CN102181442B - Molecular label relevant with cotton fiber strength and arising from high-quality variety Xinluzao No.24 - Google Patents
Molecular label relevant with cotton fiber strength and arising from high-quality variety Xinluzao No.24 Download PDFInfo
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Abstract
The invention discloses a molecular label linked with a major gene of a high-strength cotton fiber, which is obtained by the following method: taking the Xinluzao No.24 as a male parent, taking Lunianyan No.28 and Ji cotton 516 as female parents; respectively constructing two segregation populations of F2 and F2:3; carrying out polymorphism screening between parents (Lunianyan 28 and Xinluzao 24) by utilizing an SSR (simple sequence repeat) primer; constructing a genetic linkage map by utilizing F2; carrying out two generation quantitative trait locus (QTL); selecting an SSR label linked with the high-strength cotton fiber to analyze the DNA polymorphism of the parent Xinluzao 24 and the Ji cotton 516; and constructing the genetic linkage map and positioning QTL; the fiber strength major QTL (qFS-62) can be stably detected in two populations and generations, and is positioned between the label interval of DPL0757-DPL0920 of chromosome c7; a synergtic gene is from the parent Xinluzao No.24; and the linking label of qFS-6-2 is DPL0757130, DPL0852170 and DPL0920130. By utilizing the label provided by the invention, molecule label assisted selection is carried out, thus improving the selection efficiency of the high-strength cotton fibre.
Description
Technical field
The present invention relates to a kind of from SSR (the Simple Sequence Repeat chain with the new land of high-quality middle long stapled cotton kind 24 fibre strength major genes morning, simple repeated sequence) molecule marker, be used for improving the molecular marker assisted selection of cotton fiber quality, to improve the efficient of selecting, accelerate the breeding paces.
Background technology
Cotton is important textile industry raw material.The production of cotton occupies an important position in national economy.The production of Cotton in China mainly concentrates on before the eighties in the raising of output, does not pay attention to the improvement of fibrous quality, makes the master of China plant the higher and inferior quality of the most output of cotton variety.Compare with the U.S., have superiority on the Cotton in China staple length, but the fiber specific tenacity than the U.S. poor slightly (Yang Wei China, Xiang Shikang .2001 such as Tang Shurong, China's self-fertile cotton variety fibrous quality analysis over 20 years. cotton journal .13 (6): 377-384).Develop rapidly along with textile industry, the speed of cotton variety improvement lags behind the development of textile technology, at present the Cotton in China fiber quality can satisfy textile industry spin in, the requirement of low-grade cotton yarn (32～40 yarns), the raw cotton fiber proportion of high-count yarn spun (60～80) is less.In recent years, China has authorized some length more than 31mm, the upland cotton new variety of fiber specific tenacity more than 30cN/tex, but Quality Type is single, and the index coupling is undesirable between the cotton fibre product, and most of upland cotton Cultivar fiber specific tenacity is on the low side, therefore, cultivate the cotton variety of high-quality, especially the high new variety of fiber specific tenacity become the task of top priority of cotton breeding work.
Adopt traditional breeding method to improve the fibrous quality level of existing upland cotton kind, at first to carry out a series of hybridization and backcross with having germplasm materials that high fibre strength gene gradually oozes and existing upland cotton kind, to break the negative correlation between fibrous quality proterties and the yield traits, but all to detect fibrous quality in backcrossing every the wheel, cost is very high, workload is very big, and can only receive bow structure bundle by the time could knowledge of result after for some time, in addition, environment is also influential to fibrous quality.This just requires that bigger breeding population must be arranged, and blindness also strengthens, and wastes time and energy, and cost is increased, and therefore, quality breeding is made slow progress.
