CN103409531A - Molecular marker method for rapid identification of variety authenticity and purity of rice Nanjing 46 - Google Patents

Abstract

The invention relates to a molecular marker method for rapid identification of variety authenticity and purity of rice Nanjing 46, and belongs to the technical field of agro-biological engineering. The method is as below: first extracting genome DNA from single seed rice endosperm; then carrying out amplification by using a specific molecular marker JS-Wx-mq of a gene Wx-mq with low amylose content, and screening a low amylose content rice variety with homozygous Wx-mq gene containing 439 bp and 292 bp characteristic strip; and utilizing five micro satellite markers to carry out PCR amplification on the low amylose content rice variety DNA with characteristic strips, and confirming the rice variety as Nanjing 46, if the above five micro satellite markers all have the characteristic strips. The method provided by the invention can realize rapid identification of variety authenticity and purity of rice Nanjing 46 with excellent taste, thereby guaranteeing rice quality of Nanjing 46 and benefiting brand maintenance and industrialization development of the variety.

Description

The molecule marking method of a kind of Rapid identification rice " southern round-grained rice 46 " kind true and false and purity

One, technical field

The present invention relates to the molecule marking method of a kind of Rapid identification rice " southern round-grained rice 46 " kind true and false and purity, belong to the biotechnology engineering field, be exclusively used in the judgement of excellent flavour rice " southern round-grained rice 46 " the kind true and false, purity, thereby guarantee the rice quality of " southern round-grained rice 46 ", be conducive to its kind Brand Maintaining and industrialization development.

Two, background technology

Rice is the main grain ration of China urban and rural residents, and the whole nation has 60% above population to take rice to be staple food, and the rice consumption accounts for 40% of national grain consumption.Therefore, rice production to ensure China's grain security and the national economic development significant (Sui its people, Liu Bo, Li Yuedong. brief talk China's rice industry current situation and development to strategy, rice in north china, 2010,40(3): 1-8).In recent years, along with the raising of the national economic development and living standards of the people, the human consumer has higher requirement to the quality of rice, and the high-grade rice of some excellent flavours gains great popularity.Take Japanese polished rice " more light " and " falling in love at first sight " is example, although its selling price is the several times of general rice, but still supply falls short of demand in market (Gong Yingjun, Feng Jie, Xiao Ke, Dong Yanjun, Lin Dongzhi. the Japanese Rice kind more QTL heredity of the Appearance Quality Traits of light rice dissects, China's agronomy circular, 2008,24 (12): 96-101).Therefore, the fine quality rice particularly industrialization development of excellent flavour rice not only has wide market outlook, be also simultaneously promote China's rice international competitiveness important channel (Zeng Songting. rice industry Business operation pattern and development countermeasure, hybrid rice, 2010, S1:550-552).。

Realize the prerequisite of excellent flavour rice industry at first must possess the high-grade rice kind of independent intellectual property right (Liu Yuzhong. slightly opinion rice variety, germplasm, brand and market, Grain in China economy, 2010,12:24-25).The Eleventh Five-Year Plan period, China be bred as GB more than three grades the high-grade rice kind a lot, but the Cooking Quality of most of kinds is not really up to the mark, on market, is difficult to compete mutually with Thailand, Japanese rice.Therefore, the Cooking Quality improvement has become the bottleneck of restriction China fine quality rice industrialized development.Except kind, the key that realizes the excellent flavour rice industry also is the establishment of rice brand.The well-known rice brand of China is a lot of at present, and the some of them brand has entered supermarket as " Jinjian ", " Panjin ", " good fortune near the house ", " 5 constant virtues " etc. and sold, and on market, has stronger occupation rate and influence power at home.Yet these rice brands are all brand names or the place of production brands of rice production unit registration, the rice of its sale is mixed and processed by a plurality of rice varieties often, and its Cooking Quality is difficult to be guaranteed.In fact, the Basic Evaluation standard of excellent flavour rice is mainly nice, therefore, only has single rice variety just can embody characteristic and the local flavor of rice, only has two kinds as the rice variety of China import Japan, is respectively " more light " and " falling in love at first sight "; And, in thailand scented rice, can allow to export and the rice varieties that enjoys high reputation also only is " Kd105 " and " KL60 ".As can be seen here, the rice variety brand to the development of fine quality rice industrialization have vital effect (rectify river. how rice enterprise builds brand, processing of farm products, 2009,3:4).

Along with the quick propelling of market economy, the development of rice variety brand also faces some new problems.Under the temptation of tremendous economic interests, the fraud event of fine quality rice brand emerges in an endless stream in recent years, indivedual illegal retailers pretend to be polished rice with rice of poor quality, such behavior brings serious economy, interests loss not only for Rice Milling Enterprises and human consumer, causes serious negative impact also for simultaneously the brand image of high-grade rice.Take Heilungkiang famous fine quality rice brand " 5 constant virtues rice " is example, its processing variety is mainly " five excellent rice No. 1 ", " five rice No. 3 ”,“ Fuji light " and " " the false rice " sold on No. 2 ”，Er markets of the rice fragrance of a flower is the poor quality of some retails rice in bulk often.Due to the method that lacks the fine quality rice kind true and false and purity detecting, its quality of brand name can not be guaranteed always, cause the industrialized development of " 5 constant virtues rice " to get into a difficult position (English plough, Sun Xiaodong. 5 constant virtues rice is pain, Green China, 2010,14:50-52; Yang Lijian. from " 5 constant virtues rice ", see grain check System Construction, forum of the association for science and technology, 2011,152).Therefore, the Rapid identification that how to realize the commercially available rice variety true and false and purity has become the problem that R&D institution, quality supervised department, Rice Milling Enterprises and human consumer etc. pay close attention in many ways.

