CN108315465A - A kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings and its primer and application - Google Patents
A kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings and its primer and application Download PDFInfo
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Abstract
The invention discloses a kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings and its primer and applications.A kind of InDel molecular labelings with cowpea salt tolerant correlated traits close linkage, the InDel molecular labelings are to carry out nucleotide sequence shown in the SEQ ID NO.2 that PCR amplification obtains to purple 41 genomic DNAs of salt tolerant cowpea variety Soviet Union with downstream primer shown in sense primer shown in SEQ ID NO.1 and SEQ ID NO.2.Primer pair for expanding InDel molecular labelings of the present invention, sense primer is as shown in SEQ ID NO.1, and downstream primer is as shown in SEQ ID NO.2.Assisted Selection is carried out using the label and its primer, cowpea salt-resistance strain can be quickly screened for cowpea salt tolerance breeding, improve the accuracy and efficiency of selection of breeding, it is cost-effective.
Description
Technical field
The invention belongs to molecular biology and Biotechnology in Genetic Breeding fields, more particularly to a kind of and cowpea salt tolerant correlated traits
The InDel molecular labelings and its primer of close linkage and application.
Background technology
According to UNESCO (UNESCO) and FAO (Food and Agriculture Organization of the United Nation) (FAO) incomplete statistics, whole world salt marsh
About 1,000,000,000 hm of native area2(Wang Zunqin, 1993).About 100,000,000 hm of China's salinized soil area2, these salinized soil are the important soils in China
A part for resource, especially northern area and the coastal south (when Tianjin rosy clouds etc., 2004；Stapies,1984).
The cowpea cultivated area of China maintains 330,000 hm throughout the year2More than, major production areas is Hebei, Henan, Jiangsu, Zhejiang
River, Anhui, Sichuan, Chongqing, Hubei, Hunan, Guangxi etc. save (autonomous region) (Pan Lei etc., 2014).Cowpea economic value is higher,
Bean sprouts, seedling, green bean all can be for vegetable consumption one of the important vegetables for solving the supply of summer and autumn off-season vegetable.China edge
Haiti area is the important locality of cowpea, and the salinity in the seawater of coastal area enters soil by approach above and below the ground, and locates
More in the moisture evaporation of lower latitude, soil, there is also soil alkaline problem (boats etc., 2014).
In the molecule marking research of cowpea, based on PCR (Polymerase chain reaction, polymerization are mostly used greatly
Enzyme chain reaction) DNA molecular marker.Using dominant molecular labeling, such as RAPD, ISSR, acquisition a batch is screened and has repeated
Good, the reliable and stable molecular labeling primer of property (old buddhist friend etc., 2008；Chen C Y,et al.,2010).In recent years, SSR and
The application of the codominant markers such as SNP receives favor.1 375 SNP sites based on est sequence are reported within 2009 for the first time
(Muchero W, et al., 2009).Li et al. (2001) takes the lead in developing 27 SSR markers applied to cowpea research.Gupta etc.
In 2010 from ncbi database (http://archive-dtd.ncbi.nlm.nih.gov/) cowpea unigenes sequences
Middle design filters out 102 SSR markers.Xu is equal to 2010 then from cowpea genome database HarvEST (http://
) and CGKB (http www.harvest-web.org/://cowpeagenomics.med.virginia.edu/CGKB) in hair
Excavate 172 polymorphism cowpea SSR molecular markers (45 EST-SSR labels and 127 gSSR are marked).InDe1 is marked
Insertion by base on nucleotide level or deletion polymorphism have higher distribution frequency in the plant genome, have heredity
The advantages that stability is high, distribution is wide, polymorphism is strong, and InDel distribution densities are far above SSR, and SSR marker includes theoretically
In InDel label ranges.Marker-assisted breeding needs high density genetic linkage map, but utilizes cowpea transcript profile data mining
The research of InDel labels has not been reported.
QTL Position Research relevant to cowpea salt tolerant is less at present, and the QTL numbers usually detected are more, but effect value
It is smaller and repeated bad, it is more difficult to be applied in Asparagus Bean Breeding.This research passes through QTL positioning, it is intended to find in the sections QTL and cowpea
The molecular labeling of beans salt tolerant correlated traits close linkage, while by molecular labeling in cowpea salt tolerant breeding.This research passes through
QTL is positioned, it is intended to filter out the sites QTLs with cowpea salt tolerant correlated traits salt tolerant rank (STR) and chlorophyll content (SPAD)
With InDel molecular labelings, it to be used for the molecular marker assisted selection of cowpea salt tolerant breeding.