Utilize molecular marking technique to detect colony in the short period of time, and genotype can be identified.Utilize the linkage relationship between objective trait and molecule marker, just can select objective trait, improve efficiency of selection greatly.The molecule marker that all takes much count of both at home and abroad carrying out high-quality fibrous quality gene selects to be used for marker assisted selection.(1998) such as Shappley etc. (1998), Jiang utilize RFLP to be marked at F
2In detect more than 40 a QTL relevant with fibrous quality; (2003) such as Yuan Youlu etc. (2000-2001) and Zhang utilize unusual cotton high-intensity fiber gradually to ooze to be 7235 with upland cotton heredity standard be that TM-1 has made up F for the parent
2And F
2:3Segregating population identifies the main effect QTL of a fibre strength, explains the phenotypic variation 30% or more, and all can detect in a plurality of environment, for marker assisted selection.Mei (2004) is F between the kind of extra large land
2Found the QTLs of 5 fibrous qualities in the colony, though number is less, the phenotypic variation of explaining is bigger, between 22-42%.Shen Xinlian etc. (2005) utilize 7235, PD6992 and three upland cotton high-intensity fibers of HS427 germplasm line have made up linkage map in 3 kinds, screen 38 QTLs relevant with fibrous quality altogether.Hu Wenjing etc. (2008) utilize the F of Chongqing cotton No. 1 and 7235
2And F
2:3Segregating population screens 36 fibrous quality QTL altogether.Chen Li (2008) utilize the upland cotton middle cotton 35 and the F of cotton No. 1 of Chongqing
2With F
2:3Segregating population is that material detects 5 fibrous quality proterties QTL altogether, i.e. 1 staple length, 2 fiber specific tenacitys and 2 fiber finenesses.Qin etc. (2008) utilize 4 upland cotton kinds, divide two groups of hybridization to obtain two F
1Continue hybridization, obtained a cover F at last
2Segregating population has obtained a genetic map that comprises 285 SSR marks and a morphology mark, and 2113.3cM covers 42% of cotton gene group altogether, has located 31 QTL altogether, and wherein 24 is significance QTL; 20 fibrous quality QTL are arranged among 31 QTL, and wherein 5 is the QTL of fibre strength.Zhu etc. (2008) utilize the cross combination of HS46 and MARCABUCAG8US-1-88 to dispose the RIL colony of 188 systems, and having made up length overall with 125 SSR marks is the genetic linkage maps of 965cM.Under the situation of LOD＞3.0, detect the QTL of an elongate fiber rate at LGU01 (uncertain to karyomit(e)), detect 17 fibrous quality QTL at A subgroup karyomit(e), D subgroup karyomit(e) detects 16 fibrous quality QTL, and this is comprising the QTL of 6 fibre strengths.Chen etc. (2009) utilize gradually ooze be 7235 with upland cotton heredity standard be TM-1 hybridization, backcross with TM-1 again and obtain 3 7TR-133 of RIL colony, 7TR-132, and 7TR-214, utilize 3 colonies to locate the QTL of 5 fibre strengths at D8 karyomit(e), wherein qFS-1 can detect in 3 colonies, and qFS-2 and qFS-3 can detect in two colonies.It is 270 F that Zhang etc. (2009) utilize cross combination T586 * Yumian 1 to obtain size
2:7RIL, and utilize the genetic linkage maps in 4 kinds of marks such as SSR, SRAP, morphology mark and IT-ISJ (509 SSR, 58 IT-ISJ, 29 SRAP and 8 morphology marks) individual site that made up 604, length overall 3140.9cmM, coverage rate Hua total genomic 70.6%.Under 5 environment, detect the QTL of 13 fibrous qualities altogether, explain the phenotypic variation of 7.4%-43.1%, comprising the QTL of two fibre strengths.An etc. (2009) utilize photon strain MD17 to hybridize with Westerner Cultivar FM966 and photon strain 181 respectively, make up two cover F
2Segregating population wherein detects 2 micron values in MD17 * FM966 colony, 1 fibre strength, and 2 fiber 50% span lengths and 2 fiber 2.5% span lengths' QTL, the QTL of fibre strength (qT1-c16-1) is consistent with the research of (2007) such as Guo.Yuan Youlu, Sun Fuding etc. (2009) patent application (molecule marker chain with the cotton fiber strength major gene), utilizing 41 choosings of middle cotton institute is that sGK9708 and upland cotton high-quality strain 0-153 combination make up RIL colony, filter out 6 synergy genes from the fibre strength QTL of high value parent 0-153, wherein stablize (patent application publication number: CN 101613761A) under 5 many environment in QTL site.Zhang Ke etc. (2009) utilize upland cotton 34B and sea island cotton Giza70 hybridization, are recurrent parent with upland cotton 34B, make up BC