Research shows, in the rice variety true and false and Purity, generally adopts and is still some physics and chemistry methods at present, and as morphologic observation, milling quality method, boiling food flavor method, protein analysis method etc.Although these methods have certain distinguishing ability, reliability, operability are not strong.Comparatively speaking, the molecular marking technique based on the DNA variation, have inheritance stability, is not subjected to the advantages such as envrionment conditions restriction, and what its banding pattern changed reflection is genotypic real difference between kind.Therefore, at aspects such as the evaluation of paddy rice resource, purity detecting and assortments, be used widely (Xin Yeyun, the exhibition, Xiong Yiping, the Yuan Longping. the super hybrid rice combination of application SSR molecular markers for identification and purity thereof, rice in China science, 2005,19 (2): 95 – 100; Chen Shulin, Wang Haiyan, Ding Zhenqian, Wang Xiue, the structure of the bright .2 of a Chen Zhong indica Hybrid Rice and 2 japonica rice variety SSR finger printings and the preliminary study of double PCR technology, the rice in China science, 2011,25(1): 19-24).Yet these researchs at present are all to take paddy rice band semina and plant to be object, and do not relate to the evaluation of the rice variety true and false and purity.Its reason mainly contains 2 points, social factor on the one hand, the purpose of the production of China's rice, processing is for solving the people's problem of food and clothing for a long time, fine quality rice industrialization, standardization level are very low, marketable value understanding to the rice variety brand is abundant not, has ignored the development of rice correlation detection technology; Be technical factor on the other hand, at first with blade, the positions such as stem stalk are compared, and accurately machined rice finished product only contains endosperm, and the reserve substances such as much starch, albumen make the extraction of endosperm of single rice grains DNA also have many difficulties to the inhibition of nucleic acid extraction.Secondly, the molecular marking technique in paddy rice, the single nucleotide variations loci gene type detected is perfect not enough, some labeling patterns such as cleaved amplified polymorphic sequence marker (Cleaved Amplified Polymorphic Sequence Marker, CAPS Marker) etc. also exist in actual applications operation loaded down with trivial details, the drawbacks such as somewhat expensive.But in any case, utilize molecular marking technique to carry out the effective way that the rice variety true and false and Purity are still at present the most worth exploration.

" southern round-grained rice 46 " is that Cereal Crops Research Inst., Jiangsu Agricultural Science Academy utilizes having of military fragrant round-grained rice 14 selection cross of 194Yu Jiangsu, Japanese high-grade rice Northeast high-yield variety Wx-mqThe low amylose content paddy rice of homozygous genotype.This kind is in 2008,2009 respectively by Jiangsu, the authorization of the Shanghai variety of crops council, and its outstanding advantages is that rice quality is excellent, and not only rice is glittering and translucent, and the mouthfeel softness is lubricious, high resilience, cold and not hard, Cooking Quality is splendid, is described as " rice that Jiangsu is the niciest "." southern round-grained rice 46 " acquisition excellent prize in the conference of national japonica rice rice in 2008 is unique front ten the rice varieties that enters of southern area; In the 9th national rice transaction expo of held in Hangzhou, obtained " gold medal rice " title, and be the unique selected rice varieties in Jiangsu in 2010; In the 5th, the 6th the national japonica rice conference that hold in Changchun 2010 and 2011, " southern round-grained rice 46 " is cited as respectively " high-quality food flavor polished rice " and " gold medal rice ", its kind brand value has obtained human consumer's extensive approval (Wang Cailin, Zhang Yadong ，Zhu town, Zhao Ling, Chen Tao, Lin Jing. feature and the cultivation technique of anti-stripe virus disease excellent flavour japonica rice new variety in evening south round-grained rice 46, Jiangsu agricultural sciences, 2008,2:91-92; Wang Cailin, Chen Tao, Zhang Yadong ，Zhu town, Zhao Ling, Lin Jing. by molecular marker assisted selection, cultivate the excellent flavour rice new variety, the rice in China science, 2009,23(1): 25-50.).

Therefore, for guaranteeing the fine quality of " southern round-grained rice 46 " rice, make better its rice variety brand, safeguard Rice Milling Enterprises and human consumer's legitimate rights and interests, be necessary to set up the kind true and false and the purity that a kind of detection method is fast and accurately effectively identified " southern round-grained rice 46 " rice.

Three, summary of the invention

Technical problem:The present invention is directed to the fraud problem that may occur in excellent flavour rice " southern round-grained rice 46 " kind brand initiative process, " the southern round-grained rice 46 " rice varieties of autonomous seed selection of take is core, by simple grain without embryo rice genomic dna rapid extraction, low amylose content gene Wx-mqThe screening of specific mark exploitation and a series of microsatellite markers, set up a kind of molecule marking method that is exclusively used in rice " southern round-grained rice 46 " the kind true and false and Purity, for its characteristic rice variety branding, industrialization development provide technical support.