Invention content
Technical problem to be solved by the present invention lies in two cowpea varieties of utilization salt tolerant and salt density value to configure cross combinations,
F2 groups are obtained after F1 generation selfing, the phenotypic evaluation of salt tolerant is carried out to F2 groups.The two parent materials carry out transcript profile sequencing,
Transcript profile assembling sequence is subjected to BLAST comparisons, screening has the transcript of insertion/deletion (InDel), exploitation InDel labels.Profit
Genetic map is built with the InDel molecular labelings of independent development, and QTL positioning is carried out according to salt-tolerant phenotype data.This is cowpea
The assignment of genes gene mapping of salt tolerant correlated traits and economical character, analysis of genetic diversity, fingerprint map construction, whole-genome association
More new InDel are provided with the structure of high density genetic linkage maps and molecular marker assisted selection breeding to mark, and make up mesh
Preceding cowpea InDel marks the deficiency more lacked.
A kind of InDel molecular labelings with cowpea salt tolerant correlated traits close linkage, the InDel molecular labelings are with SEQ
Downstream primer shown in sense primer shown in ID NO.1 and SEQ ID NO.2 revives purple 41 genomic DNAs to salt tolerant cowpea variety
Carry out nucleotide sequence shown in the SEQ ID NO.2 that PCR amplification obtains.
Primer pair for expanding InDel molecular labelings of the present invention.
The preferred sense primer of the primer pair is as shown in SEQ ID NO.1, and downstream primer is as shown in SEQ ID NO.2.
With the InDel molecular labelings of cowpea salt tolerant correlated traits close linkage cowpea genetic map construction, QTL positioning,
Application in marker assisted selection and analysis of genetic diversity.
With the InDel molecular labelings of cowpea salt tolerant correlated traits close linkage salt tolerant and/or Gao Ye are screened in molecular breeding
Application in chlorophyll contents cowpea variety.
The primer pair is in cowpea genetic map construction, QTL positioning, marker assisted selection and analysis of genetic diversity
Application.
Application of the primer pair in molecular breeding screens salt tolerant and/or high chlorophyll content cowpea variety.
A kind of development approach with the InDel molecular labelings of cowpea salt tolerant correlated traits close linkage of the present invention,
Include the following steps：
1) be maternal and salt density value Soviet Union cowpea 1419 is that male parent is hybridized using salt tolerant cowpea variety purple 41 of reviving, F1 generation is selfed
F2 is obtained for segregating population；
2) use cetyl trimethylammonium bromide method extraction Soviet Union purple 41, Soviet Union's cowpea 1419, F1 generation and F2 for segregating population list
The blade total DNA of strain；
3) transcript profile sequencing is carried out to Soviet Union's purple 41 and Soviet Union's cowpea 1419 using lllumina Hiseq2000 platforms；
4) transcript profile assembling sequence is subjected to BLAST comparisons, screening has the transcript of insertion/deletion；
5) the relatively large sequence of sequence length difference for selecting insertion/deletion develops InDel in conjunction with primer-design software
Labeled primer；
6) the InDel primers for utilizing independent development carry out PCR amplification to two parents and F1 generation, and product is solidifying in 8% polypropylene
Electrophoresis, development, dyeing and banding pattern identification, screening parent mark with polymorphism and F1 generation with codominant InDel in glue；
7) molecular marker gene type analysis is carried out to F2 segregating populations using InDel labels 5), utilizes Joinmap3.0
Software carries out F2 for the structure of population genetic collection of illustrative plates, and recombination fraction is set as graph parameter<0.4, LOD>1.0；WinQTL is used in combination
2.5 softwares of Cartographer carry out quantitative trait locus positioning, and LOD threshold values are test with 1000 displacements and determined, probability water
Flat P=0.05 detects the presence of main effect QTL site qSTR-11 and qSPAD-11 in the 11st linkage group, explains salt tolerant respectively
The 7.0% and 7.1% performance variation of rank STR and chlorophyll content SPAD, away from main effect QTL site qSTR-11 and qSPAD-11
It is labeled as VUIn282 apart from nearest Indel, distance is 13.7 and 9.23；The primer sequence of the molecular labeling is
VUIn282_F:SEQ ID NO.1, VUIn282_R:SEQ ID NO.2；This InDel label Soviet Union purple 41 amplified fragments be
131 bp:AGCCTGGATATAATCCAAACACTTTTTTTCCGAAGGCGATTGACAACACTTTCTTTTTGCACTAGATAA
TATGCTTCTAGTAATTTGATAAAAATCATTGAAAATTCTTGTTCTTTGCTTTGGTTGCTTTC(SEQ ID NO.3)。
Advantageous effect：
Cross combination is configured using two cowpea varieties of salt tolerant and salt density value, F2 groups are obtained after F1 generation selfing, to F2 groups
Body carries out the phenotypic evaluation of salt tolerant.The two parent materials carry out transcript profile sequencing, and transcript profile assembling sequence is carried out BLAST ratios
Right, screening has the transcript of insertion/deletion (InDel), exploitation InDel labels.The present invention by the InDel of exploitation label and its
Primer pair obtains the QTL of one and cowpea salt tolerant correlated traits salt tolerant rank (STR) and chlorophyll content (SPAD).Therefore, sharp
Assisted Selection is carried out with the label and its primer, cowpea salt-resistance strain can be quickly screened for cowpea salt tolerance breeding, improve
The accuracy and efficiency of selection of breeding, it is cost-effective.
