CN103468791B - Molecular marker assisted method used for variety selection of cowpea variety with grain-filling resistance, and PCR primer set used in molecular marker assisted method - Google Patents
Molecular marker assisted method used for variety selection of cowpea variety with grain-filling resistance, and PCR primer set used in molecular marker assisted method Download PDFInfo
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Abstract
The invention belongs to the field of plant biotechnology, and especially relates to a molecular marker assisted method used for variety selection of a cowpea variety with grain-filling resistance and a PCR primer set used in the molecular marker assisted method. Upstream primer sequence of the primer set is 5'-TTTCCATGGTAAAATTTTTTACC-3', and downstream primer sequence of the primer set is 5'-CCATTCCAAGAACGATTTGGCT-3'. Advantages of the molecular marker assisted method are that: 1, work amount and cost of seedling stage identification are reduced; and 2, accuracy of variety selection is improved.
Description
Technical field
The present invention relates to plant biotechnology field, particularly relate to the method for the resistance to bulging grain kind of a kind of molecular marking supplementary breeding cowpea.
Background technology
Asparagus bean (
vigna unguiculatassp
. sesquipedalis(L.) Verd.) be subordinate to Papillionoideae (
fabaceae) Phaseoleae (Trib.
phasoleaedC.) Vigna (
vigna Savi), be important one of the legume vegetable in summer of China.Because mainly edible part is tender pod, pod wall is plump, seed is little, bar pod uniformity becomes one of most important economic target of the tender pod of asparagus bean commodity.But usually run in asparagus bean production reality in tender pod growth course and cause tender pod in the protruding and phenomenon of other area depression of seed attachment region because the unbalanced growth, particularly seed development of pod wall and seed are too fast too early, be referred to as drum grain.Tender pod drum grain has a strong impact on the exterior quality of tender pod, reduces commodity value.The essence of cowpea tender pod drum grain is that in beanpod, seed development is too fast too early, causes seed to account for the ratio of whole beanpod too high.Breed and production practice shows, different cowpea variety tender pod drum grain degree is different, and the kind that Breeding and application not easily rouses grain is produced significant to cowpea.
The growth of seed and fruit is especially expanded and nutrient accumulation mainly regulated and controled with utilization by the distribution of photosynthate.Sucrose is the principal mode that plant photosynthesis product transports and distributes between plant tissue, and it plays vital effect as nutrient and signaling molecule in the growing of plant.Research shows, legume crop tender pod pod wall and seed also exist significant competitive relation to the absorption of sucrose and utilization, and sucrose decides seed ratio shared in beanpod at pod wall and interseminal distribution, accumulation and metabolism.Sucrose Metabolism and distribution, comprise phloem sucrose unloading, sucrose inversion be hexose, and the process of hexose accumulation is subject to the regulation and control of the many factors such as sucrose synthase, invertase and sucrose, hexose protein called membrane transporters.Sucrose, must be first after sucrose synthase and saccharase are decomposed into hexose in plant materials, could be used for the g and D of plant.
Physiologic Studies finds that easily drum grain cowpea variety has the active and higher protoblast mitotic activity of higher seed coat cell walls invertase (CWIN) than not easily rousing grain kind in early days in tender pod growth.Subsequently, cause its embryo more early terminate cell division phase and more early enter cell expanding stage owing to easily rousing grain kind seed coat CWIN quick decline that is active and embryo hexose/sucrose ratio, this causes easily drum grain kind protoblast to have the major reason of more volume.Therefore, seed coat CWIN plays an important role in asparagus bean tender pod drum grain, can as the target of resistance to bulging grain breeding.
Summary of the invention
For rely on merely in the resistance to bulging grain breeding ordinary method of cowpea field carry out the beanpod drum range estimation of graininess or instrument measure subjectivity existing for qualification by force, the easy defect such as affected by environment, a large amount of soil of time and effort consuming, needs and manpower, an object of the present invention is the PCR primer group of the resistance to bulging grain kind of molecular marking supplementary breeding cowpea, another object of the present invention is to provide the method adopting the resistance to bulging grain kind of above-mentioned primer sets molecular marking supplementary breeding cowpea.Method of the present invention has feature accurately and fast.
In order to realize first above-mentioned object, present invention employs following technical scheme:
The PCR primer group of the resistance to bulging grain kind of molecular marking supplementary breeding cowpea, the sequence of this primer sets is upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3'; Downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3'.
