CN101113468B - Molecule labelling method for soft rice gene locus with low-content of amylose - Google Patents
Molecule labelling method for soft rice gene locus with low-content of amylose Download PDFInfo
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- CN101113468B CN101113468B CN2007100252498A CN200710025249A CN101113468B CN 101113468 B CN101113468 B CN 101113468B CN 2007100252498 A CN2007100252498 A CN 2007100252498A CN 200710025249 A CN200710025249 A CN 200710025249A CN 101113468 B CN101113468 B CN 101113468B
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Abstract
The invention provides a molecular marking method for low amylose content gene locus of Haopi of Yunnan softpanel rice variety, pertaining to molecular genetics field. The DNA of Haopi of Yunnan softpanel rice variety is increased by a marking primer dCAPs-SalI-hp and then restriction enzyme digestion is done with limited endonuclease SalI. If two fragments respectively with a length of 151bp and 145bp can be obtained, the existence of low amylose content gene locus of Haopi of Yunnan softpanel rice variety is proved. The molecular marking for low amylose content gene locus of Haopi of Yunnan softpanel rice variety is adopted to detect whether Haopi and derivative varieties thereof contain the gene locus and the method can predict the amylose content and greatly improve the efficiency of selection of rice with low amylose content.
Description
One, technical field
The invention provides the molecule marking method of Yunnan softpanel rice variety milli wind gene locus with low-content of amylose, belong to the molecular genetics field, be exclusively used in the seed selection of paddy rice low amylose content and the utilization of germ plasm resource.
Two, background technology
In recent years, along with improving constantly of rapid development of national economy and living standards of the people, rice quality has been proposed more and more higher requirement, molecular biology research, the seed selection high-grade rice kind of strengthening the formation of fine quality rice quality are most important.The main component of rice endosperm is a starch, and the rice endosperm starch can be divided into amylose starch and amylopectin, studies show that amylose content and amylose starch chain length are the determinatives of rice food flavor quality, also affects the rice quality simultaneously.Low amylose content rice hardness is little, viscosity big, the food flavor quality better, is the ideal material of improvement rice grain amylose content and food flavor quality.
Yunnan softpanel rice variety milli wind have rice softness, high resilience, glossy, mouthfeel is good, hardness is little, viscosity is big, the characteristics of food flavor quality better, is the ideal material of improveing rice grain amylose content and food flavor quality.
Wind and high amylose content rice varieties Nanjing 11 and the surprising glutinous hybridization of glutinous rice variety in the least, its F
1Amylose content all between parents, and be partial to the high parent of amylose content, F
2And BC
1F
1Amylose content meet the segregation ratio of 3: 1 and 1: 1 respectively, further milli wind is hybridized F with Wx gene mutation body Milky Queen
2The amylose content of seed is unimodal distribution near parents' mean value, show that milli wind low amylose content is that the Wx transgenation causes.
Three, summary of the invention
Technical problem
The objective of the invention is: the molecule marking method that Yunnan softpanel rice variety milli wind gene locus with low-content of amylose is provided, by detecting the molecule marker of this marker site, can predict the amylose content of rice plant, accelerate the selection progress of low amylose content paddy rice.
Technical scheme
The molecule marking method of Yunnan softpanel rice variety milli wind gene locus with low-content of amylose is characterized in that:
Use labeled primer dCAPs-SalI-hp,
Left end primer sequence 5 '-GTGGAGTCGACCGTGTGTTCGTCG-3 '
Right-hand member primer sequence 5 '-CCCCAAACCTGAAATCACCAGTGGA-3 '
Amplification milli wind genomic dna is used the SalI digestion with restriction enzyme then, if can obtain 151bp and two segments of 145bp, then indicates the existence of Yunnan softpanel rice variety gene locus with low-content of amylose.
The molecule marking method of beneficial effect Yunnan softpanel rice variety milli provided by the present invention wind gene locus with low-content of amylose has the following advantages:
(1) can obtain the paddy rice gene locus with low-content of amylose by molecule marker of the present invention;
(2) the gene locus with low-content of amylose locality specific of selecting by molecule marker mark of the present invention, it is convenient to identify.By detecting this molecule marker, promptly can predict the amylose content of rice plant, whether the genotype detection that is used for rice varieties or strain has low amylose content to judge this kind or strain, and then the kind of rapid screening low amylose content or strain are used for rice breeding.This method is easy to detect fast, and is not affected by environment;
(3) the assistant breeding select target is clear and definite, saves cost.In traditional breeding way, at first to collect parent and Cultivar and carry out a series of hybridization, and will carry out individual plant to amylose content and measure with low amylose content.Therefore the low amylose content breeding is extremely time-consuming.By detecting gene locus with low-content of amylose, can just identify the individual plant of low amylose content in seedling stage, eliminate other plant, not only save production cost but also improve the efficiency of selection of low amylose starch paddy rice greatly.
