anti-bean weevil individual plant selfing,
The more homozygous new lines of as anti-bean weevil genotype；
(4) result and analysis：The result identified with anti-bean weevil by comparing the anti-bean weevil plant of Markers for Detection, find to divide
The accuracy rate of son mark detection is more than 90%.
Claims (22)
1. for detecting primer pair of the mung bean to bean weevil resistance, for as the single stranded DNA shown in sequence in sequence table 1 and the institute of sequence 2
The primer pair of the single stranded DNA composition shown.
2. primer pair according to claim 1, it is characterised in that：The bean weevil is Callosobruchus chinensis.
It is 3. slow containing the primer pair described in claim 1, and PCR reactions for detecting kit of the mung bean to bean weevil resistance
Fliud flushing, archaeal dna polymerase and 4 kinds of dNTP.
4. kit according to claim 3, it is characterised in that：The bean weevil is Callosobruchus chinensis.
5. the primer pair described in claim 1 is in detection mung bean to the application in bean weevil resistance.
6. application according to claim 5, it is characterised in that：The bean weevil is Callosobruchus chinensis.
7. the kit described in claim 3 is in detection mung bean to the application in bean weevil resistance.
8. application according to claim 7, it is characterised in that：The bean weevil is Callosobruchus chinensis.
9. a kind of detection mung bean comprises the following steps to the method for bean weevil resistance：
(a1) using the genomic DNA of mung bean to be measured as template, performing PCR amplification is entered with the primer pair described in claim 1, obtained
PCR primer；
(a2) size of PCR primer obtained by detecting step (a1), determines that the mung bean to be measured resists to bean weevil as follows
Property：If containing the DNA fragmentation that size is 106bp in the PCR primer, the mung bean to be measured is or candidate is that anti-bean weevil is green
Beans；If the DNA fragmentation in the PCR primer containing size for 120bp, the mung bean to be measured is or candidate is that sense bean weevil is green
Beans.
10. according to the method for claim 9, it is characterised in that：The DNA fragmentation that the size is 106bp is in sequence table
DNA fragmentation shown in sequence 3；The DNA fragmentation that the size is 120bp is DNA fragmentation shown in sequence 4 in sequence table.
11. according to the method for claim 9, it is characterised in that：Using the primer pair enter performing PCR amplification annealing temperature as
50℃。
12. according to any described method in claim 9-11, it is characterised in that：The bean weevil is Callosobruchus chinensis.
13. application of the primer pair in following (I)-(IV) is any described in claim 1：
(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.
14. application according to claim 13, it is characterised in that：The bean weevil is Callosobruchus chinensis.
15. application of the kit in following (I)-(IV) is any described in claim 3：
(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.
16. application according to claim 15, it is characterised in that：The bean weevil is Callosobruchus chinensis.
17. application of any described method in following (I)-(IV) is any in claim 9-11：
(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.
18. application according to claim 17, it is characterised in that：The bean weevil is Callosobruchus chinensis.
19. cultivating the method for anti-bean weevil mung bean variety, including use and detected by any methods described in claim 9-11
The step of anti-bean weevil mung bean variety arrived carries out breeding as parent.
20. according to the method for claim 19, it is characterised in that：The bean weevil is Callosobruchus chinensis.
21. cultivating the method for sense bean weevil mung bean variety, including use and detected by any methods described in claim 9-11
The step of sense bean weevil mung bean variety arrived carries out breeding as parent.
22. according to the method for claim 21, it is characterised in that：The bean weevil is Callosobruchus chinensis.
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| CN105368956B (en) * | 2015-12-11 | 2019-01-08 | 河北省农林科学院粮油作物研究所 | A kind of molecular labeling and its application for the anti-bean weevil gene Br3 assisted Selection of mung bean |
| CN105950736B (en) * | 2016-05-26 | 2020-06-09 | 中国农业科学院作物科学研究所 | Molecular marker co-separated from bruchid-resistant gene VrPGIP of mung bean and application thereof |
| 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 |
| CN112746119B (en) * | 2020-11-06 | 2022-02-15 | 江苏省中国科学院植物研究所 | Development and application of lycoris radiata fertility-related EST-SSR (expressed sequence tag-simple sequence repeat) marker |
| CN114032321B (en) * | 2021-10-29 | 2023-08-18 | 海南大学 | SSR (simple sequence repeat) marker for detecting broad bean anti-bean weevil variety and application thereof |
| CN116287390B (en) * | 2023-02-24 | 2023-10-03 | 江苏省农业科学院 | Molecular marker linked with pea anti-bean image character and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100769366B1 (en) * | 2006-09-28 | 2007-10-31 | 대한민국 | Ssr primer derived from mungbean and use thereof |
| CN101358232A (en) * | 2007-08-03 | 2009-02-04 | 中国农业科学院作物科学研究所 | Molecular marker linked with bruchid resistance gene in mung bean |
| CN101358233A (en) * | 2007-08-03 | 2009-02-04 | 中国农业科学院作物科学研究所 | QTL mapping range of bruchid resistance in mung bean |
| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
| US20140235459A1 (en) * | 2007-12-21 | 2014-08-21 | Tmg - Tropical Melhoramento E Genetica Ltda. | Genotypes, Alleles and Molecular Markers Associated With Asian Soybean Rust, as Well as Methods, Processes and Uses Thereof |
-
2014
- 2014-10-22 CN CN201410566900.2A patent/CN105087768B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100769366B1 (en) * | 2006-09-28 | 2007-10-31 | 대한민국 | Ssr primer derived from mungbean and use thereof |
| CN101358232A (en) * | 2007-08-03 | 2009-02-04 | 中国农业科学院作物科学研究所 | Molecular marker linked with bruchid resistance gene in mung bean |
| CN101358233A (en) * | 2007-08-03 | 2009-02-04 | 中国农业科学院作物科学研究所 | QTL mapping range of bruchid resistance in mung bean |
| US20140235459A1 (en) * | 2007-12-21 | 2014-08-21 | Tmg - Tropical Melhoramento E Genetica Ltda. | Genotypes, Alleles and Molecular Markers Associated With Asian Soybean Rust, as Well as Methods, Processes and Uses Thereof |
| CN103642912A (en) * | 2013-11-29 | 2014-03-19 | 中国农业科学院作物科学研究所 | Method for developing mung bean simple sequence repeat (SSR) primer based on transcriptome sequencing |
Non-Patent Citations (5)
| Title |
|---|
| Development of a molecular marker for a bruchid (Callosobruchus chinensis L.) resistance gene in mungbean;Huei-Mei Chen等;《Euphytica》;20070404;第157卷;第113-122页 * |
| Development of a mungbean (Vigna radiata) RFLP linkage map and its comparison with lablab (Lablab purpureus) reveals a high level of colinearity between the two genomes;M.E.Humphry等;《Theor Appl Genet》;20020523;第105卷;第160-166页 * |
| 绿豆遗传连锁图谱构建及抗豆象基因定位;钟敏;《中国优秀硕士学位论文全文数据库 农业科技辑》;20101015(第10期);D047-109 * |
| 绿豆遗传连锁图谱的整合;赵丹 等;《作物学报》;20100612;第36卷(第6期);第932-939页 * |
| 绿豆遗传连锁图谱的构建及其分析利用;赵丹;《中国优秀硕士学位论文全文数据库 农业科技辑》;20110215(第2期);D047-108 * |
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