CN105368956B - A kind of molecular labeling and its application for the anti-bean weevil gene Br3 assisted Selection of mung bean - Google Patents
A kind of molecular labeling and its application for the anti-bean weevil gene Br3 assisted Selection of mung bean Download PDFInfo
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Abstract
The invention discloses a kind of molecular labeling for the anti-bean weevil gene Br3 assisted Selection of mung bean and its applications, molecular labeling of the present invention, it is named as SYD404, it is using the genomic DNA of mung bean variety V1128 as template, it is obtained after PCR amplification by using 1 pair of SSR primer, the genetic distance with anti-bean weevil gene Br3 is 4.0cM.A kind of molecular labeling of the invention provides a good approach for the anti-bean weevil breeding of mung bean variety V1128, selection can be advanceed to the progress of growth of mung beans seedling stage, both a large amount of time-consuming and laborious anti-bean weevil identification works can have been exempted, early generation selection can be carried out to breeding material again, and target gene can be carried out accurately tracking by generation.
Description
Technical field
The present invention relates to a kind of with the molecular labeling of anti-bean weevil gene Br3 close linkage and its in the anti-bean weevil breeding of mung bean
Purposes, the invention belongs to Crop Genetic Breeding technical fields.
Background technique
Bean weevil is the main storage pest for endangering the Food Legumes crop such as mung bean.Edible beans weight after being endangered reduces,
Nutritional losses, quality degradation.Currently, the method for prevention and treatment bean weevil harm is mainly aluminum phosphide fumigation method in production, this is not only
The cost of edible beans production is increased, and is easy to cause pesticide residue, causes environmental pollution, influences eater's health.
The method of prevention and treatment bean weevil harm the most cost-effective and environmentally friendly is to cultivate anti-bean weevil to eat beans kind.
TC1966 and ACC41 is that 2 parts of most anti-wild mung bean resources of bean weevil are utilized in previous research.Researcher or
Using itself, or using their hybridization or backcross progeny as analysis of material, carry out anti-bean weevil heredity, anti-bean weevil machine
The research of reason, anti-bean weevil molecular labeling and anti-bean weevil breeding etc..However these wild materials often have fried pod, overgrow,
Unfavorable character in the breedings such as late-maturing, granule, and it is more difficult overcome, more importantly in the backcross progeny of TC1966 there may be
Certain ingredient [Miura K, 1996, Effects of bruchid-resistant mungbean being harmful to human health
meal on growth and blood-biochemical values in mice.Japan International
Research Center for Agricultural Sciences Journal, 3:23-31], therefore to anti-in cultivation mung bean
The screening of bean weevil resource is utilized and is got more and more attention.So far, the reported anti-bean weevil resource of cultivation mung bean has 4 parts, wherein
V2709 and V1128 is all from India, and V2802 and V2817 are respectively from Philippine and Nigeria.V2709 and V2802 are to beans
As with medium resistance, V1128 and V2817 object bean weevil have complete resistance [Talekar N S, 1992,
Characterization of Callosobruchus chinensis(Coleoptera:Bruchidae)resistance
in mungbean.Journal of Economic,85:1150-1153；Somta C,2008,Characterization of
new sources of mungbean(Vigna radiata(L.)Wilczek)resistance to bruchids,
Callosobruchus spp.(Coleoptera:Bruchidae).Journal of Stored Products
Research,44:316-321].Young etc. (1992) is with the hybridization F of TC1966 and sense bean weevil mung bean2Group is material use
RFLP label antagonism gene is positioned, and anti-bean weevil gene (Br) is located in the section of 10cM in the 8th linkage group, two sides mark
Remember pA882 and pM151 away from a distance from Br gene be 3.6cM and 6.5cM.The backcrossing group that Kaga etc. (1998) is constructed with TC1966
Body further positions Br gene, Br has been navigated in the section of 0.7cM, and wherein Bng143 label is with Br at a distance of only
0.2cM.Miyagi etc. (2004) earliest confrontation the wild mung bean ACC41 of bean weevil resistant gene studied, have found 2 with
Anti- bean weevil gene Br1 chain STS label: STSbr1 and STSbr2.Hereafter beautiful (2007) He Zhaodan (2011) of plum is again in succession right
The resistant gene of ACC41 is studied, by Br1 gene to the section for navigating to about 11cM, wherein with right flank SSR marker C220's
Distance about 2.6cM, left wing mark RAPD to mark C78 about 8.4cM.The F that Sun Lei etc. (2008) is constructed using VC1973A and V27092
Group has carried out Primary Location to the anti-bean weevil gene Br2 of V2709, by the Br2 assignment of genes gene mapping in the section of 16.8cM, away from its two sides
RAPD label and STS label be respectively 11.0cM and 5.8cM.
