CN109897909A - One kind molecular labeling relevant to corn kernel size and its application - Google Patents

Abstract

The invention belongs to corn breeding and molecular biology field, one kind molecular labeling relevant to corn kernel size and its application.The molecular labeling is cornskmThe DNA sequence dna as shown in SEQ No:1 on gene, there are a SNP sites at the DNA sequence dna 165bp, and base is G or C herein, and base is the big kernel corn of correspondence of G herein, and base is the correspondence fine grain corn of C herein.The alternative traditional method that corn kernel size is judged after corn is mature of the present invention, excludes the influence of human factor completely, keeps result more accurate and reliable.It is more simple and effective with conventional method, Saving in time costs and economic benefit can be greatlyd improve, and can be used for Large-scale Screening breeding material, greatly speed up the process of corn breeding research.

Description

One kind molecular labeling relevant to corn kernel size and its application

Technical field

The invention belongs to corn breedings and molecular biology field, more particularly to relevant to corn kernel size point of one kind Son label and its application.

Background technique

Source of the corn as grain, animal feed and industrial materials has become generation in plantation extensively all over the world at present Boundary the first generalized grain crop.With the appearance of the rapid growth of population, the decrease of cultivated land and food shortage, high yield will be corn The main target of breeding.Corn yield is mainly determined by corn per hectare spike number, number of grain per ear and every average weight three parts It is fixed.And grain is the quantitative inheritance character controlled by many genes again, influence it is smaller, mainly by grain length, grain is wide and grain is thick determines, People have carried out a large amount of research to the hereditary basis of seed size and kernel weight.

Grain recast is that important agronomy character and Yield Components can be used for promoting corn yield, and lose in recent years in molecule It passes more and more attractive in learning.Molecular labeling apply in map-based cloning be it is essential, it can help people Target gene is positioned.The type of molecular labeling has very much, such as simple sequence repeats (Simple sequence Repeat, SSR), single nucleotide polymorphism (Single nucleotide polymorphisms, SNP), randomly amplified polymorphic Property DNA (Random amplified polymorphic DNA, RAPD), DNA cloning fingerprint (DNA amplified Fingerprints, DAF) etc..The website MaizeGDB (http://www.maizegdb.org/) discloses a large amount of SSR mark Note, is distributed in each section of every chromosome.Even if in this way, in the map based cloning later period in order to be accurately positioned out candidate gene, It may also need to develop new molecular labeling.It, can preferential development in view of SSR marker principle is simple and convenient to operate.Due to B73 Whole genome sequence is it is known that can use it as reference sequences, the position SSR in the recycling software SSRHunter region of search Point, and new primer is designed according to output result, therefrom there are the molecular labelings of polymorphism to be used for the assignment of genes gene mapping for detection.If working as Prelocalization section is smaller, and without available SSR marker, then needs to consider exploitation SNP marker, set in current positioning section Meter primer uses the segment of the DNA cloning 1-2Kb of two kinds of self-mating systems respectively, and the two sequencing result is compared, and therefrom finds SNP site designs amplimer.But at present independent of the report of the relevant SNP marker of corn kernel size.

Summary of the invention

For the research lacked about the relevant SNP marker of corn kernel size, the present invention provides a kind of and beautiful The relevant molecular labeling of rice seed size.

It is a further object of the present invention to provide the applications of SNP marker relevant to corn kernel size.

The present inventor is in 2014 in Beijing Shunyi planting base, it was found that a seed size meets oneself of 3:1 separation Right mutant is selfed by several generations and is identified, it is found that the phenotype of the mutant can stablize heredity, be named as skm.

To achieve the above object, the present invention adopts the following technical scheme that:

A kind of molecular labeling relevant to corn kernel size, the molecular labeling are on corn skm gene such as SEQ No: DNA sequence dna shown in 1, there are a SNP sites at the DNA sequence dna 165bp, and base is G or C herein, herein base For the big kernel corn of correspondence of G, base is the correspondence fine grain corn of C herein.

A kind of specific primer of above-mentioned molecular labeling, the sequence of forward primer is as shown in SEQ No:2, reverse primer Sequence is as shown in SEQ No:3.

SEQ No:2:CAAGTACGTGACGGCGAAC；

SEQ No:3:TTCTTCGCGACCTCTTTCTC；

A method of identification corn kernel genotype, using following steps:

(1) genomic DNA of corn to be measured is extracted；

(2) using the genomic DNA of corn to be measured as template, using the primer of claim 2, pcr amplification reaction is carried out；

(3) amplified production is detected.

Preferably, PCR amplification, system and the program difference of amplification are carried out using the primer of claim 2 in step (2) Are as follows:

The reaction system of the PCR are as follows: 10 μ L

The response procedures of PCR are as follows:

The relevant molecular labeling of one kind identification corn kernel genotype described in claim 1 is assisted in corn molecular labeling Application in breeding.

