CN102912010A - Method for quickly and accurately detecting purity of hybrid rice seed - Google Patents

Abstract

The invention discloses a method for quickly and accurately detecting the purity of a hybrid rice seed, which comprises the following steps: according to seed purity requirements, calculating the quantity of samples; sampling in a quantity-equal manner and mixing extracted DNA (deoxyribonucleic acid), or respectively extracting DNA and then mixing in a quantity-equal manner; designing PCR (polymerase chain reaction) primers according to the base sequence of detected sites, and amplifying the detected sites; recovering and purifying the PCR amplification products; building a library by using the amplification products, and performing high-flux sequencing; and calculating the seed purity according to the sequence proportion of the detected sites. The invention has the obvious characteristic that the requirements for detection speed and accuracy in the purity detection of a commodity hybrid seed are well satisfied.

Description

A kind of method that detects fast and accurately hybrid rice seeds purity

Technical field

The invention belongs to the genetics field, disclose a kind of method that detects fast and accurately hybrid rice seeds purity.

Background technology

Seed purity is one of sixty-four dollar question in the agriculture production, and it is related to national food safety, is most important, most crucial content in the Seed Industrialization exploitation, thereby also concerns Seed enterprises final and decisive juncture.Most of crop seeds purity requirement is all more than 95%, even be 99.9%(table 1), thereby a large amount of statistical samplings of needs could obtain accurate data, workload is very huge.For example qualified Hybrid Rice Varieties purity requirement reaches 96%, means that then 100 plant at most only allow 4 hybrid strains, will make so detected result reliable, should detect at least 500 seeds, and workload is huge.In addition, because technological deficiency, also there are the problems such as affected by environment, that speed is slow, expense is high in various seed purity detection methods, have had influence on the major issues such as accuracy, the seed identified in time go on the market, cost.

Table 1 crop seeds purity rubric (GB4404.1-2008)

Seed purity detects the development of having experienced from the field phenotypic evaluation to the laboratory molecular markers for identification.The operating process of field phenotypic evaluation is such: randomly draw a quantity of seeds from the seed of producing, field planting is also observed proterties, compares by the true proterties with kind, identifies hybrid strain.For example, in the evaluation colony that is made of 100 plant, find that 2 strain plant heights and 3 strain colors obviously are different from the plant of this variety authentication shape, then the purity of this kind is (2+3)/100=95%.The defective of field test is obvious: the one, need to plant a season, and the cycle is long, occupation of land is many, expense is high, and seed only has 2-3 month from producing to the time of selling, the field test cycle has had a strong impact on seed and has sold listing.The 2nd, tree characteristics is often affected by environment, and as under high soil fertility condition, plant is taller and bigger, thereby is mistaken for hybrid strain, makes qualification result inaccurate.The molecular markers for identification seed purity has overcome above problem to a great extent, and its theoretical foundation can simply be described as: utilize the different of specific molecular between hybrid strain and kind, identify hybrid strain, calculate seed purity.Different according to the type of molecule marker, can be divided into protein (such as isozyme etc.) and dna molecular marker evaluation.Protein molecular marker is identified also obvious defective, and the one, protein expression is affected by environment equally, makes qualification result inaccurate; The 2nd, the protein molecular mark that can be used for identifying is few, this technology is difficult to use in distinguishes the nearer kind of sibship.Dna molecular marker is to identify that according to hybrid strain and the normal sequence difference of kind on dna level this technology has overcome the deficiency of above several authentication methods to a great extent.At first, dna sequence dna difference is no longer dependent on environment, and therefore, qualification result is accurately; The second, theoretically, any hybrid strain all there are differences with normal kind DNA, therefore, can be used for identifying any kind; The 3rd, owing to seed, seedling, the DNA of strain phase are the same, therefore, identifying no longer needs the field seed, only needs indoor simple germination to extract DNA and gets final product, and has shortened sense cycle, for the time has been won in the seed popularization.Yet, whether the seed that enterprise may need to detect a plurality of production sites or the producer is qualified, the testing amount is huge, even utilize dna molecular marker to detect, usually also can can't in time finish detection owing to workload is large, simultaneously, huge workload also is accompanied by the too high problem of testing cost.For example, the state's laws regulation, the purity of hybrid rice seeds must not be lower than 96%, therefore, if require under 95% probability guarantees, the seed purity error is no more than 1%, and then each sample need to be inspected 3000 seeds by random samples and just can reach requirement, if detect 100 samples, then need to extract DNA, PCR, electrophoresis is respectively 30,00*,100,=30 ten thousand times.In the limited situation of testing staff and equipment, almost be impossible finishing testing at seed packaging and before sales, have a strong impact on listing.In the case, can only reduce amount of sampling, to reach the purpose that reduces workload.For example, 100 seeds of general enterprises sampling observation in the case, if still require purity error only 1%, only are about 15% to result's power of a test (probability assurance) so.So, even obtained detected result, do not hold and judge whether the seed of this batch is qualified, brings great risk to market sale.