The V2709 and the anti-bean weevil ability of V2802 (medium resistance) that cultivation mung bean V1128 was identified more in the past are stronger, therefore
There are bigger potentiality in the anti-bean weevil breeding of mung bean, be easy to cultivate anti-bean weevil mung bean variety with it.However, due to annual
Anti- bean weevil identification work is very many and diverse, and there is presently no the reports that mung bean variety is bred as using V1128 as anti-bean weevil genetic donor
Road.It, will significantly if assisted Selection can be carried out in growth of mung beans seedling stage using the molecular labeling with anti-bean weevil gene close linkage
The workload for reducing later period anti-bean weevil identification, accelerates the anti-bean weevil breeding process of mung bean.
Summary of the invention
The object of the present invention is to provide a kind of molecule marks for the anti-bean weevil gene Br3 assisted Selection of mung bean variety V1128
Note and its application.
Above-mentioned purpose of the invention is achieved by the following technical solution:
A kind of molecular labeling for the anti-bean weevil gene Br3 assisted Selection of mung bean variety V1128 of the invention, is named as
SYD404, it is characterised in that the molecular labeling is using the genomic DNA of mung bean variety V1128 as template, using 1 couple of SSR
Primer obtains after PCR amplification；The sequence of the SSR primer are as follows:
Upstream primer: 5 ˊ ACGGCTATTCATCGTTTTGC, 3 ˊ
Downstream primer: 5 ˊ CAACCCGAAGCCAAAAACTA 3.
The genetic distance of the label SYD404 and the anti-bean weevil gene Br3 of mung bean are as follows: 4.0cM.
In the present invention, it is preferred to, for expanding the PCR reaction system and amplification program of the molecular labeling are as follows:
PCR reaction system is 10 μ L, and the buffer containing 1 × PCR, 0.2mM dNTPs, each 0.2 μM of upstream and downstream primer, 20ng is green
The genomic DNA of beans kind V1128,0.5U Taq enzyme；
PCR amplification program: 95 DEG C of initial denaturation 5min, then each circulation: 95 degree of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C
Extend 45s, carries out 35 circulations, last 72 DEG C of extensions 10min altogether.
The invention also provides the molecular labeling and the SSR primers to educate in anti-bean weevil mung bean variety V1128 auxiliary
Purposes in kind.
Further, after the mung bean variety V1128 hybridization containing anti-bean weevil gene Br3 that the invention also provides a kind of screenings
The method in generation, comprising the following steps: using the filial generation genomic DNA of mung bean variety V1128 to be measured as template, with institute of the present invention
The SSR primer stated carries out PCR amplification, electrophoresis detection amplified production, and the mung bean material to be measured for having 113bp band in amplified production is
The candidate progeny material containing anti-bean weevil gene Br3.
Wherein, it is preferred that the reaction system and amplification program of the PCR amplification are as follows:
PCR reaction system is 10 μ L, and the buffer containing 1 × PCR, 0.2mM dNTPs, each 0.2 μM of upstream and downstream primer, 20ng is green
The filial generation genomic DNA of beans kind V1128,0.5U Taq enzyme；
PCR amplification program: 95 DEG C of initial denaturation 5min, then each circulation: 95 degree of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C
Extend 45s, carries out 35 circulations, last 72 DEG C of extensions 10min altogether.
Wherein, the electrophoresis detection is to detect amplified production using 8% native polyacrylamide gel electrophoresis.?
Add 2 μ L sample-loading buffers, 280V constant voltage electrophoresis 1h in PCR product.0.1%AgNO310min is dyed, 1.5%NaOH (contains
0.5% formaldehyde) solution development.