Beneficial effect

(1) present invention uses forward genetics method, accurately separates candidate gene；

(2) function of skm gene does not have any report in plant, and the present invention provides the bases of regulation corn kernel size Because of skm nucleotide sequence, which has the molecule mechanism scheduling theory research that parsing corn kernel size is formed important Value.

(3) present invention can identify mature F₃Homozygosis, the heterozygosis situation of fruit ear exclude artificial, growth deficiency factor completely It influences, keeps result more accurate and reliable.It can be used for Large-scale Screening breeding material, so that assignment of genes gene mapping work is more accurate, greatly The big process for accelerating corn breeding research.

Detailed description of the invention

The left side Fig. 1 is doubtful F₂Homozygous fruit ear, the right side are F₂Heterozygosis fruit ear

Fig. 2 is B73 and skm plant disparity map after sowing 20 days；

Fig. 3 is B73 and skm endosperm starch granular size statistical chart；

Fig. 4 is B73 and skm endosperm starch particle comparison diagram under scanning electron microscope；

Fig. 5 SNP primer expands the agarose gel electrophoresis detection of B73, homozygous big grain, the big grain of heterozygosis, skm respectively.

Specific embodiment

Technical solution of the present invention is further explained in following embodiment, according to above description and these implementations Example, those skilled in the art can determine essential characteristic of the invention, and without departing from spirit and scope of the invention the case where Under, various changes and modifications can be made to the present invention, so that it is applicable in various uses and condition.

Embodiment 1

1 material and method

1.1 corn material

1.1.1 the acquisition of mutant material skm

2014, in Beijing Shunyi planting base, it was found that a seed size meets the natural mutant of 3:1 separation, It is selfed and identifies by several generations, it is found that the phenotype of the mutant can stablize heredity, be named as skm,

1.1.2 the building of target group

In May, 2016 in Beijing Shunyi planting base by the heterozygote plant pollen of the small particle mutant, it is normal with B73 respectively It makes and hybridizes of self-mating system；

In November, 2016 constructs the F of B73 self-mating system background in the numerous planting base breeding skm of South of Hainan₂Segregating population, As target group；

In May, 2017 constructs the F of B73 background in Jiyang Area, Shandong planting base breeding skm₃Segregating population, as fine Target group.

1.2 mutant skm grain characters mask datas statistics

5 B73 × skm F are taken respectively₁、B73×skm F₂、B73×skm F₃The heterozygosis fruit ear of group counts wild type The segregation ratio of seed and saltant type seed, statistical data are as follows:

1 B73 of table × skmF₁Group's ASSOCIATE STATISTICS and analysis

In summary data are analyzed, it can be deduced that draw a conclusion: the small particle mutant of the corn meets classical Mendel and loses Rule is passed, the seed mutant of Recessive genes control is belonged to.

2 B73 of table × skmF₂Group's ASSOCIATE STATISTICS and analysis

3 B73 of table × skmF₃Group's ASSOCIATE STATISTICS and analysis

1.3 corn kernel mutant skm phenotypic analyses and its germination and upgrowth situation investigation

As shown in Figure 1, it is less than normal seed in the mutation seed shape of mutant skm, the contracting of kind rhicnosis, recess.

In the emergence rate experiment of mutant skm, heterozygosis fringe 4 of phenotype separation are randomly selected, it is random on each fringe The germination of 50 fine grain roll papers is chosen, then moves on in frog stone/Nutrition Soil of 1:1 and grows, when tri-leaf period counts the emergence of each tassel Rate such as table 4, it can be seen that only about 10% or so mutation seed can be with germinating to seedling.Compared with wild type, although in same One stage of development, but the seedling of mutant is weak and growth and development is obviously slow.

The test of 4 mutant skm emergence rate of table

Summer in 2017 plants the mutant seed on 5 row parent hybrid fringes in Jiyang base experimental plot, and every row 26 is double Grain sowing, germinates 7 plants, seedling is markedly less than normal seed seedling, and can not grow to heading stage, sees Fig. 2 in total.

1.4 corn kernel mutant skm scanning electron microscope analysis

Using scanning electron-microscopy to the constituent microstructure observing of the endosperm of wild type seed and mutant seed Analysis, under scanning electron microscope, endosperm forms the imaging results such as following figure:

Under microstructure, significant changes are had occurred compared to wild type grain endosperm in mutant endosperm.Mutant endosperm Constituent it is obviously loose, many little particles are adulterated between amylum body, it is not of uniform size, filler it is also obvious rare and Amylum body size is than more consistent in normal seed, and close-packed arrays, the gap of starch intergranular are filled substance and fill up.