2F
4:6And BC
2F
4:7Height carries out the test of 3 years four environment for backcrossing recombinant inbred lines, has located the QTL of 27 fibrous quality proterties altogether, wherein can detectedly have 7 under two environment, comprises two QTL that fiber is strong.17 synergy gene is arranged from sea island cotton among all QTL of fibrous quality, the contribution rate of single QTL is between 6-13%.Extensively superfine (2009) utilize that two sibships are far away, output of cotton and fibrous quality proterties all exist the upland cotton kind HS46 of larger difference and MARCABUCAGSUS-1-88 to be the parent in the hole, to cultivate the RIL colony that forms be material by improveing capable hybrid system, carried out the test of many environment for many years, located the QTL of 25 fibrous qualities altogether, comprising the QTL of 6 fibre strengths.Wang Yiqing etc. (2010) utilize upland cotton Shandong cotton grind 21 with high-quality strain NM03102 hybridization, make up F
2And F
2:3Segregating population, by interval graphing method, detected the QTL of 20 fibrous quality proterties, wherein, the QTL of the QTL of 1 fibre strength and 1 fibre uniformity is consistent with existing report, and the QTL of the QTL of 1 fibre strength and 1 micron value is stable existence in two generations.The good precocious upland cotton Cultivar middle cotton institute 36 that Liang Yan etc. (2010) utilize to produce go up promote be receptor parent, and sea island cotton sea 1 be donor parents, and selection has been cultivated one and overlapped the BC that is made up of 303 individual plants
5F
2The chromosome segment substitution line.Adopt the One-way ANOVA between proterties-mark, 33 QTLs relevant with the fibrous quality proterties have been located altogether, wherein there is certain genetic stability in these 4 QTL of qUN-14-2, qBW-2-20, qFL-2-20 and qMV-1-38, have comprised the QTL of 12 fibre strengths among the QTL of these fibrous quality proterties.Ma Jing etc. (2010) utilize cotton No. 1,7235 of upland cotton fine quality Chongqing, high yield kind middle cotton institute 35 to be the parent, disposed triparental mating colony, utilize the composite interval mapping method, detected the QTL of 10 fibrous quality proterties altogether, explain the phenotypic variation of 6.5%-29.2%, comprising the QTL of 2 fibre strengths, lay respectively on c15 and the c25 karyomit(e).
These results of study are that the assisted Selection of fibrous quality is laid a good foundation.But, the deficient in stability that the QTLs of these location has and reliability, some selects not consider to combine with the breeding selection in experiment material, be difficult to be applied to breeding practice in.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of from the new land of fine quality 24 molecule markers relevant with cotton fiber strength early, that is: by screening from the new land of the excellent fibrous quality material SSR molecule marker chain with the cotton fiber strength major gene in 24 early, carry out the early stage assisted Selection on the dna level, improve breeding efficiency.
Technical scheme provided by the invention is: a kind of molecule marker chain with the cotton fiber strength major gene loci, described cotton fiber strength major gene loci is positioned at karyomit(e) c7 to be gone up between the DPL0757-DPL0920 mark zone, chain with it DPL0757 that is labeled as
130, DPL0852
170, DPL0920
130
Among the present invention, the synergy gene is from the new land of high-quality fiber parent early 24, described cotton fiber strength major gene loci QTL called after qFS-6-2, the name of QTL is with reference to naming rule (the MCCOUCH S R on paddy rice, Cho Y G, Yano M, et al.Report on QTL nomenclature[J] .Rice Genet Newslett, 1997,14:11-13.): q (quantitative character) proterties title english abbreviation (the fracture specific tenacity, FS)+"-"+place linkage group code name (LG6)+QTL number (last the 2nd the fibre strength QTL of LG6).
