CN103789421B - The genomic SSR marker BM1 of a kind of qualification Oryza B, C - Google Patents

Abstract

The invention provides the genomic SSR marker BM1 of a kind of qualification Oryza B, C.It by nucleotide sequence as SEQ? ID? be upstream primer shown in NO.1 and nucleotide sequence as SEQ? ID? NO.2? shown downstream primer composition, SEQ? ID? the nucleotides sequence of NO.1: CAAGTTGCATTTGGACATGGSEQ? ID? the nucleotides sequence of NO.2: ATTGCCTTCACGTTTTGTCC.The genomic SSR marker BM1 of qualification Oryza B, C provided by the present invention, can overlap genome to B, C two and identify, its accuracy detected is high, simple to operate.

Description

The genomic SSR marker BM1 of a kind of qualification Oryza B, C

Technical field

The present invention relates to the genomic SSR marker BM1 of a kind of qualification Oryza B, C, belong to biology field.

Background technology

Under the severe challenge that cultivated area and water resources reduce gradually in current population in the world continuation growth, only have the yield per unit by improving paddy rice could meet the ever-increasing grain demand of the mankind.And the important breakthrough of the realization of this target and following rice breeding will depend on paddy gene storehouse (ricegenepoo1) to a great extent, particularly to exploitation and the sustainable utilization of redundant gene resource in wild-rice.This countermeasure is proved by the lot of examples in breeding practice, as: common wild-rice ( o.rufipogon) discovery of male sterile (MS) gene, transformation and three series mating, for huge contribution (Yuan, 1993) has been made in the incubation of hybrid rice and heterotic utilization; Annual common wild-rice ( o.nivara) in rosette poison (grassystuntvirus) resistant gene discovery and imported cultivated rice, incubation for anti-rosette rice varieties provides unique anti-source (Khush, 1977) value that the examples of many successful that, only above-mentioned two wild-rice genetic resourceses utilize is created has been calculate in units of hundred million yuan.In addition, the genomic species of BBCC have the characteristics such as disease-resistant, pest-resistant, waterlogging, salt tolerant, and in the exploitation and the work of paddy rice modern breeding of genetic resources, have very important significance (Brar & Khush, 1997).Comprising Oryza minuta ( o.minuta), tetraploid Oryza punctata ( o.punctata) and o.malampuzhaensis, overlap genome composition by B, C two, have with BB, CC genome and contact closely.BB genome only has a kind of seed rice, diplontic Oryza punctata ( o.punctata, 2n=24); And CC genome comprise oryza officinalis ( o.officinalis), rhizome wild-rice ( o.rhizomatis) and Oryza eichingeri ( o.eichingeri).

SSR marker is also called microsatellite marker (Microsatellitemarkers) or SSLP mark (SimpleSequenceLengthPolymorphism is called for short SSLP), widely distributed in higher organism genome.Generally form repeating unit's (core sequence) by 2-4 Nucleotide and repeat the tandem repetitive sequence that length that several times to tens formed is tens Nucleotide, as (GA) _n, (AC) _n, (GAA) _ndeng, on chromosome in stochastic distribution.Polymorphism (the Weber etc. that each seat enriches are caused owing to repeating the change of the incomplete of degree and multiplicity; 1989); therefore SSR is the very valuable molecule marker of one; be widely used in the structure of paddy DNA finger printing at present; colony and evolutionary genetics research; analysis of genetic diversity, the research field such as plasm resource protection and utilization.Although the length of iteron can change on the same loci of different genotype, but the DNA sequence dna on both sides, iteron is the single-copy sequences relatively guarded, therefore when using same primer, by PCR, the simple sequence repeats district of different genotype is increased out, the polymorphism between allelotrope can be shown.The major advantage of this molecule marker of SSR: first, SSR has extremely abundant polymorphism, quantity is enriched, cover whole genome, more much higher state property can be disclosed, karyomit(e) same position has multiple allelotrope, and numerous different allelotrope can be used as the different zones that special tag indicates homologous chromosomes; Secondly, SSR exists with high-caliber heterozygous, and this is very valuable for genetic mapping; Again, SSR marker is codominance, follows mendelian inheritance, not only very simple, and is identified also comparatively simple by PCR; Meanwhile, as neutrality mark, as a rule, SSR marker does not have phenotypic effect, does not exist the harmful of individuality or second-order effect; Further, have allelic characteristic, what can provide contains much information; Except a little, there is higher versatility.Its shortcoming is that this technology to the design of each site and will synthesize special primer, needs to carry out a large amount of examining order, thus needs to drop into a large amount of human and material resources.

Summary of the invention

The object of the present invention is to provide the genomic SSR marker BM1 of a kind of qualification Oryza B, C.For this reason, the present invention is by the following technical solutions:

The genomic SSR marker BM1 of a kind of qualification Oryza B, C, it is made up of the upstream primer of nucleotide sequence as shown in SEQIDNO.1 and the downstream primer of nucleotide sequence as shown in SEQIDNO.2.

