CN102808029B - Primer and method for identifying low phytic acid rice mutant zju-lpa2 filial generation genetypes - Google Patents
Primer and method for identifying low phytic acid rice mutant zju-lpa2 filial generation genetypes Download PDFInfo
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Abstract
The invention discloses a primer and a method for identifying low phytic acid rice mutant zju-lpa2 filial generation genetypes, wherein the primer is a primer ZHJ1, a primer ZHJ2 or a combination of the ZHJ1 with the primer ZHJ2. The method includes: (1) extracting total DNA (deoxyribonucleic acid) of leaves of rice samples at the seedling stage; (2) with the total DNA serving as a template, performing PCR (polymerase chain reaction) amplification by the aid of the primer in patent claim 1, subjecting amplified products to gel electrophoretic separation, and imaging after dyeing; and (3) judging the genetypes of the rice samples according to electrophoresis results. The primer co-separated from low phytic acid rice mutant zju-lpa2 can detect the leaves of the rice samples at the seedling stage, so that selection efficiency of LPA (low phytic acid) rice varieties can be greatly improved, and LPA rice breeding process is accelerated.
Description
Technical field
The invention belongs to the agro-biological engineering technical field, relate in particular to a kind of evaluation paddy rice low phytic acid mutant genotypic primer of zju-lpa2 filial generation and method.
Background technology
Phytic acid is the main storage form of phosphorus in the plant seed, accounts for about the 65-85% of the full phosphorus amount of seed.Common and the Zn of phytic acid
2+, Ca
2+, Fe
3+Become phytate Deng the metallic cation chelating, and further form spherical complex body with albumen, be deposited in the aleurone layer of gramineous crop seed.Owing to do not contain phytase in people and non-ruminant animal (pig, chicken, the fish etc.) body, be difficult to the phytic acid that digestibility and utilization is taken in, greatly reduced the biological effectiveness of phosphoric, phytic acid also influences the biological effectiveness of trace element, albumen and starch simultaneously, is a kind of typical antinutritional factor.In addition, the phytic acid follower defecate that is difficult to be utilized causes body eutrophication in environment on the one hand, causes serious environmental to pollute; In order to satisfy the needs of animal growth, also need extra organophosphorus or the bone meal etc. of adding in the feed on the other hand, improved feeding cost.Many scholars propose available phytase degraded phytic acid and separate out available phosphorus for animal use; Also can change phytase gene over to plant, make phytase in seed, obtain expressing, thereby improve the utilization ratio of phytate phosphorus in the feed.
Therefore, the content of phytic acid becomes the focus that breeding scholar, nutritionist and environmentalist pay close attention to day by day in the reduction crop kernel.Use physics and chemistry mutagenesis and genetic engineering technique, low phytic acid (LPA) crop that phytic acid content descends in the initiative seed can address the above problem from the source.
So far, from crops such as paddy rice, soybean, corn, barley, wheat, obtain a collection of LPA mutant both at home and abroad, cultivated some LPA crop new variety.A large amount of animal vivo tests show that the LPA crop has greatly been improved the biological effectiveness of phosphorus, have reduced the phosphorus in the environment and have polluted, and have effectively improved micronutrient element deficiency disease in the animal body.But the main path that obtains LPA mutantion line at present is physics and chemistry mutagenesis and screening naturally, and can only could detect individual seed content of inorganic phosphorus by the microdetermination method behind the harvesting crops mature seed, preliminary screening high inorganic phosphorus strain, further measure phytate phosphorus and content of tatal phosphorus by methods such as iron precipitations according to material phenotype and experiment condition then, the seed selection cycle is longer, the cost height, and efficient is low.
