CN1172005C - Molecular label linked with major gene of high-strength cotton fibre - Google Patents
Molecular label linked with major gene of high-strength cotton fibre Download PDFInfo
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- CN1172005C CN1172005C CNB001302949A CN00130294A CN1172005C CN 1172005 C CN1172005 C CN 1172005C CN B001302949 A CNB001302949 A CN B001302949A CN 00130294 A CN00130294 A CN 00130294A CN 1172005 C CN1172005 C CN 1172005C
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Abstract
The present invention relates to molecular marks linked with major genes of high strength cotton fibre, which is used for molecular breeding to enhance the quality of cotton fibre. A germplasm system 7235 is used as material, molecular marks are sieved to identify three SSR marks NAU/SSR/Fs1<130>, NAU/SSR/Fs2<190> and NAU/SSR/Fs3<220> and six RAPD marks NAU/PSPD/Fs1<933>, NAU/PSPD/Fs2<1920>, NAU/PSPD/Fs3<1320>, NAU/PSPD/Fs4<983>, NAU/PSPD/Fs5<821> and NAU/PSPD/Fs6<1047> which are linked with genes of high strength cotton fibre. The first two SSR marks and the six RAPD marks are closely linked with one major gene QTL and are on the same linkage group, and the marked QTL has stable performance in different years and different environments. NAU/SSR/Fs3<220> marks a high strength fibre QTL of another linkage group. The molecular marks linked with major genes of high strength cotton fibre lay the foundation for enhancing the fibre quality of cotton varieties in China.
Description
One, technical field
The invention discloses the molecule marker chain, be used to improve the molecular breeding of cotton fiber quality, be exclusively used in the screening of high-strength cotton degree fiber major gene with major gene of high-strength cotton fibre.
Two, technical background
Cotton fibre is important textile industry raw material.The history in existing more than 50 year of the research of american cotton quality breeding.People such as Culp are since nineteen forty-six PD germplasm improved plan, last 40 surplus year, provided more than 30 germplasm line and 3 kinds.The fibrous quality of these germplasm lines or kind, especially fibre strength is greatly improved than conventional variety, and output reaches the level of commercial variety.Since nineteen eighty-two, for adapting to the transformation of textile industry equipment, the U.S. has accelerated the improvement to cotton fiber quality.Only 1982-1986 year encourages the bonus of Cotton Fiber Strength breeding just up to 5,600,000 dollars.From 1980, the annual 0.25tex/g that improves of U.S.'s fiber specific tenacity reached 21.7cN/tex to 1991 annuals.
Along with the progress of molecular biology research, all take much count of carrying out the molecular marker screening of high-quality fiber gene to be used for marker assisted selection both at home and abroad.Yu etc. (1998) are making up on the basis of upland cotton * sea island cotton species hybrid genetic map, identify and 11 of the chain molecule markers of sea island cotton high-quality fiber gene (QTL) 3 fibre strengths wherein, 3 staple lengths, 5 fiber finenesses.Soluble (the 35%-50% of the total heritable variation of sea-island and land hybrid F2 of TM-1 * 3-79) of these QTL.The research of Jiang etc. (1998) also proves in the tetraploid cotton seed (AADD), the QTL of most of fibrous quality, output is positioned at D karyomit(e) subgroup, and the ancestors of A chromosome subgroup have fiber in the tetraploid cotton seed, and D karyomit(e) subgroup then is a photon, does not have fiber.The soluble sea-island and land hybrid F of 3 fibre strength QTL that they identify
230.9% of total heritable variation.Above-mentioned these two results of study are all utilized species hybrid F
2The individual plant fibrous quality shows and draws, and how repeatability remains to be confirmed.But, the QTL that the hereditary effect value is little, tend to detect along with the variation of environment less than.Shappley etc. (1998) identify the RFLP mark chain with fibrous quality in upland cotton interbreed offspring.These results of study are that assisted Selection and even clone's fibrous quality gene of fibrous quality lays the foundation.
