CN103468791A - 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

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

The method of the anti-drum grain of molecular marking supplementary breeding cowpea kind and the PCR primer sets of use thereof

Technical field

The present invention relates to plant biotechnology field, relate in particular to the method for the anti-drum grain of a kind of molecular marking supplementary breeding cowpea kind.

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 main edible part is tender pod, the 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.Yet usually run in asparagus bean production reality in tender pod growth course and to cause tender pod because the unbalanced growth, particularly seed development of pod wall and seed are too early too fast in seed attachment region projection and the phenomenon of other area depression is referred to as drum grain (accompanying drawing 1).Tender pod drum grain has a strong impact on the exterior quality of tender pod, reduces commodity value.The essence of the tender pod of cowpea 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.The breed and production practice shows, the tender pod drum of different cowpea varieties grain degree difference, and Breeding and application is difficult for the kind of drum grain and produces significant to cowpea.

The growth of seed and fruit is especially expanded and nutrient accumulation is regulated and controled with utilizing institute by the distribution of photosynthate mainly.Sucrose is the principal mode that the plant photosynthesis product transports and distributes between plant tissue, and it is bringing into play vital effect as nutrient and signaling molecule in the growing of plant.Research shows, legume crop tender pod pod wall and seed exist significant competitive relation to absorption and the utilization of sucrose, and sucrose is determining seed shared ratio in beanpod in pod wall and interseminal distribution, accumulation and metabolism.Sucrose Metabolism and distribution, comprise that phloem sucrose unloading, sucrose inversion are 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 in plant materials after sucrose synthase and saccharase are decomposed into hexose, could be used for the g and D of plant.

Physiologic Studies finds that easily drum grain cowpea variety has higher kind chrotoplast wall invertase (CWIN) activity and higher protoblast mitotic activity than being difficult for drum grain kind in early days in tender pod growth.Subsequently, owing to easily rousing all skin CWIN of grain product fast-descending active and embryo hexose/sucrose ratio, cause its embryo more early to finish cell division phase and more early enter the cell expanding stage, this is to cause the easy major reason that grain kind protoblast has more volume of rousing.Therefore, plant skin CWIN and play an important role in the tender pod drum of asparagus bean grain, can be used as the target of anti-drum grain breeding.

Summary of the invention

For in the anti-drum grain breeding of cowpea ordinary method, rely on merely field carry out the range estimation of beanpod drum graininess or instrument measure identify existing subjectivity by force, easily affected by environment, time and effort consuming, need the defects such as a large amount of soils and manpower, an object of the present invention is the PCR primer sets of the anti-drum grain of molecular marking supplementary breeding cowpea kind, another object of the present invention is to provide the method that adopts the anti-drum grain of above-mentioned primer sets molecular marking supplementary breeding cowpea kind.Method of the present invention has characteristics accurately and fast.

In order to realize first above-mentioned purpose, the present invention has adopted following technical scheme:

The PCR primer sets of the anti-drum grain of molecular marking supplementary breeding cowpea kind, the sequence of this primer sets is upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3'; Downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3'.

In order to realize second above-mentioned purpose, the present invention has adopted following technical scheme:

The method of the anti-drum grain of molecular marking supplementary breeding cowpea kind is characterized in that carrying out according to the following steps:

1) extraction of cowpea genomic dna: after each cowpea sample to be detected is extracted to genomic dna by the CTAB method, preserve respectively, standby;

2) pcr amplification of genomic dna: by each sample gene group of cowpea DNA 20 ng, add respectively in each PCR pipe, and in each pipe, add successively the PCR primer sets to each 0.2 μ M of final concentration, 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, after finally adding aseptic redistilled water and supplying volume to 20 μ l, at 94 ℃ of denaturation 2 min, 94 ℃ of sex change 30 sec, 58 ℃ of annealing 30 sec, 72 ℃ are extended 30sec, 36 circulations, under 72 ℃ of extension 5min, increased, 4 ℃ of preservations of product, the sequence of described primer sets is upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3', downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3',