Fast development along with the modern molecular biology theory and technology, utilize forward (map based cloning) or reverse genetics (RNAi, T-DNA inserts sudden change) technology, reported and cloned some LPA gene (MIPS, MIK, MRP, 2-PGK, IPK1), not only disclosed the molecular mechanism that the LPA sudden change takes place, also for to establish theoretical basis based on the primer of LPA genes involved nucleotide diversity aspect the seed selection of LPA crop new variety.As document (Kim, S.I., Andaya, C.B., Newman, J.W., Goyal, S.S., and Tai, T.H.2008.Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK.Theoretical and Applied Genetics 117 (8): 1291-1301.) among the disclosed rice material N15-186 phytic acid content to descend then be (the Shi that the equipotential sudden change by the MIK gene causes, J.R., Wang, H.Y., Hazebroek, J., Ertl, D.S., and Harp, T.2005.The maize low-phytic acid 3encodes a myo-inositol kinase that plays a role in phytic acid biosynthesis in developing seeds.The Plant Journal 42 (5): 708-719
), this provides a new direction for utilizing Protocols in Molecular Biology seed selection LPA new rice variety.
Paddy rice is that China reaches topmost food crop in the world.As the big country of rice production and consumption, China also is that one of more serious country takes place trace element deficiencies such as sideropenia and hypoferric anemia, can significantly improve Fe and reduce the paddy rice phytic acid content
3+, Zn
2+Biological effectiveness Deng trace element.Therefore, if can obtain with goal gene be divided into from molecule marker, effective Rapid identification contains the low phytic acid rice material of goal gene, the LPA paddy rice that cultivation has nutrition and environmental protection dual-use function just has important economy and social effect in China.
Summary of the invention
The invention provides a kind of primer, utilize this primer to identify the genotype of paddy rice low phytic acid mutant zju-lpa2 filial generation in seedling stage easily.
A kind of primer is primer ZHJ1, primer ZHJ2 or their combination; Wherein, the base sequence of primer ZHJ1 is:
Forward primer sequence: 5 '-TCCTTTAGTCTAGGACCGTTGG-3 ';
Reverse primer sequence: 5 '-AGCCGCTTCTTGGAGTGAT-3 '
With 5 '-ACCAGGGATGCCGTTGAG-3 ';
The base sequence of primer ZHJ2 is:
Forward primer sequence: 5 '-AGCCGCTTCTTGGAGTGAT-3 '
With 5 '-ACCAGGGATGCCGTTGAG-3 ';
Reverse primer sequence: 5 '-AGCCGCTTCTTGGAGTGAT-3 '.
Mutant zju-lpa2 low phytic acid proterties is controlled by MIK equipotential mutator gene ZJU-LPA2, and it has inserted the retrotransposon of a 6.2kb at the intron of sequence LOC_Os03g52760.Accordingly, (http://rice.plantbiology.msu.edu/) downloads corresponding nucleotide sequence in TIGR V7.0 website, and sequence compared, design corresponding primer ZHJ1 and ZHJ2 near utilizing the 6.2kb insertion segment area of Primer Premier 5.0 softwares in the low phytic acid gene ZJU-LPA2 of paddy rice, forward primer of this primer and reverse primer are the conservative region designs according to gene M IK, and another forward primer or reverse primer are according to the retrotransposon sequences Design of inserting among the gene ZJU-LPA2, so primer has good versatility and specificity.
The present invention also provides a kind of evaluation paddy rice low phytic acid mutant zju-lpa2 filial generation genotypic method, comprising:
(1) extracts the total DNA of paddy rice sample seedling leaf;
(2) be template with described total DNA, utilize the described primer of claim 1 to carry out pcr amplification, amplified production separates through gel electrophoresis, the imaging of taking pictures after the dyeing;
(3) according to imaging results, judge the genotype of paddy rice sample;
When utilizing primer ZHJ1 to carry out pcr amplification, if the 350bp characteristic strip only occurs, then this paddy rice sample is the homozygous material that contains low phytic acid gene ZJU-LPA2, if the 498bp characteristic strip only occurs, then this paddy rice sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2, if occur 350bp and 498bp characteristic strip simultaneously, then this paddy rice sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2;
When utilizing primer ZHJ2 to carry out pcr amplification, if the 754bp characteristic strip only occurs, then this paddy rice sample is the homozygous material that contains low phytic acid gene ZJU-LPA2, if the 498bp characteristic strip only occurs, then this paddy rice sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2, if occur 754bp and 498bp characteristic strip simultaneously, then this paddy rice sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2;
When utilizing primer ZHJ1 and primer ZHJ2 to carry out pcr amplification, if 350bp and 754bp characteristic strip only occur, then this paddy rice sample is the homozygous material that contains low phytic acid gene ZJU-LPA2, if the 498bp characteristic strip only occurs, then this paddy rice sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2, if occur 350bp, 754bp and 498bp characteristic strip simultaneously, then this paddy rice sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2;
The base sequence of described low phytic acid gene ZJU-LPA2 is shown in SEQ ID NO.1.