In order to realize second above-mentioned object, present invention employs following technical scheme:
The method of the resistance to bulging grain kind of molecular marking supplementary breeding cowpea, is characterized in that carrying out according to the following steps:
1) extraction of cowpea genomic dna: after each cowpea sample CTAB method to be detected is extracted genomic dna, preserve respectively, for subsequent use;
2) pcr amplification of genomic dna: by cowpea each sample genomic dna 20 ng, add in each PCR pipe respectively, and in each pipe, add PCR primer group to final concentration each 0.2 μM successively, dNTP is to final concentration 0.25 mM, MgCl2 is to final concentration 2.0 mM, PCR damping fluid to final concentration is 1 times, and add Taq archaeal dna polymerase 1 unit, finally add after aseptic redistilled water supplies volume to 20 μ l, at 94 DEG C of denaturation 2 min, 94 DEG C of sex change 30 sec, 58 DEG C of annealing 30 sec, 72 DEG C extend 30sec, 36 circulations, increase under 72 DEG C of extension 5min, product 4 DEG C preservation, the sequence of described primer sets is upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3', downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3',
3) gel electrophoresis analysis of digestion products: amplified production, at 8% non-denaturing polyacrylamide gel, above carries out electrophoretic separation, then silver dye develops the color, digital photo camera record result; Described non-denaturing polyacrylamide gel is acrylamide: methylene diacrylamide=29:1;
4) qualification of the resistance to bulging graininess of cowpea sample: according to the pattern of PCR primer band on gel, all amplified productions are the material that the material of 180bp fragment is containing the resistance to bulging grain gene type of cowpea, and all materials that can not obtain pcr amplification product are the material containing the not resistance to bulging grain gene type of cowpea;
5) resistance to bulging grain phenotype final election: normal water and fertilizer management is carried out to the above-mentioned plant through molecular marker screening, plant to be planted 10 buds of often selecting good strains in the field for seed flowering period are listed, by the bulging grain degree of the tender pod of the every bar of vernier caliper measurement after listing 10 days, using the mean value of every strain 10 tender pods as the bulging graininess degree value of this strain, remove the false positive caused due to Marker selection error, and resistance to bulging graininess of selecting and remain improves and the good new strain of offspring of economical character.
The invention has the beneficial effects as follows:
1. seedling stage assay, reduce workload, cost-saving: present invention obtains the PCR molecule marker according to small segment disappearance design on cowpea cell walls invertase gene, this mark associates with the resistance to bulging graininess of cowpea, and the auxiliary transformation that can be applicable to resistance to bulging grain gene detects; The method have easy to detect, disclose the advantages such as polymorphism ability is stable, low cost; Whether thus the resistance to bulging graininess of prediction plant the existence that can detect resistance to bulging grain gene in seedling stage with this mark, can be eliminated a large amount of not resistance to bulging grain separation offsprings in early days, the workload pressure of follow-up transplanting, management, qualification etc. can be minimized.Conventional incubation cowpea variety needs the cycle of 6-8, the breeding of application molecular marker assisted selection, can the multiple kind of inner analysis at one time, only need sample 1-2 time-of-week in seedling stage to the material of given generation and whether just clearly can be separated the existence of target gene in offspring, the seed selection of new variety can be completed in 3-4, greatly save man power and material;
2. improve the accuracy of selection: traditional breeding way needs to carry out field to the resistance to bulging graininess of filial generation or greenhouse is identified one by one, because tender pod drum grain phenotype is subject to the impact of environment, phenotypic evaluation often has certain error, utilizing molecule marker to carry out test in laboratory can avoid environment on the impact of tender pod drum grain phenotype, and accuracy obtains larger raising.Present method seedling stage apply Markers for Detection carry out primary dcreening operation after again centering select plant to carry out the check of phenotype at Adult plant, further increase the accuracy of selection.
Feature of the present invention (advantage or innovative point) is:
Utilize DNA sequence dna small segment to lack and develop molecule marker with remarkable association the between tender pod drum grain degree phenotypic number.Seedling stage adopts Marker-assisted selection, detects and needs DNA to measure fast and easy less, can reject most of non-targeted strain in early days, greatly reduce follow-up field work amount.Molecule marking method can be analyzed a large amount of germ plasm resource and hybridization thereof, backcross progeny, selects breeding parent material quickly from a large amount of target strain, shortens generation and the time of breeding.All have can use primer with the identical resistance to bulging grain cowpea strain of small segment deletion segment on " 121 " cell walls invertase gene
p-VuCWINcarry out marker assisted selection.