Summary of the invention

The purpose of the embodiment of the invention is the defective for above-mentioned prior art, provide a kind of can be accurately, the method for rapid detection hybrid seed purity.

The technical scheme taked of the present invention is to achieve these goals:

A kind of method that detects fast and accurately hybrid rice seeds purity may further comprise the steps: according to the seed purity requirement, calculate the sampling sample size; Equivalent sampling and mixed extraction DNA or extract respectively DNA after balanced mix again; By the base sequence of detection site, design PCR primer amplification detection site; Reclaim and the purifying pcr amplification product; Utilize amplified production to build the storehouse and carry out high-flux sequence; According to detection site sequence ratio, calculate seed purity.

The invention provides preferred technical scheme is: a kind of method that detects fast and accurately hybrid rice seeds purity may further comprise the steps:

(1) calculates minimum sampling sample size: according to the probability guarantee of commodity seed purity requirement, setting and the error of permission, determine minimum sample amount of sampling;

(2) DNA extraction: to each plant, extract DNA after choosing the equivalent material mixing of same area; Perhaps, extract the DNA of each plant, balanced mix again after again DNA quantitatively being extracted;

(3) amplification and recovery detection site: by the base sequence of detection site, the design primer carries out pcr amplification to detection site, and amplified production should comprise the diversity sequence of loci, and the PCR product is reclaimed purifying;

(4) build storehouse and high pass and measure order-checking: make up respectively parental gene DNA library, ePCR and high-flux sequence by the high-flux sequence flow process;

(5) determining of seed purity: according to the result of order-checking, obtain the segments of male parent and maternal sequence, calculate thus seed purity.

The beneficial effect of the embodiment of the invention is:

(1) reduces workload, accelerated detection speed.Adopt Markers for Detection, if 3000 plant of 1 sample sampling, then DNA extraction, pcr amplification and electrophoresis detection all need to do 3000 times, so workload is large, and speed is slow, usually can't gather in the crops packing at seed and go on the market and shortly in two or three months finish.Adopt this programme, how many plant no matter each sample is sampled all become 1 sample with these plant balanced mix, DNA extraction, found a capital storehouse and order-checking the time are only done 1 time, workload greatly reduces, and speed is greatly accelerated simultaneously, has satisfied well the requirement of the timely list marketing of seed.

(2) improved accuracy rate.As mentioned above, Markers for Detection usually can only reduce the plant number of sampling in each sample because workload is large, reaches the purpose of quickening progress, and this has just caused the inaccurate situation of detected result.Adopt this programme, existing high-flux sequence flux all more than 100G, even the gene order that detects reaches 1K, also can detect above 100,000,000 sequences, and per 2 sequences can represent 1 plant, then can detect to surpass 5,000 ten thousand plant.Large size of a sample like this, we almost can extract many arbitrarily plant and detect in each sample, guaranteed well the accuracy of detected result.