Description of the drawings
Fig. 1 one kind is salt tolerant correlated traits STR and the SPAD phenotypic variation for the F2 groups that 41 × Soviet Union of purple of Soviet Union cowpea 1419 is built
Histogram.The result shows that STR and SPAD performances are distributed in continuity, make a variation as normal distribution, illustrate STR and SPAD
Shape belongs to quantitative character.
A kind of the 11st linkage group schematic diagrames of cowpea of Fig. 2.Left-half instruction each marks corresponding genetic distance, right side
Divide the label title indicated in the linkage group.
A kind of salt tolerant correlated traits STR and the SPAD main effect in the 11st linkage group of cowpea of QTLCar2.0 software scans of Fig. 3
The LOD curve graphs of gene loci (QTL) scanning.
Fig. 4 is to be detached to two parents Soviet Union purple 41 and Soviet Union's cowpea 1419 and its F2 generations using molecular labeling VUIn282 (codominance)
The single plant of group carries out genotyping and screening is schemed.The first two P1 and P2 respectively represent parent Soviet Union purple 41 and Soviet Union's cowpea in figure
1419；1-45 numbers for F2 single plants；A, B, H respectively represent P1, P2 and the molecular labeling banding pattern of heterozygosis.
Specific implementation mode
A kind of development approach with the InDel molecular labelings of cowpea salt tolerant correlated traits close linkage of the present invention,
Preferably include following steps：
(1) hybridized using the two cowpea varieties Soviet Union purple 41 and Soviet Union's cowpea 1419 in salt tolerant and salt density value, F1 generation selfing
F2 is obtained for segregating population；
(2) asparagus bean seed sterilizes 10min with 1% hypochlorite solution, then with distilled water flushing 5 times.By parent, F1 and F2
In generation, is seeded in the plastic cup (lower port diameter 7cm, back cut diameter 9cm, high 15cm) for filling vermiculite.Parent and F1 generation broadcast 10 per basin
Grain, in F2 generations, broadcast 1 per basin, then cover 2cm vermiculites.NaCl concentration for the treatment of is 300mmolL-1；
(3) parent and F1 generation thinning after true leaf is fully deployed stays 4 plants per basin.With 100mmol L-1NaCL is handled 2 days,
Salinity is every 1 day with 100mmol L-1It is incremented by, until ultimate density reaches 300mmol L-1, salt to be replaced every 3 days molten later
Liquid, to keep the constant concentration of NaCl, continuous processing is until experiment terminates.Salt treatment PH is 6.0-6.5, and conductivity is
1.08S/m.Air pump is ventilated for 24 hours daily；
(4) until salt density value material is dead (after processing about 4 weeks), (1, plant leaf blade is strong by range estimation salt tolerant rank STR (1-5)
Health；5, plant is completely dead).It is used in combination chlorophyll meter (Konica Minolta SPAD-502) to measure plant leaf chlorophyll to contain
It measures (SPAD values)；
(5) parent Soviet Union purple 41 and Soviet Union's cowpea 1419, F1 and F2 is taken to extract DNA for plant leaf after true leaf expansion.Using 16
Alkyl trimethyl ammonium bromide method (CTAB methods) extract blade total DNA, used in the process of to reagent include extracting solution (1.4M
NaCl, 100mM Tris, pH 8.0,20mM EDTA, pH 8.0,2%CTAB), benzene expects：Chloroform：Isoamyl alcohol (25：24：1), chlorine
It is imitative：Isoamyl alcohol (24：1), absolute ethyl alcohol, 75% ethyl alcohol；
(6) transcript profile survey is carried out to cowpea variety Soviet Union purple 41 and Soviet Union's cowpea 1419 using 2000 platforms of lllumina Hiseq
Sequence；
(7) data analysis is carried out using bioinformatics software, transcript profile assembling sequence is subjected to BLAST comparisons, screening
There is the transcript of insertion/deletion (InDel)；
(8) PCR primer is designed in InDel sequences both sides, and polymorphic screening and the screening of F1 codominances is carried out to parent；