The cotton variety output that China breeds over nearly 15 years is a little more than the U.S., and fibrous quality is some gap then.The fibrous quality of China's govern-house-variety is medium, substantially can satisfy the requirement of textile industry, but quality is single, and fibre strength is on the low side, and a large amount of imports of high quality cotton need of high-count yarn spun (.1999. opinion Cotton in China quality present situations such as Xiang Shikang, Yu Nan, Hu Yuchang. cotton journal .11 (1): 1-10).Introduce efficiently spinning technique fast such as Air-Jet Spinning, air spinning along with spinning Ministry of worker door, and the people's generally raising that clothing is required, to the overall fibre quality of Cotton in China kind, especially fibre strength is had higher requirement.Price of the cotton changes height price by fibre strength with original into by the staple length price again from the world market in 1999.Therefore, it is very urgent how to increase substantially the fibre strength of Cotton in China kind in the near future.Improve the fibrous quality level of existing upland cotton (G.hirsutum L) kind, at first will collect the germplasm line with high fibre strength gene does parent and existing Cultivar and carries out a series of hybridization and hand over mutually, so that break the negative correlation between intensity and the output, but backcross every the wheel, all will test to fibrous quality, cost is very high, and to receive the florescence by the time could knowledge of result after finishing for some time, thereby, require breeding population big, blindness is also big.Therefore the cost of quality breeding is very high, time-consuming, and difficulty is also bigger, makes slow progress.By filtering out one or several and high-intensity fiber major gene close linkage and molecule marker not affected by environment is used for assisted Selection, can improve the efficiency of selection of high-intensity fiber greatly.
Three, summary of the invention
Technical problem the objective of the invention is: filter out one or several and major gene of high-strength cotton fibre close linkage and molecule marker not affected by environment, these molecule markers are used for the cotton assisted selection, can improve the efficiency of selection of high-intensity fiber greatly, thereby improve the fibrous quality level of Cotton in China kind as early as possible.
Technical scheme embodiment of the present invention are as follows.
The molecule marker chain with major gene of high-strength cotton fibre filters out by the following method:
(1) to drawing upland cotton high-intensity fiber germplasm line 7235 from academy of agricultural sciences, Jiangsu Province cash crop institute, with the hereditary standard of drawing from crop germplasm resource research department, farming research center, Plain, USDA south be that TM-1 is hybridized, F
1The seed selfing produces F
2
(2) extract (7235 * TM-1) F with the CTAB method
2The individual plant DNA of segregating population.
Adopt SSR and two kinds of molecule markers of RAPD to carry out the screening of high-strength cotton fibre mark.With 3 couples of SSR primer SSR1521, SSR2961, SSR3255, to 7235 and TM-1 parent's dna polymorphism carry out initial analysis, after the SSR reaction system is DNA94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 35 times, and last 72 ℃ are extended 7min; Increase on PE9600 amplification instrument, (FML, Maine USA) carry out isolation identification to amplified production on the glue at Metaphor agrose; The screening of RAPD mark is to use random primer UBC301, UBC431, and UBC757, OPAP01, OPAL19, OPM07, in the enterprising performing PCR amplification of PE-9600 thermal cycler, the PCR reaction volume is 20ul, wherein 10 * buffer (contains 25mM MgCl
2) 2.0ul, 2mM dNTPs 1.0ul, 10uM Primer1ul, 5mM TMACI and each 0.2ul of 10mg/ml BSA, Taq enzyme (5unit/ul) 0.2ul, template DNA 20ng adds ddH
2O to 20ul, PCR reaction heat cycling program is: after 94 ℃ of pre-sex change of 2min, then 94 ℃ of 30sec, 36 ℃ of 30sec, 72 ℃ of 70sec, 10 circulations, 89 ℃ of 20sec again, 36 ℃ of 20sec, 72 ℃ of 60sec, 35 circulations, 72 ℃ of 7min again; Pcr amplification product is electrophoresis in 1.4% sepharose, takes a picture the record result then on ultraviolet transilluminator.
(3) find 3 SSR mark NAU/SSR/Fs1
130, NAU/SSR/Fs2
190, NAU/SSR/Fs3
220With 6 RAPD marks, NAU/RAPD/Fs1
933, NAU/RAPD/Fs2
1920, NAU/RAPD/Fs3
1320, NAU/RAPD/Fs4
983, NAU/RAPD/Fs5
821, NAU/RAPD/Fs6
1047Chain with the high-intensity fiber major gene of cotton.