(3) kept the advantage of DNA detection.The technical program due to dna molecular marker detects category.Therefore, detect relatively with Fields detection and protein molecular marker, they have not affected by environment, are not subject to seasonal restrictions, not the advantage such as land occupation.

Outstanding feature of the present invention is: satisfied well the commodity hybrid seed purity detect in to the requirement of detection speed and accuracy.

Description of drawings

Fig. 1 is that hybrid rice variety " the section excellent 73 " seed purity that the embodiment of the invention provides detects synoptic diagram.

Embodiment

The invention will be further described below in conjunction with the drawings and specific embodiments, but not as a limitation of the invention.

Embodiment 1

Hybrid rice variety " section excellent 73 " seed purity detection method (referring to Fig. 1):

(1) " section excellent 73 " cenospecies sampling

" section excellent 73 " is a kind of new hybrid rice varieties of seed selection, and applied for that new variety protection, its male parent are R7723, and female parent is the golden 1A of section." section excellent 73 " seed purity sampling analysis to 127 parts batches.Utilize DPS(Data Processing System: data handling system.) " binominal distribution population mean fiducial interval " function in 2000 softwares, calculate the sampling sample and need to reach the error that 3000 samples could satisfy detection and be no more than 1% requirement.Therefore utilize the seed sampling device, the principle by random sampling from the seed of each batch, approximately extracts 4500 seeds.

(2) seed germination, sample mix and DNA extraction

5 layers of newspaper are laid in the porcelain dish of 40cm * 80cm, and it is fully moistening but till not revealing open fire to newspaper to add a small amount of moisture.4500 seed uniform broadcastings in porcelain dish, are covered to keep moisture with another porcelain dish with it.Naturally germinate in indoor (about about 30 ℃ of temperature), during check and suitably replenish moisture content.When seed germination grew to 7-10 days, existing 1 blade grew, and choose 3000 seedlings this moment, and every seedling is got 1 blade, and blade is stacked neatly, and the unified amount that cuts is about equally mixed.Press plant genome DNA and extract test kit (DP305, day more, Beijing) operational manual separation and purification genomic dna, utilize ND2000 ultramicrospectrophotometer working sample concentration and purity.Obtain altogether 127 parts of hybrid dna samples, represented respectively 127 sampling samples.

(3) pcr amplification and recovery

Male parent R7723 is different at plant height gene HD from the maternal golden 1A of section, and male parent is high bar, contains the dominant gene of this gene, its sequence is the SEQ ID NO.1 in the sequence table, and female parent is short bar, contains the recessive gene of this gene, and its sequence is the SEQ ID NO.2 in the sequence table.Wherein, dominant and recessive site is single base mutation, namely in the 203rd base site, is T among the R7723, and is G among the golden 1A of section.Utilize the difference site of Primer5 software design primer amplification HD gene between R7723 and the golden 1A of section, adopt the default parameters of software to carry out during design of primers.Wherein, Sense Primer(forward primer) be the SEQ ID NO.3 in the sequence table.Antisense Primer(reverse primer) be the SEQ ID NO.4 in the sequence table, the PCR product of acquisition is 272bp, for SEQ ID NO.5(and R7723 in the sequence table mate fully) and SEQ ID NO.6(and the golden 1A of section mate fully).The pcr amplification system is pressed test kit DreamTaq ^TMGreen PCR Master Mix (2X) (K1081, Fermentas) recommends ratio proportioning, i.e. PCR mixture 50l, template 15ng, primer 2 l(concentration: 0.5M/l), adding deionized water to cumulative volume is 100l.Amplification program is as follows: 94 ℃ of denaturations 4 minutes; 94 ℃, 1 minute, 54 ℃, 1 minute, 72 ℃, 2 minutes, circulate 20 times; 72 ℃ were extended 8 minutes.Amplified production detects and presses sepharose with the PCR product and reclaim the recovery of test kit (K0691, Fermentas) operational manual and purifying target DNA fragment with 3% sepharose.