Primary Location is sequenced in 1.5 BSA

Corn material for sequencing are as follows: parent 1:B73 (wild type), parent 2:skm (saltant type), filial generation 1:B73 × skm F₃Group's wild type seed, filial generation 2:B73 × skm F₃Group's saltant type seed.

The parent 1 for having sprouting condition, parent 2, filial generation 1,2 seed of filial generation are selected, is set and the frog of 1:1 stone/Nutrition Soil In, 28 DEG C of illumination 16h, 22 DEG C of dark 8h, in greenhouse when culture to tri-leaf period, 10 parts of parents 1 of clip equivalent, 2 blade of parent It is placed in valve bag, the filial generation 1 of 200 parts of equivalent of clip, 2 blade of filial generation are respectively placed in valve bag and number.

1.5.1 Bioexperiment is sequenced in BSA

1. extracting the genomic DNA in sample, carry out building library after electrophoresis detection is qualified.

2. the qualified DNA sample of detection first passes through the segment that Covaris is broken into 350bp at random, using TruSeq DNA LT Sample Prep kit kit carries out building library, and DNA fragmentation is repaired by end, adds ployA tail, add sequence measuring joints, is pure Change, PCR amplification, is finally completed library construction.

1.5.2 bioinformatic analysis is sequenced in BSA

Under sequencing data after machine, data filtering is carried out first, removes low quality data, obtains Clean Reads；Then will Clean Reads detects the site SNP and InDel according to comparison result and annotates with reference to genome alignment；According to SNP site Information and SNP-index carry out mutational site filtering and positioning, finally filter out candidate gene.

1.5.3 chain deciding field

In segregating population build process, filial generation can be selected according to phenotype, filter out saltant type filial generation pond and wild Type filial generation pond, according to genetic linkage commutative law, the genotype in filial generation pond can be isolated with phenotype generation, be reacted in physical map Spectrum level, the meeting of chain chromosome segment and not chain chromosome interval generate stable SNP-index difference with phenotype.

1.5.4 candidate gene screening

1.5.4.1 principle is screened

(1) when Δ SNP-index is positioned, candidate gene is screened in the chromosomal region beyond belief line

Positive peak shows: in chain section, the SNP mutation type of filial generation is consistent with mutation parent, will lead to mutation table The generation of type.Therefore, it is consistent with mutation parent to screen mutation type in filial generation, and the site conduct that SNP-index differs greatly Candidate gene；

Negative sense peak shows: in chain section, the SNP mutation type of filial generation is different from mutation parent, will lead to mutation table The generation of type.Therefore, it is different from mutation parent to screen mutation type in filial generation, and the site conduct that SNP-index differs greatly Candidate gene.

(2) when SNP-index is positioned, candidate gene is screened in chromosomal region of the SNP-index close to 1

It is consistent with parent is mutated to screen mutation type in filial generation, and site of the SNP-index close to 1 is as candidate gene；

(3) screening criteria

It during detecting SNP&InDel, needs to use with reference to genome as bridge, therefore, is examined using reference genome Each sample SNP Ratio (ratio of reads number and the total reads number in the site different from the reference base) screening measured is waited Select gene.In view of sampling error, sequencing mistake etc. can be impacted to data, the site of value >=0.9 Ratio is defined For homozygous mutation, the site of value≤0.1 Ratio is defined as homozygosis and is not mutated.Screening criteria is as follows:

Table 3.5.1 Ratio value screening criteria

Remarks: (1) character is classified: character is generally divided into qualitative character, quantitative character, lethal qualitative character, different property Shape is related to sampling mode difference；(2) the aobvious recessiveness of gene: for cause mutant character gene aobvious recessiveness；(3) PM_ratio: It is mutated parent ratio value；(4) PWT_ratio: wild type ratio value；(5) FM_ratio: muton is for ratio value；(6) FWT_ratio: wild filial generation ratio value

1.5.4.2 block information is positioned

Table 3.5.2 group1 positions block information

Remarks: (1) Chromosome: chromosome location；(2) Start_position: positioning section initial position；(3) End_position: positioning section final position；(4) Gene_number: positioning section gene number；(5) SNP_number: Position SNP, section number；(6) Indel_number: positioning Indel, section number；(7) Peak: the highest in positioning section is set Believe interval threshold.

1.6 PARMS finely positionings

(Penta-primer amplification refractory mutation system, five primers expand PARMS Increase Refracting Mutation system) it is that skill is detected by the novel SNP marker of Jing Tai Biotechnology Co., Ltd, Wuhan City independent research Art has entirely autonomous intellectual property.PARMS is that one kind combines a pair of of universal fluorescent primer, a pair of SNP allele The SNP PCR analytical technology of special primer and a reversed general primer.SNP allele base can quickly and easily be carried out Because type detects.This research carries out finely positioning using PARMS technology.