(9) genotyping is carried out using codominance primer pair F2 segregating populations, utilizes 4.0 software building F2 of Joinmap
Population genetic linkage map is set as recombination fraction as graph parameter<0.4, LOD>1.0, in conjunction with salt-tolerant phenotype data, utilize WinQTL
2.0 softwares of Cartographer carry out the QTL of cowpea salt tolerant correlated traits salt tolerant rank (STR) and chlorophyll content (SPAD)
Positioning, the wealthy values of LOD are test with 1000 displacements to be determined, probability level P=0.05；
(10) presence of main effect QTL is detected in the 11st linkage group, explains that salt tolerant rank (STR) and chlorophyll contain respectively
Measure the 7.0% and 7.1% performance variation of (SPAD).Away from main effect QTL site qSTR-11 and qSPAD-11 apart from nearest InDel
Labeled as VUIn282 (molecular labeling of independent development is named), distance is 13.7 and 9.23.
The structure and character of 1. cowpea salt tolerant segregating population of embodiment；
The group used in the present embodiment is that (salt tolerant rank STR is respectively 1 grade of purple 41 Hes of Soviet Union by salt tolerant and salt density value parent
5 grades of Soviet Union's cowpea 1419) filial generation F2 groups.STR and SPAD data distributions the result shows that:The two characters be in continuity just
State is distributed, and range of variation is very wide, it was demonstrated that STR and SPAD belongs to quantitative character (Fig. 1).
The extraction of 2. parent of embodiment Soviet Union purple 41, revive cowpea 1419, F1 and F2 segregating population blade total DNAs；
Blade total DNA is extracted using CTAB methods, is as follows：
A. it takes 0.1 gram of fresh sample of blade to be put into grinding, adds 650 microlitres of extracting solution grindings, be fitted into immediately in 1.5 milliliters of centrifuge tubes
65 DEG C of waters bath with thermostatic control 60 minutes are placed in, are mixed 2-3 times therebetween；
Plus isometric phenol B.:Chloroform:Isoamyl alcohol (25:24:1, volume ratio), gently overturning makes it mix well；
12000rpm, which is centrifuged 10 minutes, makes its layering, gentle aspiration supernatant be transferred to another 1.5 milliliters of centrifuge tubes, add isometric chlorine
It is imitative:Isoamyl alcohol (24:1, volume ratio) it extracts again once；
C. 1 milliliter of -20 DEG C of precooling absolute ethyl alcohols are added, is placed in 4 DEG C of freezings and allowed DNA to be precipitated no more than 30 minutes,
12000rpm is centrifuged 10 minutes, outwells ethanol solution in centrifuge tube；It is cleaned 2-3 times with 75% ethyl alcohol (volume ratio)；Outwell immersion
Liquid opens centrifuge tube and is placed on drying in draught cupboard；
D. TE (10mM Tris, pH8.0 are added after drying；LmM EDTA, pH8.0) dissolving DNA；Ultraviolet specrophotometer
The concentration for measuring DNA, saves backup in 4 DEG C of refrigerators.
The exploitation of embodiment 3.InDel molecular labeling primers and the screening of polymorphism：
Sequence is assembled according to Soviet Union's purple 41 and Soviet Union's 1419 transcript profile of cowpea, carries out BLAST comparisons, screening has insertion/deletion
(InDel) transcript；
The relatively large sequence of sequence length difference of insertion/deletion (InDel) is selected, Primer5.0 softwares are then used
Design InDel labeled primers.
It is right that InDel primers 845 have been synthesized by biotech firm.The selection result shows that the amplified production for having 93% primer exists
There are length polymorphism differences between parents, and there are codominances in F1 for the amplified production of 31% primer.Polymorphism screening sequence is as follows：
(1) 4 plants of DNA mixed in equal amounts are respectively randomly choosed from parent, the template as screening primer.