The screening method of the molecule marker that a kind of described and cotton fiber strength major gene loci are chain comprises the steps:
(1) grinding No. 28 with the Shandong cotton is that female parent makes up F with 24 hybridization early of new land respectively with Ji cotton 516
2, F
2:3Colony, wherein, first colony with new land early 24 be male parent and transgenic pest-resistant conventional variety Shandong cotton to grind No. 28 be hybridization of female parent, acquisition F
1Seed, F
1Selfing produces F
2, F
2Selfing gets F
2:3Another colony 24 is male parent early with new land, and Ji cotton 516 be hybridization of female parent, and the kind method for planting is with first colony;
(2) extract above-mentioned F
2Segregating population and parent's individual plant DNA;
(3) adopt SSR label screening high-strength cotton fibre mark, obtain one two from generation to generation, two colony's stable existence fibre strength main effect QTLs, it is positioned on the karyomit(e) c7, chain with it DPL0757 that is labeled as
130, DPL0852
170, DPL0920
130
Wherein, 3 pairs of SSR characteristic primer nucleotides sequences are classified as:
DPL0757 primer, its forward sequence are shown in SEQ ID NO.1, and reverse sequence is shown in SEQ ID NO.2, and with new land No. 24 specific marks morning that this primer amplification goes out, the band molecular weight is 130bp;
DPL0852 primer, its forward sequence are shown in SEQ ID NO.3, and reverse sequence is shown in SEQ ID NO.4, and with new land No. 24 specific marks morning that this primer amplification goes out, the band molecular weight is 170bp;
DPL0920 primer, its forward sequence are shown in SEQ ID NO.5, and reverse sequence is shown in SEQ ID NO.6, and with new land No. 24 specific marks morning that this primer amplification goes out, the band molecular weight is 130bp.
In above-mentioned (3) step, at first, with SSR primer screening new land morning 24 and Shandong cotton grinding No. 28 parents' dna polymorphism, obtain polymorphism primer and pleomorphism site, utilize these primers that polymorphism is arranged to F
2Colony's individual plant screens, and utilizes F
2The polymorphism result of colony's individual plant makes up genetic linkage maps, and in conjunction with F
2And F
2:3The fibrous quality measurement result, the relevant QTL in location; Then, according to positioning result, select on the 6th linkage group that QTL assemble to occur 4 pairs of SSR primers namely: DPL0757, DPL0852, DPL0920, BNL1694, dna polymorphism to new land morning 24 and cotton 516 parents in Ji is analyzed, obtaining 3 pairs has polymorphism primer namely: DPL0757, DPL0852, DPL0920 and described 3 marker sites, and make up genetic linkage maps and Mapping of QTL.
Above-mentioned with the new land of SSR primer screening early 24 and the Shandong cotton grind No. 28 parents' dna polymorphism, wherein, the pcr amplification reaction system is the 10ul system, 10 * buffer 1.0ul wherein, 10mMd NTPs 0.5ul, Taq enzyme (5U/ul) 0.1ul, template DNA 1.2ul, ddH
2O 6.2ul, the forward primer 0.5ul of 10uM, the reverse primer 0.5ul of 10uM.PCR thermal response program is: 95 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 45s, circulate 30 times, and last 72 ℃ are extended 2min, 4 ℃ of preservations; Increase at TC-512 and the imperial EDC-810 amplification of east victory instrument, pcr amplification product is electrophoresis in 8% polyacrylate hydrogel, and silver dyes colour developing, the record result.
The present invention has following beneficial effect:
The present invention relates to a site qFS-6-2 relevant with the cotton fiber strength major gene, the synergy gene is from the new land of excellent fibrous quality parent early 24, and at two generation (F
2And F
2:3), two colonies (the Shandong cotton grind No. 28 * new land early 24, Ji cotton 516 * new land early 24) in equal stable existences.The phenotypic variation of explaining is respectively 17.26%, 6.72%, 8.09%, 8.29%.Additive effect is bigger, is respectively 1.0274,1.1226,0.755,0.9241.The QTL of location, stable, reliable, for the early stage assisted Selection of carrying out on the dna level, improve breeding efficiency.