Technical scheme:

1, the molecule marking method of a kind of Rapid identification rice " southern round-grained rice 46 " kind true and false and purity is characterized in that:

(1) extract the endosperm of single rice grains genomic dna;

(2) take above-mentioned genomic dna is template, uses low amylose content gene Wx-mqSpecific mark JS- Wx-mq

Forward outer primer sequence: 5'-ATGTTGTGTTCTTGTGTTCTTTGCAGGC-3'

Reverse outer primer sequence: 5'-GTAGATCTTCTCACCGGTCTTTCCCCAA-3'

Forward inner primer sequence: 5'-GGGTGAGGTTTTTCCATTGCTACAATCG-3'

Reverse inner primer sequence: 5'-GTCGATGAACACACGGTCGACTCAAT-3'

Carry out pcr amplification, screening has 439 bp and two feature bands of 292 bp simultaneously Wx-mqThe low amylose content rice variety of gene pure;

(3) on this basis, utilize microsatellite marker JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358

Mark JS-RM206

Forward primer sequence: 5'-CCCATGCGTTTAACTATTCT-3'

Reverse primer sequence: 5'-CGTTCCATCGATCCGTATGG-3'

Mark JS-RM495

Forward primer sequence: 5'-AATCCAAGGTGCAGAGATGG-3'

Reverse primer sequence: 5'-CAACGATGACGAACACAACC-3'

Mark JS-RM505

Forward primer sequence: 5'-AGAGTTATGAGCCGGGTGTG-3'

Reverse primer sequence: 5'-GATTTGGCGATCTTAGCAGC-3'

Mark JS-RM223

Forward primer sequence: 5'-GAGTGAGCTTGGGCTGAAAC-3'

Reverse primer sequence: 5'-GAAGGCAAGTCTTGGCACTG-3'

Mark JS-RM1358

Forward primer sequence: 5'-GATCGATGCAGCAGCATATG-3'

Reverse primer sequence: 5'-ACGTGTGGCTGCTTTTGC-3'

Continuation is carried out pcr amplification to above-mentioned low amylose content rice variety DNA with 439 bp and 292 bp feature bands, screening has respectively the kind of 173 bp, 149 bp, 181 bp, 151 bp and 170 bp feature bands at JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358 marker site, be " southern round-grained rice 46 ".

The extracting method of described endosperm of single rice grains genomic dna is:

By the 1.5 mL centrifuge tubes of packing into after the Single rice grain grinding of removal rice husk, kind skin, pericarp, embryo, add 800 μ L washingss, in shaking table 120 rpm vibration 5 min, centrifugal 2 min of 4,000 rpm, abandon supernatant liquor; In precipitation, add 600 μ L Extraction buffers, be placed in 65 ° of C water-bath 45 min; Add phenol-chloroform of 600 μ L volume ratio 25:24:1-primary isoamyl alcohol mixed solution, in shaking table 120 rpm vibration 10 min, centrifugal 5 min of 12,000 rpm, draw 500 μ L supernatant liquors to another centrifuge tube; Adding 500 μ L volume ratios is the chloroform-primary isoamyl alcohol mixed solution of 24:1,120 rpm vibration 10 min on shaking table, and centrifugal 5 min of 12,000 rpm, draw 400 μ L supernatant liquors to another centrifuge tube; Add 5 mol/L liquor kalii aceticis and the 293 μ L Virahols of 40 μ L pH=5.2 to mix, 4 ° of C place 2 h, and centrifugal 10 min of 12,000 rpm, abandon supernatant liquor; In precipitation, add 1 mL volume ratio 70% ethanol in shaking table 120 rpm vibration 2 min, centrifugal 3 min of 7,000 rpm, abandon supernatant liquor drying precipitated, with 100 μ L deionized water dissolvings, obtains the endosperm of single rice grains DNA solution; Described washings is: the Tris-HCl of 100 mmol/L pH=8.0,50 mmol/L EDTA, 0.35 mol/L sorbyl alcohol, mass ratio 1% polyvinylpyrrolidone; Described Extraction buffer is: mass ratio 2% CTAB, 100 mmol/L Tris-HCl, 25 mmol/L EDTA, 15 mol/L sodium-chlor, mass ratio 1% polyvinylpyrrolidone, 0.1 mg/mL Proteinase K, mass ratio 0.5% Nonidet P40, mass ratio 0.5% polysorbas20.

Described pcr amplification reaction and gel electrophoresis method are:

1) low amylose content gene specific mark JS- Wx-mqThe pcr amplification system be 20 μ L, the DNA 2.0 μ L of 10 ng/ μ L wherein, the primer 2 .0 μ L of 4 pmol/ μ L, four each 0.5 μ L of primer, contain 25 mmol/L MgCl ₂10 * PCR Buffer, 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 13.1 μ L; PCR response procedures: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s, 65 ℃ of renaturation 30 s, 72 ℃ of extension 1 min, circulate 35 times; Then 72 ℃ are extended 7 min, after 10 ℃ of cooling 10 min, amplified production are added to loading damping fluid termination reaction; Amplified production is electrophoresis 30 min on 1.5% sepharose than concentration in quality, under gel imaging system, observes after ethidium bromide staining;