The preparation of described paddy rice low phytic acid mutant zju-lpa2 is at document (Liu, Q.L., Xu, X.H., Ren, X.L., Fu, H.W., Wu, D.X., and Shu, Q.Y. 2007.Generation and characterization of low phytic acid germplasm in rice (Oryza sativa L.) .Theoretical and Applied Genetics 114 (5): open 803-814-), the MIK transgenation that it refers to control the paddy rice phytic acid content is the LPA mutant that the ZJU-LPA2 gene produces; Described rice mutant zju-lpa2 filial generation refers to that the parent is the filial generation of the rice varieties that do not suddenly change of rice mutant zju-lpa2 and MIK gene.Compare LPA mutator gene ZJU-LPA2, the rice varieties that described MIK gene does not suddenly change refers to the rice varieties of normal phytic acid content, and is fine as Jiahe 218, Japan, 9311 etc.
Plant total DNA extraction method commonly used is the CTAB method.
The secondary metabolite of plant has interference effect to nucleic acid extraction.Therefore, the general children of choosing as far as possible cambium tender, that metabolism is vigorous is as the material that extracts DNA, and young tender cambium secondary metabolite is less, the dna content height, and be easy to fragmentation.Vegetable material is preferably fresh.
The reaction system of described pcr amplification is 20 μ L, and wherein, 2 * PCR Master Mix damping fluid, 10 μ L (contain 1.5mM Mg
2+, 200 μ M dNTP, 1U Taq enzyme), each 0.4 μ L of the forward and reverse primer of 10 μ M, total DNA 1 μ L, sterilization deionized water mend to 20 μ L.
The response procedures of described pcr amplification is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s, 57 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 7min.
Compared with prior art, beneficial effect of the present invention is:
1, the primer that obtains of the present invention is synthetic in the difference design that nucleotides sequence lists according to zju-lpa2 mutant and normal water rice varieties, does not therefore have the exchange in the heredity, does not also need the further checking of phenotype.
2, utilize two cover primers that the present invention obtains to screen a large amount of zju-lpa2 filial generation colony, thereby precise and high efficiency identify the LPA rice material that contains the low phytic acid gene ZJU-LPA2 of paddy rice.
3, usually the evaluation of LPA rice material must wait until that the rice paddy seed maturation just can carry out later on, and utilize and paddy rice hang down phytic acid gene ZJU-LPA2 be divided into from primer can its blade be detected in seedling stage, can improve the efficiency of selection of LPA rice varieties greatly, accelerate LPA rice breeding process.
Description of drawings
Fig. 1 is for being used for the PCR design of primers strategy that low phytic acid mutant gene ZJU-LPA2 detects, wherein black box represents gene coding region, white box represents UTR, dotted line represents intron, the dna fragmentation that double-headed arrow represents 6.2kb among the low phytic acid mutant zju-lpa2 inserts, and single arrow represents primer location.
Fig. 2 is that the ZHJ1 primer is to zju-lpa2 * Jiahe 218F
2Selection effect for individual plant.Wherein M is dna molecular amount standard; 1 swimming lane is Jiahe 218; 2 swimming lanes are zju-lpa2; The 3-7 swimming lane is not for containing the normal phytic acid content individual plant of ZJU-LPA2 gene pure type; The 8-12 swimming lane is the normal phytic acid content individual plant of ZJU-LPA2 gene hybridizing type; The 13-17 swimming lane is the low phytic acid individual plant of ZJU-LPA2 gene pure type.