That beneficial effect the present invention selects for use is high-quality fiber germplasm line 7235 (Qian Siying, Huang Junqi, the Peng Yuejin etc. that unusual cotton (G.anomalum) gene gradually oozes, has Acala3080 and PD4381 genetic background.1992。Upland cotton (G.hirsutum L.) and cotton unusually (G.anomalum Wawr.﹠amp; Peyr.) research of species hybrid and the application in breeding thereof.Scientia Agricultura Sinica, 25 (6): 44-51) carry out the QTL molecular marker screening of high-strength cotton fibre.Compare with present technology, its advantage is:
(1) mark is stable.This research identifies on the genetic background of upland cotton and 3 of the chain SSR marks of two fibre strength QTLs, 6 of RAPD marks, especially mark the heritable variation that main effect QTL is shared is big, (7235 * TM-1) are combined in 1998,1999 respectively in Nanjing of China, Hainan, the configuration of Texas ,Usa continent produces the F2 segregating population, detects with these marks, show that this mark performance is stable, be not subjected to the time, the SSR primer has been added in the influence of envrionment conditions, assisted Selection is convenient, should be the beneficial gene resource that improves Cotton in China kind fibre strength level.
(2) the assistant breeding select target is clear and definite, saves cost.Improve powerful and breeding method that lint yield combines in, at first to collect parent and carry out a series of hybridization and mutual friendship, and carry out backcrossing and composite hybridization repeatedly, so that break the negative correlation between brute force and the output with Cultivar with powerful gene.But all to carry out individual plant in backcrossing and select fiber strength every the wheel.Carry out the mensuration of fiber strength with the home-made instrument, efficient is very low, even a skilled labor also can only survey 10-20 sample, and the HV900 instrument of import (costs an arm and a leg, general unit cannot afford) the efficient height, but cost is also high, and to receive by the time the florescence finish for some time could knowledge of result, require colony just greatly like this, blindness is also big, therefore the cost of quality breeding is very high, time-consuming, and difficulty is also bigger.What filter out by the present invention is used for assisted Selection with high-intensity fiber gene close linkage and molecule marker not affected by environment, can just identify the individual plant of high fibre strength in seedling stage, thereby improve the efficiency of selection of high-strength cotton fibre greatly.
Four, embodiment
Implementation procedure of the present invention is: to drawing the upland cotton high-intensity fiber germplasm line 7235 from academy of agricultural sciences, Jiangsu Province cash crop institute, select by individual plant, the 7235 strains system of isozygotying is selected in the evaluation of plant economical character and fibrous quality check.According to our measurement result in 1998,7235 fibre strengths are 27.3cN/tex, and staple length is 35mm, and fiber fineness (micron value, down together) is 4.1; Upland cotton heredity standard is that the fibre strength of TM-1 then is 20.7cN/tex, and staple length is 30.5mm, and fiber fineness is 5.0.TM-1 draws from crop germplasm resource research department, farming research center, Plain, USDA south.
Hybridized with TM-1 in 1997 7235, the end of the year F
1Seed is delivered to Hainan Island, and selfing produces F
2Seed.1998 with (7235 * TM-1) F
2The segregating population kind is in testing station, Agricultural University Of Nanjing Jiangpu.Extract the individual plant DNA of this segregating population with the CTAB method.Behind the blow-of-cottons, individual plant is received and is spent, takes a sample, carries out the fibrous quality test.Winter in 1998 and summer in 1999 are respectively at Sanya, Hainan Island and testing station, Agricultural University Of Nanjing Jiangpu plantation (7235 * TM-1) F
3Plant is so that estimate (7235 * TM-1) F with the mean value of the fibre strength of individual plant in the F3 plant
2The individual plant level.In order to identify the heredity performance of fibrous quality in the strange land, 1999 also will be from (7235 * TM-1) F of same colony
2Plant in experiment field, farming research center, Plain, Texas, USA university city (College Station) Ministry of Agriculture south.The interior cotton sample of host country all delivers to Earthquake of Anyang station in Henan Ministry of Agriculture fibrous quality and seed quality inspection center carries out the fibrous quality check.Molecule marker mainly utilizes microsatellite marker (SSR) and RAPD mark.