2) the pcr amplification system of microsatellite marker is 10 uL, the DNA 1.0 μ L of 10 ng/ μ L wherein, and the primer 0.5 μ L of 4 pmol/ μ L, wherein each 0.25 μ L of positive and negative primer, contain 25 mmol/L MgCl ₂10 * PCR Buffer, 1.0 μ L, the dNTP 0.25 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 6.75 μ L; The PCR response procedures comprises: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s, 55 ℃ of renaturation 30 s, 72 ℃ of extension 30 s, circulate 35 times; Then 72 ℃ are extended 7 min, after 10 ℃ of cooling 10 min, amplified production are added to loading damping fluid termination reaction; Amplified production is electrophoresis on 9% polyacrylamide gel than concentration in quality, and 180V electrophoresis 2 h, silver are observing after dying colour developing under visible lamp box.

Beneficial effect

The molecule marking method of a kind of Rapid identification rice provided by the invention " southern round-grained rice 46 " kind true and false and purity has the following advantages:

(1) genomic dna that in the inventive method, the molecule marker amplification is used is from extracting and obtain the rice endosperm, such method of drawing material is most important to identifying the rice variety true and false and purity, correlated quality superintendent office and feeler mechanism can directly take a sample, identify the rice of market sale, the action of practicing fraud of farthest avoiding Rice Milling Enterprises to occur in the offering sample process, thereby the verity of assurance qualification result.

(2) in the inventive method Markers for Detection genomic dna from Single rice grain, extracting, can judge the true and false of every rice like this, then by the method for statistical sampling, calculate the purity of " southern round-grained rice 46 " rice in certain batch products, make detected result have higher confidence level.

(3) the inventive method is to utilize the PCR molecule marker to identify the true and false and the purity of rice " southern round-grained rice 46 " kind, with morphologic observation, conventional physical, the chemical processes such as milling quality method, boiling food flavor method, protein analysis method are compared, its advantage is that speed is fast, simple to operate, accuracy is high, result is stable and is not subject to the shadow of environment and human factor, successfully solved the guardian technique problem of the commercially available rice variety true and false and Purity.

Four, accompanying drawing explanation

Fig. 1 is for detection of the rice low amylose content gene Wx-mqThe molecule marker design diagram

(annotate: underscore means WxThe variant sites of G to A occurs in the 497th Nucleotide of site the 4th exon; Wx- Mq-O-F means the forward outer primer, Wx- Mq-O-R means reverse outer primer, Wx- Mq-I-F means the forward inner primer, Wx- Mq-I-R means reverse inner primer)

Fig. 2 low amylose content gene Wx-mqSpecific mark JS- Wx-mqAgarose gel electrophoresis figure to the amplification of different rice varieties (strain) endosperm genomic dna

(M:DNA molecular weight standard, 100-2,000 bp; Swimming lane 1-5: do not contain Wx-mqThe normal amylose content rice variety of gene (strain), town rice 88, Huaidao 9, town rice 14, southern round-grained rice 44, force are transported round-grained rice No. 23; Swimming lane 6-10: contain Wx-mqHomozygous genotype low amylose content rice variety (strain), southern round-grained rice 46, southern round-grained rice 5055, peaceful 5059, peaceful 8107, southern round-grained rice 9108)

Fig. 3 microsatellite marker JS-RM206, JS-RM495, JS-RM505, JS-RM223 and the JS-RM1358 polyacrylamide gel electrophoresis figure to the amplification of low amylose content rice variety (strain) endosperm genomic dna

(A: microsatellite marker JS-RM206; B: microsatellite marker JS-RM495; C: microsatellite marker JS-RM505; D: microsatellite marker JS-RM223; E: microsatellite marker JS-RM1358; The M:DNA molecular weight standard, 50-450 bp; Swimming lane 1-5: contain Wx-mqHomozygous genotype low amylose content rice variety (strain), southern round-grained rice 46, southern round-grained rice 5055, peaceful 5059, peaceful 8107, southern round-grained rice 9108)

Fig. 4 low amylose content gene Wx-mqSpecific mark JS- Wx-mqAgarose gel electrophoresis figure to rice " southern round-grained rice 46 " the kind true and false and Purity

(M:DNA molecular weight standard, 100-2,000 bp; Swimming lane 2-10: contain Wx-mqHomozygous genotype low amylose content rice variety " southern round-grained rice 46 "; Swimming lane 1: do not contain Wx-mqThe normal amylose content rice variety of gene " southern round-grained rice 45 ")

Fig. 5 microsatellite marker JS-RM206, JS-RM495, JS-RM505, JS-RM223 and the JS-RM1358 polyacrylamide gel electrophoresis figure to rice " southern round-grained rice 46 " the kind true and false and Purity

(A: microsatellite marker JS-RM206; B: microsatellite marker JS-RM495; C: microsatellite marker JS-RM505; D: microsatellite marker JS-RM223; E: microsatellite marker JS-RM1358; The M:DNA molecular weight standard, 50-450 bp; Swimming lane 2,3,4,5,7,8,9,10: contain Wx-mqHomozygous genotype low amylose content rice variety " southern round-grained rice 46 "; Swimming lane 1: do not contain Wx-mqThe normal amylose content rice variety of gene " southern round-grained rice 45 "; Swimming lane 6: contain Wx-mqHomozygous genotype low amylose content rice variety " southern round-grained rice 50 ")

Five, embodiment

For fully disclosing the molecule marking method of a kind of Rapid identification rice of the present invention " southern round-grained rice 46 " kind true and false and purity, be illustrated below in conjunction with method validation and embodiment.Its concrete implementation step is as follows:

(1) test materials

Do not contain Wx-mqThe normal amylose content rice variety of gene (strain): town rice 88, Huaidao 9, town rice 14, southern round-grained rice 44, military No. 23, round-grained rice of fortune, southern round-grained rice 45.