Four, description of drawings
Fig. 1 dCAPs marker detection milli wind, milli wood is thin and milliamperes vexed 1,3,5,7 and 9 be respectively with milli wind, milli skin, milli wood PCR product S alI enzyme thin, that milliampere vexed and Japanese fine genomic dna is a template and cut preceding electrophoretic band, and 2,4,6,8 and 10 are respectively the electrophoretic band after cutting with milli wind, milli skin, milli wood PCR product S alI enzyme thin, that milliampere vexed and Japanese fine genomic dna is a template.
Fig. 2 dCAPs marker detection Hunan, Guangxi, Fujian, Jiangxi, Zhejiang, Heilungkiang and Jilin variety source
Five, embodiment
Genetic analysis and allelism test show that milli wind low amylose content is controlled by Wx allelotrope.According to paddy rice whole genome sequence information design primer, utilize the overlapping PCR method, and the PCR product directly checked order, obtain the genome sequence of the about 7kb of milli wind Wx, The sequencing results is: the 4th exon in the coding region (+497) locates to exist single base mutation (base A sports G), coding sports GGC by GAC, has caused aspartic acid to sport glycine.Single nucleotide mutation site design dCAPs mark according to the 4th exon when utilizing the soft rice of milli wind to carry out low amylose starch rice molecular breeding, can carry out molecular marker assisted selection.
(1) label screening:
(1) Yunnan softpanel rice variety milli wind and the 11 positive and negative hybridization of high amylose starch rice varieties Nanjing, F
1The seed amylose content all is partial to Nanjing 11.F
2And BC
1F
1In the seed, high amylose content meets 3: 1 respectively with the low amylose content seed number and separated ratio with 1: 1, shows that its low amylose starch controlled by a pair of recessive nuclear gene.
(2) the soft rice in Yunnan kind milli wind and the surprising glutinous hybridization of the glutinous property of Wx gene mutant, F
1The seed amylose content is between amphiphilic, near milli wind.F
2And BC
1F
1In the seed, the low amylose content seed meets 3: 1 respectively and 1: 1 ratio with separating of glutinous property seed, further according to F
2: 3Family is known each F by inference
2Middle dominance is isozygotied: heterozygosis: the ratio of recessive homozygous individual meets 1: 2: 1, shows milli wind low amylose starch gene and Wx equipotential.
(3) further wind and Wx gene mutation body Milky Queen hybridization in the least, F
2The seed amylose content is unimodal distribution near parents' mean value, show that also milli wind low amylose content is controlled by Wx allelotrope.
(4) according to paddy rice whole genome sequence information design primer, utilize overlapping PCR method (GenomeRes.1997,7:389-398), and the PCR product directly checked order, obtain the genome sequence of the about 7kb of milli wind Wx, The sequencing results is: a single base mutation (base A sports G) is located to exist at the 4th exon (+497) in the coding region, and coding sports GGC by GAC, has caused aspartic acid to sport glycine.
(5) according to single nucleotide mutation site (+497 A of place sport G) the design dCAPs mark of the 4th exon, the dCAPs-SalI-hp primer sequence is:
Sense primer, 5 '-GTGGAGTCGACCGTGTGTTCGTCG-3 ',
Antisense primer, 5 '-CCCCAAACCTGAAATCACCAGTGGA-3 '.
The sequence of table 1. labeled primer and amplified fragments size
(2) materials and methods:
Yunnan softpanel rice variety: milli wind;
Japonica rice variety: Japan is fine;
Yunnan local variety source: 101 parts; Each 5 parts of Hunan, Guangxi, Fujian, Jiangxi, Zhejiang, Heilungkiang and Jilin variety sources.
Utilize the SDS method (Plant Mol Bioi Rep, 1983,1:19-21) extract the DNA of above rice varieties, utilize dCAPs mark (The Plant Journal, 1998,14 (3), 381-385,387-392; Theor Appl Genet, 2004,108:1200-1204) detect single nucleotide polymorphism.
Used mark dCAPs-SalI-hp primer sequence is:
Sense primer, 5 '-GTGGAGTCGACCGTGTGTTCGTCG-3 ',
Antisense primer, 5 '-CCCCAAACCTGAAATCACCAGTGGA-3 '.。
Reaction system comprises: the about 10ng of dna profiling, 10 * LA buffer, 1 μ l, LA Taq (5u/ μ l) 0.1 μ l, MgCl
2(25mM) 1 μ l, dNTPs (2.5mM) 1 μ l, justice, each 1.5 μ l of antisense primer (2mM) add ddH then
2O makes volume arrive 10 μ l, and the PCR program is: 94 ℃ of 4min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 3min, 30 circulations; 72 ℃ of 10min.The enzyme system of cutting is, 5 μ l pcr amplification products, and 2 μ l, 10 * buffer, 0.1 μ lSal I restriction enzyme, it is 20 μ l that enzyme is cut cumulative volume, enzyme is cut behind the 3h electrophoresis on 8% polyacrylamide gel.