(4) build storehouse and high-flux sequence

Because therefore the not enough 400bp length of PCR product, build the storehouse to 127 parts of DNA cloning products that obtain by the amplicon series process in the SOLiD 5500 high-flux sequence operational manuals.Build in the process of storehouse, adopt Barcoding (barcode) that sample is distinguished, adopt altogether 64 barcodes.Take altogether 2 order-checking passages during order-checking, obtain data 22.7G, on average each sample obtains data 0.18G, and the variation amplitude of 127 samples is 0.15G-0.21G.Add the sequences such as top connection, every 500bp can represent 1 and detect segment, and then average each sample has been detected 37.48 ten thousand times, and the variation amplitude of 127 samples is 31.45 ten thousand times-44.04 ten thousand times.So large detection frequency has guaranteed the accuracy of detected result.

(5) calculating of seed purity

By the complete principle of coupling, with among the R7723 with the golden 1A of section in diversity sequence and sequencing result compare acquisition and R7723 and the Jin Ke 1A number of fragments of mating fully respectively in each sample.Represent respectively the number of fragments that R7723 and the golden 1A of section detect by seed purity=2x/ (x+y) * 100%(x and y) calculate the seed purity of each sample.Calculate by above-mentioned formula, the average purity of these 127 kinds is 98.67%, and variation amplitude is 94.72%-99.98%.The purity that has 4 samples is lower than 96%, is defective seed, should transfer commodity grain to and sell, and all the other 123 sample purity can enter normal seed sales section all greater than 96%.

Embodiment 2

The detection method of present embodiment is substantially the same manner as Example 1, and difference is:

The step of DNA extraction is in the step (2):

When seed germination grows to 7-10 days, existing 1 blade grows, choose 3000 seedlings this moment, every seedling is got 1 blade, press plant genome DNA and extract test kit (DP305, day more, Beijing) operational manual separation and purification genomic dna, extract the DNA of each blade, balanced mix again after again DNA quantitatively being extracted; Utilize ND2000 ultramicrospectrophotometer working sample concentration and purity.Obtain altogether 127 parts of hybrid dna samples, represented respectively 127 sampling samples.

Above-described embodiment, the present invention embodiment a kind of more preferably just, the common variation that those skilled in the art carries out in the technical solution of the present invention scope and replacing all should be included in protection scope of the present invention.

Claims (2)

1. a method that detects fast and accurately hybrid rice seeds purity is characterized in that, may further comprise the steps: according to the seed purity requirement, calculate the sampling sample size; Equivalent sampling and mixed extraction DNA or extract respectively DNA after balanced mix again; By the base sequence of detection site, design PCR primer amplification detection site; Reclaim and the purifying pcr amplification product; Utilize amplified production to build the storehouse and carry out high-flux sequence; According to detection site sequence ratio, calculate seed purity.

2. the method that detects fast and accurately hybrid rice seeds purity according to claim 1 is characterized in that, may further comprise the steps:

(1) calculates minimum sampling sample size: according to the probability guarantee of commodity seed purity requirement, setting and the error of permission, determine minimum sample amount of sampling;

(2) DNA extraction: to each plant, extract DNA after choosing the equivalent material mixing of same area; Perhaps, extract the DNA of each plant, balanced mix again after again DNA quantitatively being extracted;

(3) amplification and recovery detection site: by the base sequence of detection site, the design primer carries out pcr amplification to detection site, and amplified production should comprise the diversity sequence of loci, and the PCR product is reclaimed purifying;

(4) build storehouse and high pass and measure order-checking: make up respectively parental gene DNA library, ePCR and high-flux sequence by the high-flux sequence flow process;

(5) determining of seed purity: according to the result of order-checking, obtain the segments of male parent and maternal sequence, calculate thus seed purity.