Contain Wx-mqHomozygous genotype low amylose content rice variety (strain): southern round-grained rice 46, southern round-grained rice 5055, peaceful 5059, peaceful 8107, southern round-grained rice 9108, southern round-grained rice 50.

Above material is public material, and agricultural germ plasm resource storehouse in mid-term, Jiangsu Province can provide free.Concrete reference is: Sheng Shenglan, Hu Chunming, Zhang Jiben, Yang Tunan. the seed selection of town rice 88 and cultivation main points, Jiangsu agricultural sciences, 1998,3:11-13; Yuan Shengtang, Yuan Caiyong, Wang Jian, Wen Zhenghuai. the seed selection of Huaidao 9 and application, Chinese rice, 2006,12(4): 21-22; The road, Jingde, Pan Guobao, Diao Liping, Lin Tianzi, Hu Chunming, Gong Hongbing, Zhou Yiwen, Qian Huafei, Lee rushes, repercussions, Zeng Shengyuan, Sheng Shenglan. seed selection and high-yield culture technique that slow Shu Zhongjing town rice is No. 14, the Jiangsu agricultural sciences, 2011,39(6): 186-188; Wang Cailin, Zhang Yadong ，Zhu town, Zhao Ling, Zhong Weigong, Chen Zhide, Yang Jie. the breeding and application of anti-stripe virus disease new rice variety south round-grained rice 44, Chinese rice, 2007,13(2): 33-34; Zhu Banghui, Xu Xiaojie, Shi Shijie, Xu Jie, Xu Yufeng, the button leaf is red, Xu Jiefen. seed selection and the cultivation technique that No. 23, the military fortune of anti-stripe virus disease new variety round-grained rice, Chinese rice, 2010,16(4): 60-61; Zhong Weigong, Chen Zhide, Yang Jie, Wang Jun, yellow transhipment, seed selection and the cultivation technique of japonica rice new variety south round-grained rice 45, Jiangsu agricultural sciences, 2009,5:123-125; Wang Cailin, Zhang Yadong ，Zhu town, Zhao Ling, Chen Tao, Lin Jing. feature and the cultivation technique of anti-stripe virus disease excellent flavour japonica rice new variety in evening south round-grained rice 46, Jiangsu agricultural sciences, 2008,2:91-92; Wang Cailin, Zhang Yadong ，Zhu town, Chen Tao, Zhao Qingyong, Zhao Ling, Zhou Lihui, Yao Shu. seed selection and the utilization of excellent flavour japonica rice new variety south round-grained rice 5055, agricultural science and technology communication, 2012,2:84-88; Wang Cailin, Zhang Yadong ，Zhu town, Chen Tao, Zhao Qingyong, Zhou Lihui, Yao Shu, Zhao Ling. anti-stripe virus disease excellent flavour japonica rice breeding of new variety research, rice in north china, 41(1): 67-71.

(2) exploitation of molecule marker and selection

(1) low amylose content gene Wx-mqThe exploitation of specific molecular marker

To contain and not contain Wx-mqTrans-genetic hybrid rice kind " southern round-grained rice 46 " and " southern round-grained rice 44 " are material, according to paddy rice WxThe genomic information in site, this site sequence of design primer pair carries out pcr amplification order-checking.South round-grained rice 46 exists WxThe 497th of coding region, site Nucleotide (the 4th exon) sports A by G, makes the 158th bit codon become CAT by CGT, thereby causes southern round-grained rice 46 WxGene expression amount descends, and amylose content reduces.According to this mononucleotide difference, designed evaluation Wx-mqThe tetra-primer ARMS-PCR PCR mark JS-of different genotype Wx-mq(Fig. 1).

(2) selection of microsatellite marker

According to microsatellite marker, be to be formed by 2-6 Nucleotide series connection repeating unit, and multiplicity is the genetic characteristics of variability, each karyomit(e) base sequence (www.gramene.org) of download paddy rice between individuality.Utilize SSRHunter software (Li Qiang, ten thousand build people .SSRHunter, the exploitation of the SSR site search software of a localization, heredity, 2005,27(5): 808-810) search is evenly distributed on the microsatellite locus of rice chromosome, utilizes Primer Premier 5.0(http: //www.premierbiosoft.com) 150 of software design microsatellite markers, every average 10 marks of karyomit(e) left and right.