(2) PCR reaction systems：
(3) PCR response procedures：
(4) gel electrophoresis
Preparation of reagents：
10% ammonium persulfate is prepared：
Ammonium persulfate 10g is weighed, after being dissolved in water, 100ml is settled to ultra-pure water；
(6) 6 × loading Buffer (being added in amplified production)
A small amount of ultrapure water dissolution is added, adds 180 milliliters of glycerine, adjusts PH to 7.0, is settled to 500 milliliters；
(7) 1 × TBE (electrophoretic buffer)：100 milliliters of 10 × TBE is settled to 1 liter with 900 milliliters of ultra-pure water；
(8) dyeing liquor：Silver nitrate 1g with ultrapure water dissolution and is settled to 1 liter；
(9) developing solution：Sodium hydroxide 15g with ultrapure water dissolution and is settled to 1 liter, the formaldehyde of 3 milliliters of addition；
(10) prepared by polyacrylamide gel glue
1. each component of electrophoresis tank is both needed to clean up before use.Especially gel glass plate, it is necessary to use foam sponge
It is stained with a little soap or detergent cleaning, is rinsed well detergent with tap water after cleaning, with ultrapure water 2 times, is dried in the air
It is dry spare.
2. selecting levels operation table top, upper glass plates and lower glass plate are closed, with one at glass edges of boards half
It is fixed to two clips；
(11) electrophoresis
1. pouring into 70 milliliters of 8% non-deformed polyacrylamide in beaker, then it is separately added into 1 milliliter of 10% persulfuric acid
Ammonium and 25 microlitres of TEMED, quickly stir evenly；
2. configured 8% non-deformed polyacrylamide gel solution is slowly injected into along glass plate upper edge (have been prevented
Bubble), injecting height is flushed away from lower edge；Corresponding sample-adding comb is inserted into gel；
3. after gelling is solid, binder clip is removed, gel slab is installed on electrophoresis tank, then steps up to prevent from oozing with clip again
Leakage；Electrophoretic buffer i.e. 1 times TBE is filled in upper/lower electrode slot, lower tank liquor face must not be higher than concave glass plate recess bottom edge 5-
10 millimeters.Lower tank liquor face must not be higher than maximum water level " MAX ".Both hands gently take out comb, you can see adding for boundary clear
Certain sample solution is drawn in sample hole with liquid-transfering gun, is added in concave gel sample hole；
4. the correctly positive and negative anodes of connection electrophoresis tank, connect electrophoresis apparatus, according to glue surface size and other requirements, selection is suitable
Voltage or electric current carry out electrophoresis.
(12) it dyes and develops the color
1. the good gel of electrophoresis is taken out, it is transferred in staining utensil.The silver nitrate solution configured is added, is shaken small-sized
It is dyed 10 minutes on bed.Silver nitrate solution is poured out, clear water is added and rinses one time.Sodium hydroxide and formalin colour developing is added
5 minutes.Developing solution is outwelled, is rinsed twice with clear water.
2. on the table by preservative film tiling, being then laid in glue is neat on preservative film and wrapping again, room temperature is dried,
Continue to employ after taking pictures.
Distribution and linksystem analysis of 4. polymorphism primer of embodiment in F2 groups
It picks out and shows that 143 single plants of codominant primer pair F2 segregating populations carry out molecular marker gene type in F1
Analysis obtains molecular marker gene type data.Such as Fig. 4,45 single plants in F2 groups are carried out using codominant marker VUIn282
The result of genotyping.Using Joinmap4.0 softwares to the molecular marker gene type data of F2 groups carry out linkage analysis with
Molecular markers linkage map spectrum is built, 9 linkage groups (containing 107 molecular labelings) are obtained.Based on the genetic map, F2 groups
Genotype data and STR and SPAD trait phenotypes data, it is fixed to carry out QTL using 2.0 softwares of WinQTL Cartographer
Position, in the 11st linkage group (Fig. 2) of cowpea molecular labeling VUIn282 near (table 1), detect respectively 1 control STR with
The QTL (Fig. 3) of SPAD, contribution rate are 7.0% and 7.1% (table 2).