With 211 pairs of SSR primers at first to 7235 and TM-1 parent's dna polymorphism carry out initial analysis.211 pairs of little satellites (SSR) labeled primer is available from U.S. Research Genetics company.The Taq enzyme, dNTPs, other reagent of PCR reaction are all available from Sigma company.After the SSR reaction system is DNA94 ℃ of sex change 4min, 94 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 35 times, and last 72 ℃ are extended 7min.Increase on PE9600 amplification instrument, (FML, Maine USA) carry out isolation identification to amplified production on the glue at Metaphoragrose.The random primer that this test is adopted is the 10bp oligonucleotide sequence of U.S. OPERON company exploitation, and PCR is reflected in the PE-9600 thermal cycler and carries out.The PCR reaction volume is 20ul, and wherein 10 * buffer (contains 25mMMgCl
2) 2.0ul, 2mM dNTPs 1.0ul, 10uM Primer 1ul, 5mM TMACI and each 0.2ul of 10mg/ml BSA, Taq enzyme (5unit/ul) 0.2ul, template DNA 20ng adds ddH
2O to 20ul.PCR reaction heat cycling program is: after 94 ℃ of pre-sex change of 2min, and then 94 ℃ of 30sec, 36 ℃ of 30sec, 72 ℃ of 70sec, 10 circulations, 89 ℃ of 20sec again, 36 ℃ of 20sec, 72 ℃ of 60sec, 35 circulations, 72 ℃ of 7min again.Pcr amplification product is electrophoresis in 1.4% sepharose, takes a picture the record experimental result then on ultraviolet transilluminator.To 7235 SSR that the specific amplified band arranged, the RAPD primer is selected the evaluation of further increasing of high fibre strength/low fibre strength pond respectively for use.When making up high/low fibre strength pond, simultaneously with reference to (7235 * TM-1) F in 1998
2In the individual plant performance in Nanjing, winter in 1998 and summer in 1999 plant respectively at Hainan Island and Jiangpu experiment centre (7235 * TM-1) F of Agricultural University Of Nanjing
3The average performance of plant individual plant fibre strength respectively selects 5～6 strains to constitute.7235 parent's specific amplified bands can repeat in the high-intensity fiber pond, and in low strong pond unexpressed primer, just with (7235 * TM-1) F in 1998
2Individual plant DNA increases.
Molecular marker screening is the result show, has 26 variant on parents to (12.32%) SSR primer.Find to have 3 SSR mark NAU/SSR/Fs1 altogether
130(can amplify the long dna fragmentation of 130 pairs of Nucleotide) with the SSR1521 primer, NAU/SSR/Fs2
190(can amplify the long dna fragmentation of 190 pairs of Nucleotide) with the SSR2961 primer, NAU/SSR/Fs3
220(can amplify the long dna fragmentation of 220 pairs of Nucleotide) and 6 RAPD marks with the SSR3255 primer, NAU/RAPD/Fs1
933(can amplify the long dna fragmentation of 933 pairs of Nucleotide) with the UBC301 primer, NAU/RAPD/Fs2
1920(can amplify the long dna fragmentation of 1920 pairs of Nucleotide) with the UBC431 primer, NAU/RAPD/Fs3
1320(can amplify the long dna fragmentation of 1320 pairs of Nucleotide) with the UBC757 primer, NAU/RAPD/Fs4
983(can amplify the long dna fragmentation of 983 pairs of Nucleotide) with the OPAP01 primer, NAU/RAPD/Fs5
821(can amplify the long dna fragmentation of 821 pairs of Nucleotide) with the OPAL19 primer, NAU/RAPD/Fs6
1047(can amplify the long dna fragmentation of 1047 pairs of Nucleotide with the OPM07 primer) is chain with the high-intensity fiber of cotton.They carry out chain detection to utilize the MAPMAKER/QTL3.0 program, find to have 2 SSR mark: NAU/SSR/Fs1
130, NAU/SSR/Fs2190 and above 6 RAPD marks and a main effect QTL compact linkage are on this linkage group; The QTL of mark accounts for (7235 * TM-1) F
2More than 30% of the total heritable variation of segregating population.In addition, the QTL of this mark is in different year, and the varying environment performance is stable, therefore, and the main site of imitating that this control high-strength cotton fibre that is we find shows.This QTL is based on additivity and recessive inheritance effect.Therefore, can carry out the high-intensity fiber breeding of cotton effectively with molecular marker assisted selection.Utilize the QTL of this mark, expectation can improve Cotton in China kind fibre strength 2-3cN/tex rapidly.NAU/SSR/Fs3
220The high-intensity fiber QTL of another linkage group of mark.