The nucleotide sequence of described SEQIDNO.1 and SEQIDNO.2 is as follows:

SEQIDNO.1：CAAGTTGCATTTGGACATGG

SEQIDNO.2：ATTGCCTTCACGTTTTGTCC。

Owing to adopting technical scheme of the present invention, the invention provides the genomic SSR marker BM1 of a kind of qualification Oryza B, C, can overlap genome identify B, C two, its accuracy detected is high, simple to operate.

Accompanying drawing explanation

Fig. 1 is that molecule marker BM1 of the present invention has to 22 parts the AFLP system that Oryza B and C two overlaps genome germ plasm resource;

To be molecule marker BM1 of the present invention only have to 1 part the AFLP system that Oryza 1 B gene group and 15 parts only have Oryza C genome germ plasm resource to Fig. 2; 0 for only having the germ plasm resource of Oryza 1 B gene group; 1-15 is for only to have the genomic germ plasm resource of Oryza C.

Embodiment

Explain the present invention further below in conjunction with embodiment, but embodiment does not limit in any form to the present invention.

The experimental technique used in following embodiment if no special instructions, is ordinary method.

The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

One, the search of SSR marker and the design of primer

(i) Oryza BBCC genome sequence is obtained

Utilize shotgun (shotgunsequencing) in conjunction with Illumina high throughput sequencing technologies (IlluminaHiSeq2000sequencer, IlluminaInc., SanDiego, CA, USA) overlap genome kind W303 carry out genome sequencing to having Oryza B, C two;

(ii) adopt Phrap to run under DOS, sequence assembly is carried out to W303;

(iii) adopt MISA.perl script (http://pgrc.ipk-gatersleben.de/misa/) to carry out SSR site to W303 sequence to search:

1. perl is installed: the activeperl downloading required version;

2. target sequence, form is fasta;

3. est_timmer.pl(http is downloaded: //pgrc.ipk-gatersleben.de/misa/), est_timmer is with removing sequence (<100bp) too short in est sequence and polyA and the polyT tail of long sequence (>700bp) and mRNA;

4. CD_HIT batch de redundant sequence is utilized: http://www.bioinformatics.org/cd-hit/ downloads CD_HIT;

5. misa.pl searches for SSR;

(iv) according to the SSR site searched, adopt bioinformatics software primer3.0 to carry out design of primers, detect through pcr amplification, obtain primer and be SSR marker.

Two, primer PCR augmentation detection

(i) material

Germ plasm resource used comprises 22 parts and has the genomic germ plasm resource of Oryza B and C simultaneously, 1 part of Rice Resources only with Oryza 1 B gene group, only have the genomic Rice Resources of Oryza C with 15 parts, all germ plasm resource all can obtain (table 1) from International Rice germ plasm resource center, International Rice Research Institute.

The essential information of table 1 germ plasm resource used

(ii) experimental technique

The extraction of 1.DNA

According to the method for (1995) such as Zheng and the method in conjunction with (1992) such as Lu Yang rivers revise a little, concrete operation step is as follows:

(1) get the young shoot that 5 strain culture dish germinate, put into little mortar after shredding with scissors, add appropriate liquid nitrogen grinding powdered, add the DNA extraction liquid of twice 450 μ l, then transfer in the centrifuge tube of 1.5mL;

(2) in 65 DEG C of water-baths, be incubated 30min, vibrate once between soak;

(3) add 400 μ l chloroforms: primary isoamyl alcohol (24:1), several to the lower floor's liquid phase that turns upside down is deep green;

(4) the centrifugal 3min of full speed;

(5) get 450 μ L supernatant liquors in another sterilized 1.5mL centrifuge tube, then add 900 μ L dehydrated alcohols, left at room temperature 10min after mixing;

(6) the centrifugal 5min of full speed;

(7) remove supernatant liquor, by 70% washing with alcohol 3 times, seasoning;

(8) add 75 μ LTE solution after drying, make it to dissolve with have gentle hands bullet, centrifugal 1min;

(9)-20 DEG C of preservations.

2.DNA quality examination

The DNA agarose gel electrophoresis extracted detects.

Detection concrete grammar is as follows:

(1) prepare agarose solution: take 3g agar Icing Sugar and pour in erlenmeyer flask, then add 1 × tbe buffer liquid 150mL, loosely Kimwipes paper beyond the Great Wall on the bottleneck of conical flask, is heated to agarose and dissolves in boiling water bath or microwave oven;

(2) even by magnetic stirrer, treat that agarose solution is cooled to warm, be poured in rubber moulding, the thickness of gel between 3-5mm, under guaranteeing the tooth of comb or between cog there is no bubble;

(3) after gel solidifies completely, put it in electrophoresis chamber, in electrophoresis chamber, add 1 × tbe buffer liquid, carefully remove comb;

(4) draw 2 μ LDNA and 2 μ L3 × tetrabromophenol sulfonphthalein mixes;

(5) each well adds 2 μ L samples;

(6) after electrophoresis completes, gel is immersed in the water containing ethidium bromide (0.5 μ g/mL), in room temperature dyeing 20min, then puts into water 20min;

(7) carry out photographing and observe bar and bring detection DNA.