Primer sequence in the 11st linkage group of table 1 with the molecular labeling of STR and SPAD character close linkages
The essential information of the major gene loci (QTL) of STR and SPAD characters is controlled in the 11st linkage group of table 2
Sequence table
<110>Jiangsu Province Agriculture Science Institute
<120>A kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings and its primer and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
agcctggata taatccaaac ac 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gaaagcaacc aaagcaaaga ac 22
<210> 3
<211> 131
<212> DNA
<213>Cowpea (Vigna unguiculata)
<400> 3
agcctggata taatccaaac actttttttc cgaaggcgat tgacaacact ttctttttgc 60
actagataat atgcttctag taatttgata aaaatcattg aaaattcttg ttctttgctt 120
tggttgcttt c 131
Claims (8)
1. a kind of and cowpea salt tolerant correlated traits close linkage InDel molecular labelings, it is characterised in that the InDel molecular labelings
For with downstream primer shown in sense primer shown in SEQ ID NO.1 and SEQ ID NO.2 to salt tolerant cowpea variety Soviet Union purple 41
Genomic DNA carries out nucleotide sequence shown in the SEQ ID NO.3 that PCR amplification obtains.
2. the primer pair for expanding InDel molecular labelings described in claim 1.
3. primer pair according to claim 1, it is characterised in that sense primer is as shown in SEQ ID NO.1, downstream primer
As shown in SEQ ID NO.2.
4. with the InDel molecular labelings of cowpea salt tolerant correlated traits close linkage cowpea genetic map construction, QTL positioning, point
Application in sub- assistant breeding and analysis of genetic diversity.
5. application according to claim 4, it is characterised in that described with cowpea salt tolerant correlated traits close linkage
Application of the InDel molecular labelings in marker assisted selection screens salt tolerant and/or high chlorophyll content cowpea variety.
6. primer pair according to claim 2 or 3 is more in cowpea genetic map construction, QTL positioning, marker assisted selection and heredity
Application in sample analysis.
7. application according to claim 6, it is characterised in that primer pair according to claim 2 or 3 is sieved in molecular breeding
Select the application in salt tolerant and/or high chlorophyll content cowpea variety.
8. a kind of development approach with the InDel molecular labelings of cowpea salt tolerant correlated traits close linkage described in claim 1,
Characterized by the following steps：
1) be maternal and salt density value Soviet Union cowpea 1419 is that male parent is hybridized, F1 generation selfing acquisition using salt tolerant cowpea variety purple 41 of reviving
F2 is for segregating population；
2) use cetyl trimethylammonium bromide method extraction Soviet Union purple 41, Soviet Union's cowpea 1419, F1 generation and F2 for segregating population single plant
Blade total DNA；
3) transcript profile sequencing is carried out to Soviet Union's purple 41 and Soviet Union's cowpea 1419 using lllumina Hiseq2000 platforms；
4) transcript profile assembling sequence is subjected to BLAST comparisons, screening has the transcript of insertion/deletion；
5) the relatively large sequence of sequence length difference for selecting insertion/deletion, in conjunction with primer-design software exploitation InDel labels
Primer；
6) the InDel primers for utilizing independent development carry out PCR amplification to two parents and F1 generation, and product is in 8% polyacrylate hydrogel
Electrophoresis, development, dyeing and banding pattern identification, screening parent mark with polymorphism and F1 generation with codominant InDel；
7) molecular marker gene type analysis is carried out to F2 segregating populations using InDel labels 5), utilizes Joinmap3.0 softwares
F2 is carried out for the structure of population genetic collection of illustrative plates, recombination fraction is set as graph parameter<0.4, LOD>1.0；WinQTL is used in combination
2.5 softwares of Cartographer carry out quantitative trait locus positioning, and LOD threshold values are test with 1000 displacements and determined, probability water
Flat P=0.05 detects the presence of main effect QTL site qSTR-11 and qSPAD-11 in the 11st linkage group, explains salt tolerant respectively
The 7.0% and 7.1% performance variation of rank STR and chlorophyll content SPAD, away from main effect QTL site qSTR-11 and qSPAD-11
It is labeled as VUIn282 apart from nearest Indel, distance is 13.7 and 9.23；The primer sequence of the molecular labeling is
VUIn282_F:SEQ ID NO.1, VUIn282_R:SEQ ID NO.2；This InDel label Soviet Union purple 41 amplified fragments be
131 bp, nucleotides sequence are classified as SEQ ID NO.3.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810258440.5A CN108315465B (en) | 2018-03-27 | 2018-03-27 | InDel molecular marker closely linked with cowpea salt tolerance related characters, primers and application thereof |
Applications Claiming Priority (1)
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| CN111394498A (en) * | 2020-04-10 | 2020-07-10 | 河北省农林科学院粮油作物研究所 | SSR molecular marker closely linked with vigna unguiculata QT L site and application of SSR molecular marker in vigna unguiculata resistant breeding |
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