Compared to the prior art the invention has the benefit that
Obtain the molecular labeling with the anti-bean weevil gene Br3 close linkage of anti-bean weevil mung bean resource V1128 for the first time in the world:
The present invention screens 8 labels chain with anti-bean weevil gene Br3 from 2882 SSR markers, wherein label SYD404 and Br3
Genetic distance be 4.0cM.Above-mentioned label is, and with Br3 gene close linkage, can be used for molecular labeling auxiliary
Selection and use.
A kind of molecular labeling of the invention provides a good approach for the anti-bean weevil breeding of mung bean variety V1128,
Selection can be advanceed to the progress of growth of mung beans seedling stage, can not only exempt a large amount of time-consuming and laborious anti-bean weevil identification works, but also can
To carry out early generation selection to breeding material, and target gene can be carried out accurately tracking by generation.
Detailed description of the invention
Fig. 1 is anti-bean weevil parent (V1128) and sense bean weevil parent (green No. 7 of Ji) and its hybridization F1The anti-bean weevil in generation identifies knot
Fruit；
Wherein V1128 and F1Generation completely anti-bean weevil, green No. 7 of Ji sense bean weevil completely；
Fig. 2 is molecular labeling SYD404 in anti-sense pond, parent and part F2Amplification in group；
Wherein M is maker, and 1 is sense pond, and 2 be anti-pond, and 3 be green No. 7 of parent Ji, and 4 be parent V1128, and 5-49 is part F2
Group；
Fig. 3 is Br3 gene close linkage map；
Fig. 4 is amplification of the molecular labeling SYD404 in recombinant inbred lines (RILs) offspring；
Wherein M is maker, and P1 is green No. 7 of parent Ji, and P2 is parent V1128, and S is sense bean weevil offspring, and R is after anti-bean weevil
Generation.
Specific embodiment
The present invention is further described with attached drawing combined with specific embodiments below, the advantages and features of the present invention will be with
Description and it is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications and replacement are fallen within the protection scope of the present invention.
The determination of 1 SYD404 molecular labeling of embodiment
Experimental material:
Feel the green No. 7 works female parent in bean weevil material Ji, which educates for Food and Oil Crops Inst., Hebei Agriculture and Forestry Academy
At kind；Anti- bean weevil material V1128 makees male parent, and the mung bean germplasm is quoted from Asia vegetables center (AVRDC).By green No. 7 of Ji and
V1128 hybridization, single plant harvest F1Generation.It will be from same F1The seminal propagation of single plant is at F2For segregating population, 158 are obtained altogether
F2Single plant.Simultaneously by F1Single plant is returned for green No. 7 with recurrent parent Ji, obtains 105 BC1Single plant.F2At random take 30 kinds at
Plant obtains F2:3Family.
Anti- bean weevil identification method:
To parent, F1、F2And BC1And F2:3Family carries out anti-bean weevil identification.Each single plant takes 90 seeds at random, is divided into 3
A repetition, each repetition 30 are put in diameter 5cm, in the small plastic box of high 2.5cm (uncovered), then by small plastic box respectively
(specification: 660 × 440 × 180mm) is put into big plastic casing.Two layers of black cloth of upper cover, is placed in insectary, insectary temperature control
System is at 28 ± 2 DEG C, relative humidity 70%-80%.The bean weevil adult of 400-500 rigid emergence 1-3d is put into each big plastic casing
Lay eggs, when on every seed ovum amount up to 5 or more when remove adult.Using green No. 7 of worm parent Ji of sense as compareing, to
When compareing the aggrieved rate of seed up to 100%, the aggrieved rate of every part of material is investigated.Anti- sense standard: aggrieved rate is highly resistance less than 20%, by
Evil rate 20%-80% is medium resistance, and aggrieved rate is greater than 80% for sense.
Molecular labeling primer design and polymorphism mark screening:
The mung bean transcript profile Unigenes sequence database obtained using autonomous sequencing finds the site SSR using MISA, and
SSR primer is designed using Primer3, product segment ranges 150-300bp obtains 2882 pairs of SSR primers altogether.According to F2:3In generation, is anti-
Bean weevil qualification result, from F2The middle DNA mixed in equal amounts for choosing 10 plants of homozygous sense bean weevil single plants and the anti-bean weevil single plant of 10 plants of homozygosis, structure
Build anti-sense pond.Polymorphism SSR marker is screened using anti-sense pond and two parent DNA.