(3) result and analysis:
When being template amplification with the fine genomic dna of Japan, owing in forward primer, there is a mispairing, just form a SalI restriction enzyme site with the SNP site, also there is another one SalI restriction enzyme site in forward primer self in addition, enzyme is cut the rear electrophoresis analysis can detect three bands, and size is respectively 151bp, 145bp, 130bp.(Fig. 1)
And when being template, only in forward primer, have a SalI restriction enzyme site with milli wind genomic dna, and enzyme is cut rear electrophoresis can detect two bands, and size is respectively 151bp and 145bp (Fig. 1).
Utilize the other 101 parts of Yunnan variety sources of this marker detection, wherein milli skin, milli wood carefully have identical genotype (Fig. 1) at marker site with milli wind with milliampere vexed Wx gene.Utilize the Wx gene order that obtains these three kinds with the identical method of milli wind then, sequential analysis shows that these four soft rice have the on all four Wx gene order of sequence, have verified dCAPs marker detection result's exactness.
Utilize this mark to detect each 5 parts of Hunan, Guangxi, Fujian, Jiangxi, Zhejiang, Heilungkiang and Jilin variety sources respectively in addition, the result shows, these variety sources Wx gene all has different genotype with milli wind at marker site, and milli wind gene locus with low-content of amylose extensively is not present in (Fig. 2) in the kind of various places.
Predict the rice varieties low amylose content by above-mentioned molecular markers for identification gene locus with low-content of amylose, expectation can improve the breeding process of China's paddy rice low amylose content kind rapidly.
Material involved in the present invention is public material: Yunnan soft rice kind milli wind (Scientia Agricultura Sinica, 2004,37 (2): 157-162), high amylose starch rice varieties Nanjing 11 (Jiangsu Agricultural College's journals, 1989,10 (1): 7-12), the glutinous property of Wx gene mutant surprising glutinous (Acta Genetica Sinica, 2003,30 (7): 641-645), Wx gene mutation body MilkyQueen (Breeding Science, 2002,52:131-135), the fine (Acta Agronomica Sinica of japonica rice variety Japan, 2007,33 (6): 979-986).
Claims (1)
1. the molecule marking method of Yunnan softpanel rice variety milli wind gene locus with low-content of amylose is characterized in that, uses labeled primer dCAPs-SalI-hp
Left end primer sequence 5 '-GTGGAGTCGACCGTGTGTTCGTCG-3 '
Right-hand member primer sequence 5 '-CCCCAAACCTGAAATCACCAGTGGA-3 '
The DNA of amplification Yunnan softpanel rice variety milli wind uses the SalI digestion with restriction enzyme then, if can obtain 151bp and two segments of 145bp, then indicates the existence of Yunnan softpanel rice variety milli wind gene locus with low-content of amylose.
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| CN110628930A (en) * | 2019-09-03 | 2019-12-31 | 广西壮族自治区农业科学院 | dCAPS molecular marker for identifying panicle length character of rice and application thereof |
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| CN102146381B (en) * | 2011-01-18 | 2012-08-15 | 浙江师范大学 | Molecular marker for assisted breeding of Xanthomonas oryzae resistance gene Xa7 and application thereof |
| CN103725774B (en) * | 2013-10-18 | 2015-06-24 | 湖北省农业科学院粮食作物研究所 | Molecular marker Wx (waxy gene)-YMZC for rapidly detecting rice amylase Wx genotype and application method |
| CN108148927A (en) * | 2018-03-16 | 2018-06-12 | 深圳盛宝联合谷物股份有限公司 | The molecular labeling and its screening technique of QTL close linkages related to rice meal taste |
| CN108148928A (en) * | 2018-03-16 | 2018-06-12 | 深圳盛宝联合谷物股份有限公司 | The molecular labeling and its screening technique of QTL close linkages related to rice meal taste |
| CN114525359B (en) * | 2022-02-28 | 2023-08-22 | 扬州大学 | DNA molecular marker and method for cultivating low-amylose-content good-taste rice by using same |
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| CN1837373A (en) * | 2005-03-21 | 2006-09-27 | 中国科学院成都生物研究所 | Molecule marking method for identifying rice glutinous trait gene by specific DNA fragment |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110628930A (en) * | 2019-09-03 | 2019-12-31 | 广西壮族自治区农业科学院 | dCAPS molecular marker for identifying panicle length character of rice and application thereof |
| CN110628930B (en) * | 2019-09-03 | 2020-09-18 | 广西壮族自治区农业科学院 | dCAPS molecular marker for identifying panicle length character of rice and application thereof |
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