(3) extraction of endosperm of single rice grains genomic dna

Get the Single rice grain of town rice 88, Huaidao 9, town rice 14, southern round-grained rice 44, military No. 23, round-grained rice of fortune, southern round-grained rice 46, southern round-grained rice 5055, peaceful 5059, peaceful 8107,9,108 10 different rice varieties of southern round-grained rice (strain), after removing rice husk, kind skin, pericarp, embryo grinding, be respectively charged into 1.5 mL centrifuge tubes, add the 800 μ L washings (Tris-HCl of 100 mmol/L pH=8.0,50 mmol/L EDTA, 0.35 mol/L sorbyl alcohol, mass ratio 1% polyvinylpyrrolidone), in shaking table 120 rpm vibration 5 min, 4, centrifugal 2 min of 000 rpm, abandon supernatant liquor; In precipitation, add 600 μ L Extraction buffer (mass ratio 2% CTAB, 100 mmol/L Tris-HCl, 25 mmol/L EDTA, 15 mol/L sodium-chlor, mass ratio 1% polyvinylpyrrolidone, 0.1 mg/mL Proteinase K, mass ratio 0.5% Nonidet P40, mass ratio 0.5% polysorbas20), be placed in 65 ° of C water-bath 45 min; Add phenol-chloroform of 600 μ L volume ratio 25:24:1-primary isoamyl alcohol mixed solution, in shaking table 120 rpm vibration 10 min, centrifugal 5 min of 12,000 rpm, draw 500 μ L supernatant liquors to another centrifuge tube; Adding 500 μ L volume ratios is the chloroform-primary isoamyl alcohol mixed solution of 24:1,120 rpm vibration 10 min on shaking table, and centrifugal 5 min of 12,000 rpm, draw 400 μ L supernatant liquors to another centrifuge tube; Add 5 mol/L liquor kalii aceticis and the 293 μ L Virahols of 40 μ L pH=5.2 to mix, 4 ° of C place 2 h, and centrifugal 10 min of 12,000 rpm, abandon supernatant liquor; In precipitation, add 1 mL volume ratio 70% ethanol in shaking table 120 rpm vibration 2 min, 7, centrifugal 3 min of 000 rpm, abandon supernatant liquor drying precipitated, with 100 μ L deionized water dissolvings, (endosperm of single rice grains extracting genome DNA process is drawn from this seminar mandate patent of invention and is slightly simplified to obtain the endosperm of single rice grains DNA solution.A kind of from endosperm of single rice grains, extracting the method for high quality genomic dna, the patent No.: ZL201110434777.5, open (bulletin) number: CN102433323A).

(4) pcr amplification reaction and gel electrophoresis

(1) low amylose content gene specific mark JS- Wx-mqThe pcr amplification system be 20 μ L, the DNA 2.0 μ L of 10 ng/ μ L wherein, the primer 2 .0 μ L of 4 pmol/ μ L, four each 0.5 μ L of primer, contain 25 mmol/L MgCl ₂10 * PCR Buffer, 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 13.1 μ L; PCR response procedures: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s, 65 ℃ of renaturation 30 s, 72 ℃ of extension 1 min, circulate 35 times; Then 72 ℃ are extended 7 min, after 10 ℃ of cooling 10 min, amplified production are added to loading damping fluid termination reaction; Amplified production is electrophoresis 30 min on 1.5% sepharose than concentration in quality, under gel imaging system, observes after ethidium bromide staining.

(2) the pcr amplification system of microsatellite marker is 10 uL, the DNA 1.0 μ L of 10 ng/ μ L wherein, and the primer 0.5 μ L of 4 pmol/ μ L, wherein each 0.25 μ L of positive and negative primer, contain 25 mmol/L MgCl ₂10 * PCR Buffer, 1.0 μ L, the dNTP 0.25 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 6.75 μ L; The PCR response procedures comprises: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s, 55 ℃ of renaturation 30 s, 72 ℃ of extension 30 s, circulate 35 times; Then 72 ℃ are extended 7 min, after 10 ℃ of cooling 10 min, amplified production are added to loading damping fluid termination reaction; Amplified production is electrophoresis on 9% polyacrylamide gel than concentration in quality, and 180V electrophoresis 2 h, silver are observing after dying colour developing under visible lamp box.

(5) mark JS- Wx-mqTo different rice varieties (strain) low amylose content gene Wx-mqGenotypic detection

The simple grain endosperm genomic dna of above-mentioned 10 different rice varieties (strain) of take is template, uses low amylose content gene Wx-mqSpecific mark JS- Wx-mqCarry out pcr amplification, screening has 439 bp and two feature bands of 292 bp simultaneously Wx-mqThe low amylose content rice variety (Fig. 2) of gene pure.

Specific mark JS- Wx-mqPrimer sequence:

Forward outer primer sequence: 5'-ATGTTGTGTTCTTGTGTTCTTTGCAGGC-3',

Reverse outer primer sequence: 5'-GTAGATCTTCTCACCGGTCTTTCCCCAA-3',

Forward inner primer sequence: 5'-GGGTGAGGTTTTTCCATTGCTACAATCG-3',

Reverse inner primer sequence: 5'-GTCGATGAACACACGGTCGACTCAAT-3'.

(6) microsatellite marker of Screening and Identification rice " southern round-grained rice 46 " the kind true and false and purity

At mark JS- Wx- MqOn the basis of detecting, utilization is evenly distributed on 150 microsatellite markers of 12 karyomit(e)s of paddy rice and continues to have 439 bp and 292 bp feature bands to 5 Wx-mqThe southern round-grained rice 46 of gene pure low amylose content rice variety (strain), southern round-grained rice 5055, peaceful 5059, peaceful 8107 and the DNA of southern round-grained rice 9108 carry out pcr amplification, screening altogether 5 of the microsatellite markers that band is clear, reproducible, reliability is high, resolving ability is strong, is respectively JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358.The determining nucleic acid sequence result shows, rice variety " southern round-grained rice 46 " has respectively the specific band (Fig. 3) of 173 bp, 149 bp, 181 bp, 151 bp and 170 bp at JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358 marker site.