Extracting genome DNA:
Fresh trifoliolate leaf 0.1g liquid nitrogen frozen and grinds are taken, the plant genome DNA using Tiangeng company is fast
Fast extracts kit extracts genomic DNA, detects DNA mass with 1% Ago-Gel, measures through ultraviolet specrophotometer dense
After degree, add ddH2It is spare that O is diluted to 10ng/ μ L.
PCR amplification and electrophoresis detection:
Pcr amplification reaction is expanded in ABI Applied Biosystems Veriti 96Well Thermol Cycler
Carried out on instrument, reaction system be 10 μ L, include 1 × PCR buffer, 0.2mM dNTPs, each 0.2 μM of upstream and downstream primer, 20ng
Genomic DNA, 0.5U Taq enzyme.Response procedures are 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C are prolonged
45s is stretched, is recycled 35 times；72 DEG C of extension 10min.Amplified production is separated through 8% native polyacrylamide gel electrophoresis,
Electrophoresis under 280V constant pressure adjusts electrophoresis time according to the distinguishable degree of PCR product molecular size range and difference banding pattern.By gel from
Glass plate is transferred to colouration box, is washed with distilled water 2 times, then uses 0.1%AgNO3Solution dyes 10min, and distilled water quickly floats
It washes 2 times, adds 1.5%NaOH (0.5% formaldehyde) solution development clear to band, then wash off remaining development on gel with distilled water
Liquid, tape reading of taking pictures.
Anti- bean weevil gene genetic analysis:
Parent V1128 shows highly resistance, the high sense of green No. 7 performances in parent Ji, F to Callosobruchus chinensis1Represent existing highly resistance (Fig. 1).F2In generation, is anti-
Feeling segregation ratio is 115 anti-: 37 senses, and Chi-square Test meets the theoretical ratio of 3:1, χ2=0.035
(χ20.05=3.54, df=1).BC1It is 59 anti-: 46 senses for anti-sense segregation ratio, Chi-square Test meets the theory of 1:1
Ratio, χ2=1.61 (χ20.05=3.54, df=1).The anti-bean weevil gene for speculating V1128 is single Major dominant gene.Continue
The naming rule of forefathers names the gene for Br3.
The anti-bean weevil assignment of genes gene mapping:
From the label for filtering out 13 performance polymorphisms between anti-sense pond in 2882 SSR markers.Utilize 13 polymorphisms
SSR marker is to F2It separates group and carries out Genotyping (Fig. 2), and calculate each molecular labeling using QTL IciMapping V4.0
With the genetic distance between anti-bean weevil gene, as a result there are 8 SSR markers to be successfully building up in linkage group, wherein from anti-bean weevil base
Because Br3 nearest molecular labeling is SYD404, genetic distance is 4.00cM (Fig. 3), the sequence of corresponding SSR primer are as follows:
Upstream primer: 5 ˊ ACGGCTATTCATCGTTTTGC, 3 ˊ
Downstream primer: 5 ˊ CAACCCGAAGCCAAAAACTA, 3 ˊ.
The verifying of 2 molecular labeling of embodiment
In order to verify the selection effect of molecular labeling SYD404 in actual breeding, we according to anti-bean weevil qualification result,
From the green No. 7 × V1128F in Ji8For randomly choosed respectively in recombinant inbred lines (RILs) 20 anti-bean weevil strains and sense beans
As strain, using SSR primer (upstream primer: 5 ˊ ACGGCTATTCATCGTTTTGC3 ˊ；Downstream primer: 5 ˊ
3 ˊ of CAACCCGAAGCCAAAAACTA) carry out PCR amplification verifying, respectively with feel bean weevil parent Ji green No. 7 (non-resistant genes) and
Anti- bean weevil parent V1128 (having anti-bean weevil Br3 gene) is as control.
PCR reaction system be 10 μ L, the buffer containing 1 × PCR, 0.2mM dNTPs, each 0.2 μM of upstream and downstream primer, the Ji 20ng
Green No. 7 × V1128F8For strain genomic DNA, 0.5U Taq enzyme；
PCR amplification program: 95 DEG C of initial denaturation 5min, then each circulation: 95 degree of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C
Extend 45s, carries out 35 circulations, last 72 DEG C of extensions 10min altogether.