Description of drawings
Fig. 1 is F of the present invention
2Colony (Shandong cotton grind No. 28 * new land early 24) part molecular marker linkage maps and QTL position.
Fig. 2 is F
2Colony (Ji cotton 516 * new land early 24) part molecular marker linkage maps and QTL position.
Mark among the figure adopts the form of " molecular weight of primer title/specific mark respective strap ", and the molecular weight after "/" is corresponding with the index number of primer title in the literary composition.
QFS-6-2 is positioned in two figure between the DPL0757-DPL0920 mark zone of karyomit(e) c7, chain with it DPL0757 that is labeled as
130, DPL0852
170, DPL0920
130Grind in the genetic linkage map of No. 28 colonies in the Shandong cotton, the peak value of this QTL is positioned at 56cM, with DPL0757
130, DPL0852
170, DPL0920
130Genetic distance Deng 3 marks is respectively 1.5cM, 0.3cM and 8.7cM.In the genetic linkage map of cotton 516 colonies in Ji, the peak value of this QTL is positioned at 6cM, with DPL0757
130, DPL0852
170, DPL0920
130Genetic distance Deng 3 marks is respectively 3.2cM, 21cM and 6.2cM.
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does the example explanation.
The molecule marker that the present invention and cotton fiber strength major gene are chain filters out by the following method:
(1) first colony is with late-maturing, and the new land of long stapled cotton early 24 is ground No. 28 for maternal for male parent and transgenic pest-resistant conventional variety Shandong cotton in the high-quality, and preparing hybrid makes up, the end of the year F
1In generation, obtain F from accompanying each other
2Seed, 1 year spring will (Shandong cotton grind No. 28 * new land early 24) F
2Plantation obtains 238 individual plants.With all F
2Individual plant selfing obtains F
2:3Seed.238 F of plantation in the 3rd year
2:3The family seed.Each family is selected 15 strains at random.Take a sample behind the autumn blow-of-cottons respectively at 1 year, the 3rd year, each family is gathered in the crops 30 normal blow-of-cottons bells at random.To parent F
2Individual plant and F
2:3Cotton sample is carried out fibrous quality and is detected.Detect index and comprise fracture specific tenacity, upper half mean length, micron value, reguarity exponential sum elongate fiber rate, adopt the HVICC calibrated horizontal.
Second colony with high yield, disease-resistant, high-quality cotton new variety Ji cotton 516 serve as maternal and new land early 24 preparing hybrids make up totally 256 F
2Individual plant, corresponding 256 F
2:3Family.It is the same to plant method for planting.
(2) (Shandong cotton grind No. 28 * new land early 24) and (Ji cotton 516 * new land morning 24) F
2CTAB method (Paterson et al., 1993) is adopted in the extraction of the individual plant DNA of segregating population
(3) adopt the SSR mark to carry out the screening of high-strength cotton fibre mark.Select 7638 pairs of SSR primers altogether for use, screen new land morning 24 and Shandong cotton and grind No. 28 parents' dna polymorphism, wherein the primer with NAU numbering is Agricultural University Of Nanjing's exploitation, and totally 2278 to (primer source: 1. http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_NAU.xls; 2. Han ZG, Guo WZ, et al., Genetic mapping of EST-derived microsatellites from the diploid Gossypium arboreum in allotetraploid cotton.Mol Gen Genomics 2004,272:308-327); Primer with the BNL numbering is U.S.'s BNL exploitation, and totally 687 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_data/BNL_ primer_marker_info.xls); Be Monsanto Company's exploitation with the primer of numberings such as CGR, DPL, SHIN, DC, totally 2937 to (primer source: 1. http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_MON.xls; 2. Xiao J, Wu K, Fang DD, et al., New SSR markers for use in cotton (Gossypium spp.) improvement, Journal of Cotton Science, 2009,13:75-157.); Primer with the HAU numbering is Hua Zhong Agriculture