3) enzyme is cut the gel electrophoresis analysis of product: amplified production, at 8% non-denaturing polyacrylamide gel, above carries out electrophoretic separation, and then silver dyes and developed the color, digital camera photographic recording result; Described non-denaturing polyacrylamide gel is acrylamide: methylene diacrylamide=29:1;

4) evaluation of the anti-bulging graininess of cowpea sample: according to the pattern of PCR product band on gel, the material that all amplified productions are the 180bp fragment is the material containing the anti-bulging grain gene type of cowpea, and all materials that can not obtain pcr amplification product are the material containing the not anti-bulging grain gene type of cowpea;

5) anti-drum grain phenotype final election: the above-mentioned plant through molecular marker screening is carried out to normal water and fertilizer management, plant to be planted 10 buds of often selecting good strains in the field for seed flowering period are listed, after listing 10 days by the drum grain degree of every tender pod of vernier caliper measurement, using the mean value of 10 tender pods of every strain as the bulging graininess degree value of this strain, the false positive that removal causes due to the Marker selection error, and the new strain of offspring that anti-bulging graininess improves and economical character is good of selecting and remain.

The invention has the beneficial effects as follows:

1. identify seedling stage, reduce workload, cost-saving: the present invention has obtained the PCR molecule marker according to small segment disappearance design on cowpea cell walls invertase gene, and this mark is associated with the anti-bulging graininess of cowpea, and the auxiliary transformation that can be applicable to anti-bulging grain gene detects; That the method has is easy to detect, disclose the advantages such as the polymorphism ability is stable, expense is cheap; Thereby the existence that with this mark, can detect anti-bulging grain gene in seedling stage is the anti-bulging graininess of prediction plant whether, can eliminate in early days a large amount of not anti-drum grain separation offsprings, the workload pressure of follow-up transplanting, management, evaluation etc. can be minimized.Conventional incubation cowpea variety needs the cycle of 6-8, the breeding of application molecular marker assisted selection, a plurality of kinds of inner analysis at one time, to the material of given generation only need seedling stage sample the 1-2 time-of-week just can clearly separate the offspring in target gene existence whether, the seed selection of new variety can be completed in 3-4, greatly saved the man power and material;

2. improved the accuracy of selecting: traditional breeding way need to carry out field or greenhouse is identified one by one to the anti-bulging graininess of filial generation, because tender pod is roused the impact that the grain phenotype is subject to environment, phenotypic evaluation often has certain error, utilize molecule marker to carry out the laboratory detection and can avoid the impact of environment on tender pod drum grain phenotype, accuracy obtains larger raising.Present method after applying Markers for Detection to carry out primary dcreening operation seedling stage again centering select plant to carry out the check of phenotype in the strain phase, further improved the accuracy of selecting.

Characteristics of the present invention (advantage or innovative point) are:

Utilize DNA sequence dna small segment disappearance and tender pod to rouse the remarkable associated exploitation molecule marker between grain degree phenotypic number.Adopt seedling stage molecule marker to select, detecting needs the DNA amount less and fast and easy can be rejected most of non-target strains in early days, has greatly reduced follow-up field workload.Molecule marking method can be analyzed a large amount of germ plasm resource and hybridization thereof, backcross progeny, selects the breeding parent material quickly from a large amount of target strains, shortens generation and the time of breeding.All have with " 121 " cell walls invertase gene on the identical anti-drum grain cowpea strain of small segment deletion segment can use primer p-VuCWINcarry out marker assisted selection.

[0010]?

embodiment

The present invention is described in further detail by following examples, but should be appreciated that the present invention is not placed restrictions on by following content.

embodiment 1:

The molecule marker of the anti-bulging graininess of special differentiation cowpea and the anti-bulging graininess screening method of cowpea based on molecule marker, the method comprises the following steps:

(1) screening in the molecule marker site associated with the anti-bulging graininess of cowpea: extract in a small amount test kit by the DNA of plants of Qiagen company and extract the DNA of 36 parts of different cowpea strains of anti-drum grain degree according to the described method of specification sheets; Utilize primer 5 '-CACTTCAGAGGATGGTTGG-3 ' and 5 '-CCTTTCTATCTTATCACTAAACTACC-3 ' amplification cowpea cell walls invertase gene fragment and carry out the routine order-checking.To from each strain amplification sequence that obtains, comparing and identify the small segment pleomorphism site P-VuCWIN that length is 4bp;