Electrophoresis detection detects amplified production using 8% native polyacrylamide gel electrophoresis.Add 2 μ L in PCR product
Sample-loading buffer, 280V constant voltage electrophoresis 1h.0.1%AgNO310min is dyed, 1.5%NaOH (containing 0.5% formaldehyde), solution was aobvious
Shadow.
Amplification show feel bean weevil offspring all amplify with the green No. 7 identical banding patterns in Ji, anti-bean weevil offspring strain
There is 113bp band in amplified production, shows as banding pattern (Fig. 4) identical with V1128.Therefore, illustrate that SYD404 primer can be with
As the molecular labeling of the screening anti-bean weevil gene Br3 of V1128, the molecular labeling auxiliary for anti-bean weevil mung bean variety V1128 is educated
Kind.
Claims (7)
1. a kind of molecular labeling for the anti-bean weevil gene Br3 assisted Selection of mung bean variety V1128, is named as SYD404, special
Sign is that the molecular labeling is expanded using 1 pair of SSR primer through PCR using the genomic DNA of mung bean variety V1128 as template
It is obtained after increasing；The sequence of the SSR primer are as follows:
Upstream primer: 5 ˊ ACGGCTATTCATCGTTTTGC, 3 ˊ
Downstream primer: 5 ˊ CAACCCGAAGCCAAAAACTA 3.
2. molecular labeling as described in claim 1, it is characterised in that for expand the molecular labeling PCR reaction system and
Amplification program are as follows:
PCR reaction system be 10 μ L, the buffer containing 1 × PCR, 0.2mM dNTPs, each 0.2 μM of upstream and downstream primer, 20ng mung bean product
The genomic DNA of kind V1128,0.5U Taq enzyme；
PCR amplification program: 95 DEG C of initial denaturation 5min, then each circulation: 95 degree of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extensions
45s carries out 35 circulations, last 72 DEG C of extensions 10min altogether.
3. purposes of the molecular labeling of any of claims 1 or 2 in anti-bean weevil mung bean variety V1128 assistant breeding.
4. a kind of SSR primer for the anti-bean weevil gene Br3 assisted Selection of mung bean variety V1128, it is characterised in that the SSR
The sequence of primer are as follows:
Upstream primer: 5 ˊ ACGGCTATTCATCGTTTTGC, 3 ˊ
Downstream primer: 5 ˊ CAACCCGAAGCCAAAAACTA, 3 ˊ.
5. purposes of the SSR primer as claimed in claim 4 in anti-bean weevil mung bean variety V1128 assistant breeding.
6. it is a kind of screening containing anti-bean weevil gene Br3 mung bean variety V1128 filial generation method, it is characterised in that including with
Lower step: using the filial generation genomic DNA of mung bean variety V1128 to be measured as template, with SSR primer as claimed in claim 4
PCR amplification is carried out, electrophoresis detection amplified production, having the mung bean material to be measured of 113bp band in amplified production is candidate containing
The progeny material of anti-bean weevil gene Br3.
7. method as claimed in claim 6, it is characterised in that the reaction system and amplification program of the PCR amplification are as follows:
PCR reaction system be 10 μ L, the buffer containing 1 × PCR, 0.2mM dNTPs, each 0.2 μM of upstream and downstream primer, 20ng mung bean product
The filial generation genomic DNA of kind V1128,0.5U Taq enzyme；
PCR amplification program: 95 DEG C of initial denaturation 5min, then each circulation: 95 degree of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extensions
45s carries out 35 circulations, last 72 DEG C of extensions 10min altogether.
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| CN106399566B (en) * | 2016-11-23 | 2019-06-28 | 河北省农林科学院粮油作物研究所 | A set of combination of SSR primer and its application for being suitable for cultivating mung bean variety identification |
| CN108642062B (en) * | 2018-05-18 | 2021-01-15 | 江苏省农业科学院 | Mung bean weevil resistance gene VrPGIP allele VrPGIP1-ACC41Molecular marker and application |
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| CN114292953B (en) * | 2022-01-25 | 2023-04-11 | 河北省农林科学院粮油作物研究所 | Molecular marker closely linked with mung bean wilt-resistant major QTL locus Fov2-1 and application thereof |
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