Embodiment
The present invention is described in further detail by following examples, but should be appreciated that the present invention not placed restrictions on by following content.
embodiment 1:
The molecule marker of the resistance to bulging graininess of special differentiation cowpea and the resistance to bulging graininess screening method of cowpea based on molecule marker, the method comprises the following steps:
(1) screening in molecule marker site that associates of bulging graininess resistance to cowpea: the DNA extracting 36 parts of different cowpea strains of resistance to bulging grain degree with the DNA of plants Mini Kit of Qiagen company according to method described in specification sheets; Utilize primer 5 '-CACTTCAGAGGATGGTTGG-3 ' and 5 '-CCTTTCTATCTTATCACTAAACTACC-3 ' amplification cowpea cell walls invertase gene fragment and carry out routine order-checking.To from the amplification of each strain obtain sequence and compare and identify the small segment pleomorphism site P-VuCWIN that length is 4bp;
(2) mixed linear model genotype data in above-mentioned P-CWIN site and 36 parts of cowpea product same as above being tied up to the MLM of the bulging grain degree value Tassel software obtained in field investigation over the years carries out association analysis, obtaining this site with the cognation P value of drum kernel Characters is 0.04, meet the statistical threshold of P<0.05, show that the resistance to bulging graininess of this marker site and cowpea significantly associates.
(3) specific detection primer
p-VuCWINdesign: the upstream and downstream primer in Indel-CWIN site is crossed in the cowpea cell walls invertase gene fragment design according to amplifying, and sequence is as follows:
Upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3';
Downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3'.
(4) pcr amplification of special primer and electrophoresis detection: amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (acrylamide: methylene diacrylamide=29:1), then silver dye develops the color, digital photo camera record result.All amplified productions are the material that the material of 180bp fragment is containing the resistance to bulging grain gene type of cowpea; All materials containing the not resistance to bulging grain gene type of cowpea, because its gene order and upstream primer 3 ' hold 3 in the base of 4, end not mate, therefore all can not obtain amplified production.
embodiment 2
Utilize mark
indel-CWINassisting sifting " 121 " and ' the resistance to bulging grain filial generation of cowpea 282 '
(1) not resistance to bulging grain main breed and resistance to bulging grain resource hybridization and backcross: select full non-resistance to bulging grain recurrent parent (' cowpea 282 ', economical character is good, easily rouses grain) and resistance to bulging grain donor parents ("
121", tender pod not easily rouses grain, but economical character is poor) be 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
1(about 5) and recurrent parent seed are sowed at land for growing field crops, treat that Post flowering is hybridized, and collect seed, be after pod maturation
bC 1 f 1 .3rd year by BC
1f
1(about 30) and recurrent parent seed are sowed at land for growing field crops, treat that Post flowering is hybridized, and collect seed, be after pod maturation by strain
bC 2 f 1 , every strain receives about 20 seeds.
(2) seedling stage Marker-assisted selection: upper step is obtained
600left and right BC
2f
1be separated progeny seed, a point individual plant is seeded in land for growing field crops and lists one by one.Seed germination gets 0.1 gram, blade sample after 20 days, grind into powder in liquid nitrogen uses CTAB method
extract DNA.First CTAB damping fluid is prepared: 100 mL 1 molL
-1tris pH 7.5,140 mL 5 molL
-1naCl, 20 mL 0.5 molL
-1eDTA pH 8.0,740 mL MiliQ H
2o, 20 g CTAB.The fresh blade getting 10mg during DNA extraction puts into mortar, adds 150 μ L CTAB damping fluids, grinds gently with alms bowl pestle, and then adds 150 μ L CTAB damping fluid mixings; 65 DEG C of water-bath 40min; Add 300 μ L 24:1 chloroform/primary isoamyl alcohol, mix 5 min up and down, make sample and chloroform fully mixed; 13000 rmin
-1centrifugal 5min; Get supernatant liquor 150 μ L, be added in the Virahol 200 μ L of-20 DEG C of precoolings, mixing of turning upside down gently; 10000 ~ 12000 rmin
-1centrifugal 3 ~ 4 min, abandon supernatant liquor; Allow Virahol volatilize clean, add the ddH of 50 ~ 100 μ L containing RNase
2o dissolving DNA; Then detect by the agarose electrophoresis of 1%.With in the present invention
moleculemark carries out pcr amplification,
reaction volumebe 25 microlitres, wherein 10 × buffer 2.5 microlitre, 25mM MgCl
21.5 microlitres, 2.5mM dNTPs 2 microlitre, Taq enzyme (5 units/microlitre) 0.2 microlitre, template DNA 10 nanogram, adds water to 25 microlitres.
pCR reaction systemafter DNA 94 DEG C of denaturation 3min, 94 DEG C of sex change 30sec, 58 DEG C of annealing 30sec, 72 DEG C extend exhibition 30sec, circulate 35 times, and last 72 DEG C extend 10min.In the enterprising performing PCR amplification of PTC-225 amplification instrument.Amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel (acrylamide: methylene diacrylamide=29:1), and then silver dye develops the color, digital photo camera record result.All amplified productions are that the material that the material of 180bp fragment is containing the resistance to bulging grain gene type of cowpea is retained, and the material that all materials that can not obtain amplified production are containing the not resistance to bulging grain gene type of cowpea is abandoned.