(2) genotype data in above-mentioned P-CWIN site and 36 parts of cowpea product same as above are tied up to the drum grain degree value obtained in field investigation over the years and carry out association analysis with the mixed linear model of the MLM of Tassel software, obtaining this site is 0.04 with the cognation P value of drum kernel Characters, the statistical threshold that meets P<0.05, show that the anti-bulging graininess of this marker site and cowpea is significantly associated.

(3) specific detection primer p-VuCWINdesign: according to the cowpea cell walls invertase gene fragment that amplifies, the upstream and downstream primer in Indel-CWIN site is crossed in design, and sequence is as follows:

Upstream primer: 5'-TTTCCATGGTAAAATTTTTTACC-3';

Downstream primer: 5'-CCATTCCAAGAACGATTTGGCT-3'.

(4) pcr amplification of special primer and electrophoresis detection: at 8% non-denaturing polyacrylamide gel, (acrylamide: carry out electrophoretic separation methylene diacrylamide=29:1), then silver dyes and is developed the color amplified production, digital camera photographic recording result.The material that all amplified productions are the 180bp fragment is the material containing the anti-bulging grain gene type of cowpea; All materials containing the not anti-bulging grain gene type of cowpea, do not mate due to 3 in its gene order and upstream primer 3 ' 4, end of end base, so all can not obtain amplified production.

embodiment 2

Utilize mark indel-CWINassisting sifting " 121 " and ' the anti-drum grain of cowpea a 282 ' filial generation

(1) hybridization of not anti-drum grain main breed and anti-drum grain resource and backcrossing: select a full non-anti-drum grain recurrent parent (' cowpea 282 ', economical character is good, easily the drum grain) and anti-rouse a donor parents (" 121", tender pod is difficult for the drum grain, but economical character is poor) be sowed at land for growing field crops.Hybridized after blooming, after the pod maturation, collected seed, be f ₁ .Next year is by F ₁(5 left and right get final product) and recurrent parent seed are sowed at land for growing field crops, after blooming, are hybridized, and after the pod maturation, collect seed, are bC ₁ f ₁ .The 3rd year by BC ₁f ₁(30 left and right) and recurrent parent seed are sowed at land for growing field crops, after blooming, are hybridized, and after the pod maturation, by strain, collect seed, are bC ₂ f ₁ , every strain is received 20 left and right seeds.

(2) seedling stage, molecule marker was selected: upper step is obtained 600left and right BC ₂f ₁separate progeny seed, a minute individual plant is seeded in land for growing field crops and lists one by one.Seed germination is got blade sample 0.1 gram after 20 days, grind into powder in liquid nitrogen is used the CTAB method extract DNA.At first prepare the CTAB damping fluid: 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.Get the fresh blade of 10mg during DNA extraction and put into mortar, add 150 μ L CTAB damping fluids, grind gently with the alms bowl pestle, and then add 150 μ L CTAB damping fluids to mix; 65 ℃ of water-bath 40min; Add 300 μ L 24:1 chloroform/primary isoamyl alcohol, mix up and down 5 min, 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 ℃ of precoolings, turn upside down and mix gently; 10000 ~ 12000 rmin ^-1centrifugal 3 ~ 4 min, abandon supernatant liquor; Make the Virahol volatilization clean, add the ddH of 50 ~ 100 μ L containing RNase ₂the O dissolving DNA; Then by 1% agarose electrophoresis, detect.With in the present invention moleculemark carries out pcr amplification, reaction volumebe 25 microlitres, 10 * buffer, 2.5 microlitres wherein, 25mM MgCl ₂1.5 microlitre, 2.5mM dNTPs 2 microlitres, Taq enzyme (5 units/microlitre) 0.2 microlitre, template DNA 10 nanograms, add water to 25 microlitres. the PCR reaction systemafter 94 ℃ of denaturation 3min of DNA, 94 ℃ of sex change 30sec, 58 ℃ of annealing 30sec, 72 ℃ are extended exhibition 30sec, circulate 35 times, and last 72 ℃ are extended 10min.In the enterprising performing PCR amplification of PTC-225 amplification instrument.At 8% non-denaturing polyacrylamide gel, (acrylamide: carry out electrophoretic separation methylene diacrylamide=29:1), then silver dyes and is developed the color amplified production, digital camera photographic recording result.The material that the material that all amplified productions are the 180bp fragment is containing the anti-bulging grain gene type of cowpea is retained, and the material that all materials that can not obtain amplified production are containing the not anti-bulging grain gene type of cowpea is abandoned.