3.PCR reaction system and program

Reaction is carried out on ABI2720PCR instrument, reaction cumulative volume 10 μ L.Amplification condition is 94 DEG C of denaturation 2min; 94 DEG C of 45s, 60 DEG C of 45s, 72 DEG C of 1min, 30 circulations; Last 72 DEG C of downward-extension 8min.

Table 2PCR reaction system

Composition	Volume
		H 2O	3.3μL
5×SSR buffer	2.0μL
		(2.5 mmol/L) dNTP	0.8μL
(25.0 mmol/L) MgCl 2	0.6μL
		(5.0 μm of ol/L) Primer Forward(upstream primer)	1.0μL
(5.0 μm of ol/L) Primer Reverse(downstream primer)	1.0μL
		(5.0 U/ μ l) Taq DNA polysaccharase	0.3μL
DNA template	1.0μL

4. product detects

Pcr amplification product is applied 6% native polyacrylamide gel electrophoresis and is separated, and silver dye detects.

Native polyacrylamide gel electrophoresis concrete steps are as follows:

(1) prepare the polyacrylamide gel liquid of 6%: in the beaker of 100mL, add 5 × tbe buffer liquid 20mL, 40% acrylamide (BIS:acrylamide is 1:19) 15mL, after being settled to 100mL with distilled water, stirring by stirring rod, solution is mixed;

(2) the spacing piece between cleaning glass plate (block length one piece is short, and specification is respectively 15cm and 13cm) and insertion two plates;

By long and short two plate while alignment, insert the wide spacing piece of 1.0mm, with clip, offset plate two sides clamped, put into enclosed slot, fix simultaneously;

(4) in enclosed slot, pour 1% liquid agar gel sealing into, until agar solidification;

(5) in polyacrylamide gel solution, add 100 μ L10% ammonium persulphates and 48 μ LTEMED, bar magnet stirs, immediately encapsulating;

(6) insert sizeable comb immediately, now note observing not allowing below comb producing bubble, clamping comb and offset plate;

(7) gelation time is about 2h;

(8) being moved by offset plate is added on electrophoresis chamber, adds 1 × tbe buffer liquid, carefully remove comb in electrophoresis chamber;

(9) in product, add 2 μ L sample-loading buffers (3 × tetrabromophenol sulfonphthalein and the blue or green mixed solution of dimethylbenzene), mixing;

(10) each well adds 2 μ L samples;

(11), after electrophoresis, powered-down, pours out electrophoresis liquid (reusable), sheet glass distilled water is rinsed 30-60s;

(12) carefully separate two pieces of sheet glass, take off polyacrylamide gel, put into the plastic tub jog 5-10min that 400mL0.1% Silver Nitrate is housed;

(13) reclaim Silver Nitrate (reusable), distilled water rinses gel twice, often all over 30-60s;

(14) add nitrite ion 400mL colour developing to occur to band;

(15) fix with stationary liquid or drain and directly scan.

The Rice Resources having BBCC genome (i.e. BBCC-01 to the BBCC-22 of table 1) at 22 parts altogether extracts DNA, carries out above-mentioned testing process.Result shows, and SSR marker BM1 of the present invention all has pcr amplification product (Fig. 1), illustrates that this SSR marker BM1 is from BBCC genome sequence, has validity.

At 1 part, there are the Rice Resources that 1 B gene group (i.e. the BB-01 of table 1) and 15 parts have a C genome (i.e. CC-01 to the CC-15 of table 1) altogether and extract DNA, carry out above-mentioned testing process.Result shows, and SSR marker BM1 of the present invention can only exist pcr amplification product in the Rice Resources with 1 B gene group, and there is not pcr amplification product (Fig. 2) having in the genomic Rice Resources of C.Illustrate that this SSR marker BM1 can be used in distinguishing the genome Rice Resources only having B or only have C, if can there is corresponding pcr amplification product is illustrate that this Rice Resources only has 1 B gene group, and if can not to there is corresponding pcr amplification product be illustrate that this Rice Resources only has C genome.

<110> China Paddy Rice Inst

<120> mono-kind identifies the genomic SSR marker BM1 of Oryza B, C

<130>

<160>2

<170>PatentInversion3.3

<210>1

<211>20

<212>DNA

<213> artificial sequence

<400>1

caagttgcatttggacatgg20

<210>2

<211>20

<212>DNA

<213> artificial sequence

<400>2

attgccttcacgttttgtcc20

Claims (1)

1. identify Oryza B, C genomic SSR marker, it is made up of the upstream primer of nucleotide sequence as shown in SEQIDNO.1 and the downstream primer of nucleotide sequence as shown in SEQIDNO.2;

The nucleotide sequence of described SEQIDNO.1 and SEQIDNO.2 is as follows:

SEQIDNO.1：CAAGTTGCATTTGGACATGG

SEQIDNO.2：ATTGCCTTCACGTTTTGTCC。