1.6.2 PARMS SNP test experience material

In finely positioning, required number of groups may be larger, and the germination percentage of mutant is again relatively low, in order to prevent Mutant seed is wasted, and in order to quickly and easily carry out positioning experiment, therefore alkaline-heating method is taken to extract maize leaf DNA.

Corn material for PARMS SNP detection are as follows: parent 1:B73 (wild type), parent 2:skm (saltant type), son Generation: B73 × skm F₃Group's saltant type seed.

Select the parent 1 for having sprouting condition, parent 2, filial generation saltant type seed, set in the frog of 1:1 stone/Nutrition Soil, In 28 DEG C of greenhouses when culture to tri-leaf period, 2 parts of parents 1 of clip equivalent, 2 blade of parent are placed in 2mL centrifuge tube, clip 300 One's share of expenses for a joint undertaking is respectively placed in 2mL centrifuge tube and numbers for blade, and alkaline-heating method extracts maize leaf DNA；

1.6.3 alkaline-heating method extracts maize leaf DNA

1. appropriate blade (being about 2cm, wide about 0.5-1cm) is taken to be put into 2mL centrifuge tube, it is added 200 μ L 0.3M's NaOH solution, grind away is carried out in sample grinding machine, and 1200rpm is centrifuged 1min, boils 30s-1min in boiling water；

2. 300 μ L 0.75M Tris-HCl (pH7.4-7.8), boiling water boiling 30s-1min are added into 2mL centrifuge tube, 1200r centrifugation, sample of color is uniform, shows as dark green (tender leaf) or dark green (old leaf)；

3. taking 50 μ L of supernatant into PCR plate.

1.6.4 PARMS positioning result

Utilize B73 × skm F₃200 parts of mutation seed groups, filter out 9 exchange single plants, successfully determine the mutant The 152.27Mb-152.34Mb (70kb) of No. 7 chromosome of corn is arrived in position, is compared with wild-type sequence, finds Zm00001d021439 gene has bases G → C mutation in skm, causes frameshift mutation, and other genes do not have Variation.Thus infer, Zm00001d021439 may be the gene for controlling the phenotype.

Embodiment 2

Since skm seed nutritional ingredient is low, upgrowth situation is bad, in the part F of field harvest₂Fruit ear can not conclude whether For homozygous fruit ear (such as Fig. 2), seed genotype can be completed in Maize at Seedling Stage using SNP marker Zm00001d021439 Identification.

Experimental subjects is wild-type corn self-mating system B73 (10), F₂(doubtful) the homozygosis normal seed of fruit ear (10), F₂ The normal seed of heterozygosis fruit ear (10), F₂Heterozygosis fruit ear is mutated seed (10), in hot-house culture to tri-leaf period, extracts corn DNA is diluted to 20ng/uL by leaf DNA, isometric to mix, building B73 gene pool, doubtful homozygous big grain gene pond, heterozygosis Big grain gene pond, mutation seed gene pool.

The specific primer of SNP marker are as follows:

Forward primer: (CAAGTACGTGACGGCGAAC)

Reverse primer: (TTCTTCGCGACCTCTTTCTC)

Respectively using B73, doubtful homozygous big grain, the big grain of heterozygosis, mutation seed leaf DNA as template, SNP marker is utilized Specific primer carry out PCR amplification, agarose gel electrophoresis testing result such as Fig. 1 is respectively doubtful homozygous big from left to right Grain, the big grain of heterozygosis, mutation seed, B73.It is possible thereby to determine, the genotype and B73 of the doubtful homozygous big grain are homozygosis always Big grain fruit ear.

Sequence table

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Claims (4)

1. a kind of molecular labeling relevant to corn kernel size, which is characterized in that the molecular labeling is on corn skm gene The DNA sequence dna as shown in SEQ No:1, there are a SNP sites at the DNA sequence dna 165bp, and base is G or C herein, this Locate the big kernel corn of correspondence that base is G, base is the correspondence fine grain corn of C herein.

2. a kind of specific primer of molecular labeling as described in claim 1, which is characterized in that the sequence of forward primer is such as Shown in SEQ No:2, reverse primer sequences are as shown in SEQ No:3.

3. a kind of method for identifying corn kernel genotype, which is characterized in that use following steps:

(1) genomic DNA of corn to be measured is extracted；

(2) using the genomic DNA of corn to be measured as template, using the primer of claim 2, pcr amplification reaction is carried out；

(3) amplified production is detected.

4. the relevant molecular labeling of one kind identification corn kernel genotype described in claim 1 is educated in corn molecular labeling auxiliary Application in kind.