Fig. 3 is that the ZHJ2 primer is to zju-lpa2 * Jiahe 218F
2Selection effect for individual plant.Wherein M is dna molecular amount standard; 1 swimming lane is Jiahe 218; 2 swimming lanes are zju-lpa2; The 3-7 swimming lane is not for containing the normal phytic acid content individual plant of ZJU-LPA2 gene pure type; The 8-12 swimming lane is the normal phytic acid content individual plant of ZJU-LPA2 gene hybridizing type; The 13-17 swimming lane is the low phytic acid individual plant of ZJU-LPA2 gene pure type.
Fig. 4 is the pcr amplification result of ZHJ2 primer in different rice varieties materials.MDNA molecular weight standard wherein; 1 swimming lane is Jiahe 218; 2 swimming lanes are zju-lpa2; 3 is that Japan is fine; 4 is 9311; 5 is elegant water 110; 6 is bright extensive 86; 7 is middle 9B.
Embodiment
One, design of primers
Paddy rice low phytic acid mutant zju-lpa2 by
137The Cs-gamma-ray and mutagenesis is handled japonica rice variety XS110 and is obtained, phytic acid content descends 64%, content of inorganic phosphorus raises 5.6 times, genetic analysis and the assignment of genes gene mapping show, paddy rice low phytic acid mutant zju-lpa2 phytic acid content is controlled by single recessive nuclear gene, is positioned at (Liu, Q.L. on No. 3 chromosomal galianconism of paddy rice, Xu, X.H., Ren, X.L., Fu, H.W., Wu, D.X., and Shu, Q.Y. 2007.Generation and characterization of low phytic acid germplasm in rice (Oryza sativa L.) .Theoretical and Applied Genetics 114 (5): 803-814.).Further result of study shows that zju-lpa2 low phytic acid proterties is controlled by MIK equipotential mutator gene ZJU-LPA2, inserted the retrotransposon of a 6.2kb at intron, download corresponding nucleotide sequence at TIGRV7.0 (http://rice.plantbiology.msu.edu/), and sequence is compared.Design corresponding primer near utilizing the 6.2kb insertion segment area of Primer Premier 5.0 (www.premierbiosoft.com) software in the low phytic acid gene ZJU-LPA2 of paddy rice, and the primer called after ZHJ1 that will newly synthesize and ZHJ2 (Fig. 1), the sequence of two primers sees Table 1.
The exploitation of table 1.ZJU-LPA2 gene primer
Forward primer of this primer and reverse primer are the conservative region designs according to gene M IK, and another forward primer or reverse primer are according to the retrotransposon sequences Design of inserting among the gene ZJU-LPA2, so primer has good versatility and specificity.When adopting different rice varieties to carry out pcr amplification, as seen all can detect specific target stripe (Fig. 4).
Two, DNA extraction and PCR detect
Utilize above-mentioned primer to identify that concrete grammar is to the genotype of paddy rice low phytic acid mutant zju-lpa2 filial generation:
(1) extraction of total DNA
Adopt the CTAB method to extract total DNA of paddy rice sample tender leaf in seedling stage, concrete operation method is as follows: blade is shredded be placed in the 2.0mL centrifuge tube, add 800 μ L CTAB and extract damping fluid (100mM Tris-HCl pH 8.0,20mM EDTApH 8.0,500mM NaCl, 2%CTAB), with organizing the instrument of milling to grind, 65 ℃ of water-bath 40min, shake 3-4 time during this time, add isopyknic chloroform: primary isoamyl alcohol (24: 1, v/v) mixed solution, mixing turns upside down, the centrifugal 10min of 10000rpm/min shifts supernatant to new 1.5mL centrifuge tube, adds the Virahol of equal-volume precooling (20 ℃), put upside down mixing gently, put-20 ℃ of precipitation 30min down; The centrifugal 10min of 10000rpm/min abandons supernatant liquor, 70% washing with alcohol 2 times, and natural air drying is dissolved in an amount of (100-200 μ L) TE solution-20 ℃ of preservations.