University's exploitation, and totally 408 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_HAU.xls); Primer with JESPR, Gh numbering is the exploitation of the agro-industrial university of texas,U.S, and totally 1013 to (primer source: 1. http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_JESPR.xls; 2. http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_GH.xls); Primer with CIR numbering is the exploitation of international farming research centre of development, and totally 121 to (primer source: 1. http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_CIR.xls; 2. Nguyen TB, Giband M, Brottier P, et al., Wide coverage of the tetraploid cotton genome using newly developed microsatellite markers.Theor Appl Genet, 2004,109:167-175); The primer of TMB numbering is the exploitation of USDA farming research service administration, totally 194 to (primer source document: http://www.cottonmarker.org/cmd_downloads/ssr_project_data/CMD_ PRIMER_TMB.xls), obtain 225 pairs of polymorphism primers and 238 pleomorphism sites altogether, utilize these primers that polymorphism is arranged to F
2Colony's individual plant screens, and utilizes F
2The polymorphism result of colony's individual plant makes up genetic linkage maps, and in conjunction with F
2And F
2:3The fibrous quality measurement result, the relevant QTL in location, according to positioning result, select the 4 pairs of SSR primers (DPL0757, DPL0852, DPL0920, BNL1694) on the 6th linkage group that QTL assemble to occur, dna polymorphism to new land morning 24 and cotton 516 parents in Ji is analyzed, obtaining 3 pairs has polymorphism primer (DPL0757, DPL0852, DPL0920) and 3 marker sites (3 pairs primer sequence and amplified fragments size see Table 1), and makes up genetic linkage maps and Mapping of QTL.In the present embodiment, used SSR primer gives birth to worker biotechnology company limited by Shanghai Ying Jun Bioisystech Co., Ltd, Shanghai respectively and Beijing three rich polygala root biotechnology limited liability companys are synthetic.The pcr amplification reaction system is the 10ul system, 10 * buffer 1.0ul wherein, 10mMd NTPs 0.5ul, Taq enzyme (5U/ul) 0.1ul, template DNA 1.2ul, ddH
2O 6.2ul, the forward primer 0.5ul of 10uM, the reverse primer 0.5ul of 10uM.PCR thermal response program is: 95 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 45s, circulate 30 times, and last 72 ℃ are extended 2min, 4 ℃ of preservations; Increase at TC-512 and the imperial EDC-810 amplification of east victory instrument, pcr amplification product is electrophoresis in 8% polyacrylate hydrogel, and silver dyes colour developing, the record result.
Table 13 pair characteristic primer sequence and amplified fragments size
(4) make up genetic map, its structure concrete grammar is: adopt mapping software Jionmap3.0 to carry out the mark linkage analysis, and with LOD=7.0, recombination fraction=0.4, the Kosambi mapping function makes up genetic linkage maps (referring to Fig. 1,2).
(5) concrete grammar of QTL location is: in conjunction with two cover F
2Colony and F
2:3Family fibrous quality measurement result, utilize QTL mapping software Windows QTL Cartographer2.5, select for use the composite interval mapping method to carry out the QTL location, the LOD threshold value is made as 2.5, carry out the experiment of 1000 minor sorts, the high-intensity fiber main effect QTL of screening two two ambient stables existence from generation to generation.Obtain 1 QTL (qFS-6-2) altogether, it is positioned on the karyomit(e) c7, chain with it DPL0757 that is labeled as
130, DPL0852
170, DPL0920
130, near underlined BNL1694 this QTL, at a distance of about 18cM, (patent application publication number: CN 101613761A) common mark BNL1694 is arranged, these two may be chain or cluster with the result of study of Yuan Youlu, Sun Fuding.The phenotypic variation that this QTL explains is respectively 17.26%, 6.72%, 8.09%, 8.29%.Additive effect is bigger, is respectively 1.0274,1.1226,0.755,0.9241.