(3) strain phase phenotype final election: the plant through upper step molecular marker screening is carried out to normal water and fertilizer management.Plant blossom phase 10 buds of often selecting good strains in the field for seed are listed, and after listing 10 days, by the drum grain degree of every tender pod of vernier caliper measurement, using the mean value of 10 tender pods of every strain as the bulging graininess degree value of this strain.In conjunction with each strain economical character, select the individual plant that anti-bulging graininess is strong and economical character is excellent, selfing obtains BC ₃f ₂, be the new lines that more excellent, the anti-bulging graininess of economical character improves and genotype is comparatively isozygotied in theory.

(4) results and analysis: the evaluation of carrying out field drum grain degree by the separation offspring who is anti-bulging grain gene type through the molecular marker analysis prompting cell walls invertase gene of taking to 14 strains, press formula: drum grain degree (%)=(L1 L2)/L2 * 100% calculates drum grain degree; It is anti-drum grain that result shows to have 11 strain drum grain degree<30%, and 3 strains, for not anti-drum grain, show that the accuracy rate of Markers for Detection is more than 78%.Screen to make up again the error of marker assisted selection by anti-drum grain phenotype final election and economical character, finally obtained 1 good new lines of anti-drum grain, called after " FK3-1 ".

Sequence table

<110 > Zhejiang Academy of Agricultural Science

<120 > method of the anti-drum grain of molecular marking supplementary breeding cowpea kind and the PCR primer sets 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 sets of the anti-drum grain kind of molecular marking supplementary breeding cowpea, it 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 anti-drum 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 to be detected is extracted to genomic dna by the CTAB method, preserve respectively, standby;

2) pcr amplification of genomic dna: by each sample gene group of cowpea DNA 20 ng, add respectively in each PCR pipe, and in each pipe, add successively PCR primer sets claimed in claim 1 to each 0.2 μ M of final concentration, 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, after finally adding aseptic redistilled water and supplying volume to 20 μ l, at 94 ℃ of denaturation 2 min, 94 ℃ of sex change 30 sec, 58 ℃ of annealing 30 sec, 72 ℃ are extended 30sec, 36 circulations, under 72 ℃ of extension 5min, increased, 4 ℃ of preservations of product;

3) enzyme is cut the gel electrophoresis analysis of product: amplified production, at 8% non-denaturing polyacrylamide gel, above carries out electrophoretic separation, and then silver dyes and developed the color, digital camera photographic recording result; Described non-denaturing polyacrylamide gel is acrylamide: methylene diacrylamide=29:1;

4) evaluation of the anti-bulging graininess of cowpea sample: according to the pattern of PCR product band on gel, the material that all amplified productions are the 180bp fragment is the material containing the anti-bulging grain gene type of cowpea, and all materials that can not obtain pcr amplification product are the material containing the not anti-bulging grain gene type of cowpea;

5) anti-drum grain phenotype final election: the above-mentioned plant through molecular marker screening is carried out to normal water and fertilizer management, plant to be planted 10 buds of often selecting good strains in the field for seed flowering period are listed, after listing 10 days by the drum grain degree of every tender pod of vernier caliper measurement, using the mean value of 10 tender pods of every strain as the bulging graininess degree value of this strain, the false positive that removal causes due to the Marker selection error, and the new strain of offspring that anti-bulging graininess improves and economical character is good of selecting and remain.