The primer sequence of microsatellite marker JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358:

Mark JS-RM206

Forward primer sequence: 5'-CCCATGCGTTTAACTATTCT-3'

Reverse primer sequence: 5'-CGTTCCATCGATCCGTATGG-3'

Mark JS-RM495

Forward primer sequence: 5'-AATCCAAGGTGCAGAGATGG-3'

Reverse primer sequence: 5'-CAACGATGACGAACACAACC-3'

Mark JS-RM505

Forward primer sequence: 5'-AGAGTTATGAGCCGGGTGTG-3'

Reverse primer sequence: 5'-GATTTGGCGATCTTAGCAGC-3'

Mark JS-RM223

Forward primer sequence: 5'-GAGTGAGCTTGGGCTGAAAC-3'

Reverse primer sequence: 5'-GAAGGCAAGTCTTGGCACTG-3'

Mark JS-RM1358

Forward primer sequence: 5'-GATCGATGCAGCAGCATATG-3'

Reverse primer sequence: 5'-ACGTGTGGCTGCTTTTGC-3'

(7) utilize molecule marker rice " southern round-grained rice 46 " to be carried out to the Molecular Identification of the kind true and false and purity

For confirming the identification result of above-mentioned molecule marker to rice " southern round-grained rice 46 " the kind true and false and purity, artificially will not contain Wx-mqGene rice " southern round-grained rice 45 " and containing Wx-mq Each 1, homozygous genotype rice " southern round-grained rice 50 " is sneaked in 8 rice varieties " southern round-grained rice 46 " at random, built Markers for Detection required mix colony.In the Molecular Identification process of the kind true and false and purity, at first extract the endosperm of single rice grains genomic dna of each sample of colony, then utilize low amylose content gene Wx-mqSpecific mark JS- Wx-mqEndosperm genomic dna to these rice carries out pcr amplification and agarose gel electrophoresis (Fig. 4), and result shows: except the 1st sample, other all has 439 bp and two feature bands of 292 bp simultaneously, can judge that thus sample 1 is not containing of sneaking into Wx-mqThe normal amylose content rice variety of gene " southern round-grained rice 45 ".

For identifying further whether this rice that mixes colony is " southern round-grained rice 46 " entirely, continuation detects (Fig. 5) by microsatellite marker JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358 to all samples DNA, the polyacrylamide gel electrophoresis result shows, except the 1st and the 6th sample, other rice all has respectively the specific band identical with rice variety " southern round-grained rice 46 " at above-mentioned marker site, can judge that thus sample 6 is containing of sneaking into Wx-mqHomozygous genotype low amylose content rice variety " southern round-grained rice 50 ".Therefore, according to the total information that above-mentioned mark provides, can identify fast and accurately the kind true and false and the purity of rice " southern round-grained rice 46 ".

SEQUENCE LISTING

<110 > Jiangsu Province Agriculture Science Institute

<120 > molecule marking method of a kind of Rapid identification rice " southern round-grained rice 46 " kind true and false and purity

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<170> PatentIn version 3.1

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<221 > mark JS-RM495 forward primer sequence

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<221 > mark JS-RM505 forward primer sequence

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<220>

<221 > mark JS-RM505 reverse primer sequence

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<223>

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gatttggcga tcttagcagc 20

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<211> 20

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<220>

<221 > mark JS-RM223 forward primer sequence

<222> (1)..(20)

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<211> 20

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<221 > mark JS-RM223 reverse primer sequence

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gaaggcaagt cttggcactg 20

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<220>

<221 > mark JS-RM1358 forward primer sequence

<222> (1)..(20)

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<221 > mark JS-RM1358 reverse primer sequence

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acgtgtggct gcttttgc 18

Claims (3)

1. the molecule marking method of Rapid identification rice " southern round-grained rice 46 " the kind true and false and purity is characterized in that:

(1) extract the endosperm of single rice grains genomic dna;

(2) take above-mentioned genomic dna is template, uses low amylose content gene Wx-mqSpecific mark JS- Wx-mq

Forward outer primer sequence: 5'-ATGTTGTGTTCTTGTGTTCTTTGCAGGC-3'

Reverse outer primer sequence: 5'-GTAGATCTTCTCACCGGTCTTTCCCCAA-3'

Forward inner primer sequence: 5'-GGGTGAGGTTTTTCCATTGCTACAATCG-3'

Reverse inner primer sequence: 5'-GTCGATGAACACACGGTCGACTCAAT-3'

Carry out pcr amplification, screening has 439 bp and two feature bands of 292 bp simultaneously Wx-mqThe low amylose content rice variety of gene pure;

(3) on this basis, utilize microsatellite marker JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358:

Mark JS-RM206

Forward primer sequence: 5'-CCCATGCGTTTAACTATTCT-3'

Reverse primer sequence: 5'-CGTTCCATCGATCCGTATGG-3'