(2) pcr amplification of DNA sample detects
Reaction system is 20 μ L, and wherein 2 * PCR Master Mix damping fluid, 10 μ L (contain 1.5mMMg
2+, 200 μ M dNTP, 1U Taq enzyme), each 0.4 μ L of the forward and reverse primer of 10 μ M, paddy rice sample total DNA (50ng/ μ L) 1 μ L, add the sterilization deionized water and mend to 20 μ L.
The PCR response procedures is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s, 57 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 7min.
Amplified production is electrophoresis on 1% sepharose, imaging behind ethidium bromide staining.
(3) judge whether to contain target gene
When adopting a pair of primer amplification respectively, 350 or the 754bp characteristic strip if can only expand, then this sample is the homozygous material that contains low phytic acid gene ZJU-LPA2; The 498bp characteristic strip if can only expand, then this sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2; If can expand simultaneously and 498bp, 350 or the 754bp characteristic strip, then this sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2.
When adopting two pairs of primer amplifications simultaneously, 350 and the 754bp characteristic strip if can only expand, then this sample is the homozygous material that contains low phytic acid gene ZJU-LPA2; The 498bp characteristic strip if can only expand, then this sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2; If can expand simultaneously and 498bp, 350 and the 754bp characteristic strip, then this sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2.
Three, the selection effect of the genotype ZJU-LPA2 filial generation of primer ZHJ1
Utilize design synthetic primer ZHJ1, to containing ZJU-LPA2 gene pure section bar material zju-lpa2 and not containing the F in ZJU-LPA2 gene pure section bar material Jiahe 218
2Filial generation carries out the pcr gene type in seedling stage and detects, and detected result is divided into 3 kinds of genotype with material as shown in Figure 2: have the material of 350bp characteristic strip, have the material of 498bp characteristic strip and the material that both have concurrently.
Results F
2F on the plant
2:3Seed, utilize chemical analysis and ion exchange chromatography to detect the phytic acid content of each individual plant, the result shows that all individual plants with 350bp characteristic strip all are low phytic acid individual plants, is the normal individual plant of phytic acid content and have the 498bp characteristic strip with the two material that has concurrently.
Four, the selection effect of the filial generation of the genotype ZJU-LPA2 of primer ZHJ2
Utilize design synthetic primer ZHJ2, above-mentioned same filial generation colony is carried out the pcr gene type in seedling stage to be detected, detected result also is divided into material 3 kinds of genotype as shown in Figure 3: have the material of 754bp characteristic strip, have the material of 498bp characteristic strip and the material that both have concurrently.In conjunction with the further analysis revealed of phytic acid phenotype detected result, all individual plants with 754bp characteristic strip all are low phytic acid individual plants, are the normal individual plant of phytic acid content and have the 498bp characteristic strip with the two material that has concurrently,
Therefore, primer ZHJ1 and ZHJ2 are the codominant marker, conform to fully with expected results, and the mark efficiency of selection is 100%.
Claims (5)
1. a primer is characterized in that, is primer ZHJ1, primer ZHJ2 or their combination; Wherein, the base sequence of primer ZHJ1 is:
Forward primer sequence: 5 '-TCCTTTAGTCTAGGACCGTTGG-3 ';
Reverse primer sequence: 5 '-AGCCGCTTCTTGGAGTGAT-3 '
With 5 '-ACCAGGGATGCCGTTGAG-3 ';
The base sequence of primer ZHJ2 is:
Forward primer sequence: 5 '-TCCTTTAGTCTAGGACCGTTGG-3 '
With 5 '-TTCGACCTCTCTACCTTGGAAG-3 ';
Reverse primer sequence: 5 '-AGCCGCTTCTTGGAGTGAT-3 '.