Claims (2)
1, with the chain molecule marker of major gene of high-strength cotton fibre, filter out by the following method:
(1) draw upland cotton high-intensity fiber germplasm line 7235 from academy of agricultural sciences, Jiangsu Province cash crop institute, with the hereditary standard of drawing from crop germplasm resource research department, farming research center, Plain, USDA south be that TM-1 is hybridized, F
1The seed selfing produces F
2
(2) extract 7235 * TM-1 F with the CTAB method
2The individual plant DNA of segregating population;
(3) adopt SSR and two kinds of molecule markers of RAPD to carry out the screening of high-strength cotton fibre mark;
(4) filter out 3 SSR mark NAU/SSR/Fs1 altogether
130, NAU/SSR/Fs2
190, NAU/SSR/Fs3
220With 6 RAPD marks, NAU/RAPD/Fs1
933, NAU/RAPD/Fs2
1920, NAU/RAPD/Fs3
1320, NAU/RAPD/Fs4
983, NAU/RAPD/Fs5
821, NAU/RAPD/Fs6
1047Chain with the high-intensity fiber major gene of cotton.
2, the described and chain molecule marker of major gene of high-strength cotton fibre according to claim 1, wherein two kinds of molecule markers of SSR of Cai Yonging and RAPD carry out the screening method of high-strength cotton fibre mark and are: with 3 couples of SSR primer SSR1521, SSR2961, SSR3255, to 7235 and TM-1 parent's dna polymorphism carry out initial analysis, after the SSR reaction system is DNA94 ℃ of pre-sex change 4min, 94 ℃ of sex change 1min, 60 ℃ of annealing 1min, 72 ℃ are extended 2min, circulate 35 times, last 72 ℃ are extended 7min; Increase on PE 9600 amplification instrument, amplified production carries out isolation identification on Metaphor agrose glue; The screening of RAPD mark is random primer UBC301, UBC431, and UBC757, OPAP01, OPAL19, OPM07, in the enterprising performing PCR amplification of PE-9600 thermal cycler, the PCR reaction volume is 20ul, wherein contains 25mM MgCl
210 * buffer 2.0ul, 2mM dNTPs 1.0ul, 10uM Primer 1ul, 5mM TMACI and each 0.2ul of 10mg/ml BSA, 5unit/ul Taq enzyme 0.2ul, template DNA 20ng adds ddH
2O to 20ul, PCR reaction heat cycling program is: after 94 ℃ of pre-sex change of 2min, then 94 ℃ of 30sec, 36 ℃ of 30sec, 72 ℃ of 70sec, 10 circulations, 89 ℃ of 20sec again, 36 ℃ of 20sec, 72 ℃ of 60sec, 35 circulations, 72 ℃ of 7min again; Pcr amplification product is electrophoresis in 1.4% sepharose, takes a picture the record result then on ultraviolet transilluminator.
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| CNB001302949A CN1172005C (en) | 2000-11-10 | 2000-11-10 | Molecular label linked with major gene of high-strength cotton fibre |
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| CN1172005C true CN1172005C (en) | 2004-10-20 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101613761B (en) * | 2009-08-12 | 2012-11-28 | 中国农业科学院棉花研究所 | SSR markers lined with major gene of cotton fiber strength |
| CN102925436B (en) * | 2012-10-31 | 2014-04-30 | 南京农业大学 | Cotton highly-verticillium wilt resistant major QTL (quantitative trait locus) and SSR molecular marker thereof |
| CN103276100B (en) * | 2013-06-19 | 2014-09-10 | 江苏省农业科学院 | Creation method of cotton fiber length single QTL near-isogenic line |
| CN110055246B (en) * | 2019-05-09 | 2021-03-30 | 江苏省农业科学院 | Abnormal cotton chromosome segment capable of improving cotton fiber strength and molecular marker thereof |
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