(3) Adult plant phenotype final election: normal water and fertilizer management is carried out to the plant through upper step molecular marker screening.Plant blossom phase 10 buds of often selecting good strains in the field for seed are listed, by the bulging grain degree of the tender pod of the every bar of vernier caliper measurement after listing 10 days, using the mean value of every strain 10 tender pods as the bulging graininess degree value of this strain.In conjunction with each strain economical character, select resistance to bulging graininess strong and the individual plant of economical character excellence, selfing obtains BC
3f
2, be more excellent, the resistance to bulging graininess of economical character in theory and improve and the genotype new lines of comparatively isozygotying.
(4) results and analysis: be the qualification that the separation offspring of resistance to bulging grain gene type carries out field drum grain degree by taking cell walls invertase gene to 14 strains through molecular marker analysis prompting institute, by formula: drum grain degree (%)=(L1 L2)/L2 × 100% calculates and rouses grain degree; Result shows have 11 strain drum grain degree <30% to be resistance to bulging grain, and 3 strains are not resistance to bulging grain, show that the accuracy rate of Markers for Detection is more than 78%.Screened with the error making up marker assisted selection by the final election of resistance to bulging grain phenotype and economical character again, finally obtain 1 resistance to bulging grain excellent new strain, called after " FK3-1 ".
Sequence table
<110> Zhejiang Academy of Agricultural Science
The resistance to method of bulging grain kind of <120> molecular marking supplementary breeding cowpea and the PCR primer group of use thereof
<160>2
<210>1
<211>29
<212> DNA
<213> artificial sequence
<400> 1
TTTCCATGGT AAAATTTTTT ACC 23
<210>2
<211>22
<212> DNA
<213> artificial sequence
<400> 2
CCATTCCAAG AACGATTTGG CT 22
Claims (2)
1. the PCR primer group of the resistance to bulging grain kind of molecular marking supplementary breeding cowpea, is characterized in that: the sequence of this primer sets is upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3'; Downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3'.
2. the method for the resistance to bulging grain kind of molecular marking supplementary breeding cowpea, is characterized in that carrying out according to the following steps:
1) extraction of cowpea genomic dna: after each cowpea sample CTAB method to be detected is extracted genomic dna, preserve respectively, for subsequent use;
2) pcr amplification of genomic dna: by cowpea each sample genomic dna 20 ng, add respectively in each PCR pipe, and in each pipe, add PCR primer group according to claim 1 to final concentration each 0.2 μM successively, dNTP is to final concentration 0.25 mM, MgCl
2to final concentration 2.0 mM, PCR damping fluid to final concentration is 1 times, and add Taq archaeal dna polymerase 1 unit, finally add after aseptic redistilled water supplies volume to 20 μ l, at 94 DEG C of denaturation 2 min, 94 DEG C of sex change 30 sec, 58 DEG C of annealing 30 sec, 72 DEG C extend 30sec, 36 circulations, increase under 72 DEG C of extension 5min, product 4 DEG C preservation;
3) gel electrophoresis analysis of digestion products: amplified production carries out electrophoretic separation on 8% non-denaturing polyacrylamide gel, then silver dye develops the color, digital photo camera record result; Described non-denaturing polyacrylamide gel is acrylamide: methylene diacrylamide=29: 1;
4) qualification of the resistance to bulging graininess of cowpea sample: according to the pattern of PCR primer band on gel, all amplified productions are the material that the material of 180bp fragment is containing the resistance to bulging grain gene type of cowpea, and all materials that can not obtain pcr amplification product are the material containing the not resistance to bulging grain gene type of cowpea;
5) resistance to bulging grain phenotype final election: normal water and fertilizer management is carried out to the above-mentioned plant through molecular marker screening, plant to be planted 10 buds of often selecting good strains in the field for seed flowering period are listed, by the bulging grain degree of the tender pod of the every bar of vernier caliper measurement after listing 10 days, using the mean value of every strain 10 tender pods as the bulging graininess degree value of this strain, remove the false positive caused due to Marker selection error, and resistance to bulging graininess of selecting and remain improves and the good new strain of offspring of economical character.
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| CN107201404A (en) * | 2017-06-15 | 2017-09-26 | 江西省农业科学院蔬菜花卉研究所 | A kind of molecular biology identification method and its application for Asparagus dioecian plant sex |
Families Citing this family (3)
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| CN108660248B (en) * | 2018-05-25 | 2021-12-17 | 浙江省农业科学院 | Method for breeding asparagus bean variety with molecular marker assistance |
| CN110004245B (en) * | 2019-03-20 | 2022-06-14 | 江苏省农业科学院 | Universal asparagus bean InDel molecular marker in different bean crops and development method and application thereof |
| CN111793706B (en) * | 2020-06-16 | 2021-06-29 | 广东省农业科学院蔬菜研究所 | Cowpea InDel molecular marker detection primer group and kit |
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