2. identify the genotypic method of paddy rice low phytic acid mutant zju-lpa2 filial generation for one kind, comprising:
(1) extracts the total DNA of paddy rice sample seedling leaf;
(2) be template with described total DNA, utilize the described primer of claim 1 to carry out pcr amplification, amplified production separates through gel electrophoresis, the imaging of taking pictures after the dyeing;
(3) according to imaging results, judge the genotype of paddy rice sample;
When utilizing primer ZHJ1 to carry out pcr amplification, if the 350bp characteristic strip only occurs, then this paddy rice sample is the homozygous material that contains low phytic acid gene ZJU-LPA2, if the 498bp characteristic strip only occurs, then this paddy rice sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2, if occur 350bp and 498bp characteristic strip simultaneously, then this paddy rice sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2;
When utilizing primer ZHJ2 to carry out pcr amplification, if the 754bp characteristic strip only occurs, then this paddy rice sample is the homozygous material that contains low phytic acid gene ZJU-LPA2, if the 498bp characteristic strip only occurs, then this paddy rice sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2, if occur 754bp and 498bp characteristic strip simultaneously, then this paddy rice sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2;
When utilizing primer ZHJ1 and primer ZHJ2 to carry out pcr amplification, if 350bp and 754bp characteristic strip only occur, then this paddy rice sample is the homozygous material that contains low phytic acid gene ZJU-LPA2, if the 498bp characteristic strip only occurs, then this paddy rice sample is not for containing the homozygous material of low phytic acid gene ZJU-LPA2, if occur 350bp, 754bp and 498bp characteristic strip simultaneously, then this paddy rice sample is the heterozygous material that contains low phytic acid gene ZJU-LPA2;
The base sequence of described low phytic acid gene ZJU-LPA2 is shown in SEQ ID NO.1.
3. method according to claim 2 is characterized in that, adopts the CTAB method to extract total DNA.
4. method according to claim 2 is characterized in that, the reaction system of described pcr amplification is 20 μ L, and wherein, 2 * PCR Master Mix damping fluid, 10 μ L, each 0.4 μ L of the forward and reverse primer of 10 μ M, total DNA1 μ L, sterilized water complement to 20 μ L.
5. method according to claim 2 is characterized in that, the response procedures of described pcr amplification is: 94 ℃ of pre-sex change 4min; 94 ℃ of sex change 30s, 57 ℃ of annealing 45s, 72 ℃ are extended 1min, 35 circulations; Last 72 ℃ are extended 7min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005113779A2 (en) * | 2004-05-20 | 2005-12-01 | Pioneer Hi-Bred International, Inc. | Plant myo-inositol kinase polynucleotides and methods of use |
| CN102154495A (en) * | 2011-03-14 | 2011-08-17 | 浙江省农业科学院 | Molecular marking method and primer for identifying mutator gene IPK1-A of low phytic acid content in soybean seeds |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005113779A2 (en) * | 2004-05-20 | 2005-12-01 | Pioneer Hi-Bred International, Inc. | Plant myo-inositol kinase polynucleotides and methods of use |
| CN102154495A (en) * | 2011-03-14 | 2011-08-17 | 浙江省农业科学院 | Molecular marking method and primer for identifying mutator gene IPK1-A of low phytic acid content in soybean seeds |
Non-Patent Citations (4)
| Title |
|---|
| Generation and characterization of low phytic acid germplasm;Qing-Long Liu;《Theor Appl Genet》;20070112;803-814 * |
| Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK;S. I. Kim;《Theor Appl Genet》;20080826;1291-1301 * |
| Qing-Long Liu.Generation and characterization of low phytic acid germplasm.《Theor Appl Genet》.2007,803-814. |
| S. I. Kim.Isolation and characterization of a low phytic acid rice mutant reveals a mutation in the rice orthologue of maize MIK.《Theor Appl Genet》.2008,1291–1301. |
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