Mark JS-RM495

Forward primer sequence: 5'-AATCCAAGGTGCAGAGATGG-3'

Reverse primer sequence: 5'-CAACGATGACGAACACAACC-3'

Mark JS-RM505

Forward primer sequence: 5'-AGAGTTATGAGCCGGGTGTG-3'

Reverse primer sequence: 5'-GATTTGGCGATCTTAGCAGC-3'

Mark JS-RM223

Forward primer sequence: 5'-GAGTGAGCTTGGGCTGAAAC-3'

Reverse primer sequence: 5'-GAAGGCAAGTCTTGGCACTG-3'

Mark JS-RM1358

Forward primer sequence: 5'-GATCGATGCAGCAGCATATG-3'

Reverse primer sequence: 5'-ACGTGTGGCTGCTTTTGC-3'

Continuation is carried out pcr amplification to above-mentioned low amylose content rice variety DNA with 439 bp and 292 bp feature bands, screening has respectively the kind of 173 bp, 149 bp, 181 bp, 151 bp and 170 bp feature bands at JS-RM206, JS-RM495, JS-RM505, JS-RM223 and JS-RM1358 marker site, be " southern round-grained rice 46 ".

2. molecule marking method according to claim 1, is characterized in that, being extracted as of described endosperm of single rice grains genomic dna:

By the 1.5 mL centrifuge tubes of packing into after the Single rice grain grinding of removal rice husk, kind skin, pericarp, embryo, add 800 μ L washingss, in shaking table 120 rpm vibration 5 min, centrifugal 2 min of 4,000 rpm, abandon supernatant liquor; In precipitation, add 600 μ L Extraction buffers, be placed in 65 ° of C water-bath 45 min; Add phenol-chloroform of 600 μ L volume ratio 25:24:1-primary isoamyl alcohol mixed solution, in shaking table 120 rpm vibration 10 min, centrifugal 5 min of 12,000 rpm, draw 500 μ L supernatant liquors to another centrifuge tube; Adding 500 μ L volume ratios is the chloroform-primary isoamyl alcohol mixed solution of 24:1,120 rpm vibration 10 min on shaking table, and centrifugal 5 min of 12,000 rpm, draw 400 μ L supernatant liquors to another centrifuge tube; Add 5 mol/L liquor kalii aceticis and the 293 μ L Virahols of 40 μ L pH=5.2 to mix, 4 ° of C place 2 h, and centrifugal 10 min of 12,000 rpm, abandon supernatant liquor; In precipitation, add 1 mL volume ratio 70% ethanol in shaking table 120 rpm vibration 2 min, centrifugal 3 min of 7,000 rpm, abandon supernatant liquor drying precipitated, with 100 μ L deionized water dissolvings, obtains the endosperm of single rice grains DNA solution;

Described washings is: the Tris-HCl of 100 mmol/L pH=8.0,50 mmol/L EDTA, 0.35 mol/L sorbyl alcohol, mass ratio 1% polyvinylpyrrolidone;

Described Extraction buffer is: mass ratio 2% CTAB, 100 mmol/L Tris-HCl, 25 mmol/L EDTA, 15 mol/L sodium-chlor, mass ratio 1% polyvinylpyrrolidone, 0.1 mg/mL Proteinase K, mass ratio 0.5% Nonidet P40, mass ratio 0.5% polysorbas20.

3. molecule marking method according to claim 1 and 2, is characterized in that, described pcr amplification reaction and gel electrophoresis method are:

1) low amylose content gene specific mark JS- Wx-mqThe pcr amplification system be 20 μ L, the DNA 2.0 μ L of 10 ng/ μ L wherein, the primer 2 .0 μ L of 4 pmol/ μ L, four each 0.5 μ L of primer, contain 25 mmol/L MgCl ₂10 * PCR Buffer, 2.0 μ L, the dNTP 0.4 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 13.1 μ L; PCR response procedures: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s, 65 ℃ of renaturation 30 s, 72 ℃ of extension 1 min, circulate 35 times; Then 72 ℃ are extended 7 min, after 10 ℃ of cooling 10 min, amplified production are added to loading damping fluid termination reaction; Amplified production is electrophoresis 30 min on 1.5% sepharose than concentration in quality, under gel imaging system, observes after ethidium bromide staining;

2) the pcr amplification system of microsatellite marker is 10 uL, the DNA 1.0 μ L of 10 ng/ μ L wherein, and the primer 0.5 μ L of 4 pmol/ μ L, wherein each 0.25 μ L of positive and negative primer, contain 25 mmol/L MgCl ₂10 * PCR Buffer, 1.0 μ L, the dNTP 0.25 μ L of 2.5 mmol/L, 5 U/ μ L's Taq0.5 μ L and ddH ₂O 6.75 μ L; The PCR response procedures comprises: 95 ℃ of denaturation 5 min; 95 ℃ of sex change 30 s, 55 ℃ of renaturation 30 s, 72 ℃ of extension 30 s, circulate 35 times; Then 72 ℃ are extended 7 min, after 10 ℃ of cooling 10 min, amplified production are added to loading damping fluid termination reaction; Amplified production is electrophoresis on 9% polyacrylamide gel than concentration in quality, and 180V electrophoresis 2 h, silver are observing after dying colour developing under visible lamp box.

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