Claims (4)
- One kind with the new land of the upland cotton chain molecule marker of 24 fibre strength major gene locis early, it is characterized in that: described cotton fiber strength major gene loci is positioned at karyomit(e) c7 to be gone up between the DPL0757-DPL0920 mark zone, with described site chain be labeled as DPL0757 130, DPL0852 170, DPL0920 130
- A claim 1 described with the new land of the upland cotton screening method of the chain molecule marker of 24 fibre strength major gene locis early, comprise the steps:(1) grinding No. 28 with the Shandong cotton is that female parent makes up F with 24 hybridization early of new land respectively with Ji cotton 516 2, F 2:3Colony, wherein, first colony with new land early 24 be male parent and transgenic pest-resistant conventional variety Shandong cotton to grind No. 28 be hybridization of female parent, acquisition F 1Seed, F 1Selfing produces F 2, F 2Selfing gets F 2:3Another colony 24 is male parent early with new land, and Ji cotton 516 be hybridization of female parent, and the kind method for planting is with first colony;(2) extract above-mentioned F 2Segregating population and parent's individual plant DNA;(3) adopt SSR label screening high-strength cotton fibre mark, obtain one at two generations, two colony's stable existence fibre strength main effect QTLs, it is positioned on the karyomit(e) c7, the mark DPL0757 chain with it 130, DPL0852 170, DPL0920 130,The screening object: two couples of parents, new land early 24 with the Shandong cotton grind No. 28, new land early 24 with Ji cotton 516,The screening step: with different primers two couples of parent DNA are carried out pcr amplification, dye the demonstration amplified band that develops the color with polyacrylamide gel electrophoresis and silver, band there are differences between the parent, then this to primer be this to parent's polymorphism primer,The primer structure: wherein, 3 pairs of SSR characteristic primer nucleotides sequences are classified as:DPL0757 primer, its forward sequence are shown in SEQ ID NO.1, and reverse sequence is shown in SEQ ID NO.2, and with new land No. 24 specific marks morning that this primer amplification goes out, the band molecular weight is 130bp;DPL0852 primer, its forward sequence are shown in SEQ ID NO.3, and reverse sequence is shown in SEQ ID NO.4, and with new land No. 24 specific marks morning that this primer amplification goes out, the band molecular weight is 170bp;DPL0920 primer, its forward sequence are shown in SEQ ID NO.5, and reverse sequence is shown in SEQ ID NO.6, and with new land No. 24 specific marks morning that this primer amplification goes out, the band molecular weight is 130bp;QTL localization method: with the primer of polymorphism being arranged to F 2Colony's individual plant screens, and utilizes F 2The polymorphism result of colony's individual plant makes up genetic linkage maps, adopts mapping software Jionmap3.0 to carry out the mark linkage analysis; In conjunction with two cover F 2Colony and F 2:3Family fibrous quality measurement result is utilized QTL mapping software Windows QTL Cartographer2.5, selects for use the composite interval mapping method to carry out the QTL location.
- 3. screening method according to claim 2, it is characterized in that: in (3) step, at first, with SSR primer screening new land morning 24 and Shandong cotton grinding No. 28 parents' dna polymorphism, obtain polymorphism primer and pleomorphism site, utilize these primers that polymorphism is arranged to F 2Colony's individual plant screens, and utilizes F 2The polymorphism result of colony's individual plant makes up genetic linkage maps, and in conjunction with F 2And F 2:3The fibrous quality measurement result, the relevant QTL in location; Then, according to positioning result, select 4 pairs of SSR primers on the 6th linkage group that QTL assemble to occur, that is: DPL0757, DPL0852, DPL0920, BNL1694, to new land early 24 and cotton 516 parents' in Ji dna polymorphism analyze, obtaining 3 pairs has polymorphism primer namely: DPL0757, DPL0852, DPL0920 and with the mark DPL0757 of fibre strength main effect QTL linkage 130, DPL0852 170, DPL0920 130, and make up genetic linkage maps and Mapping of QTL.
- 4. screening method according to claim 3, it is characterized in that: with SSR primer screening new land morning 24 and Shandong cotton grinding No. 28 parents' dna polymorphism, the pcr amplification reaction system is 10 μ l systems, 10 * buffer, 1.0 μ l wherein, 10mM dNTPs 0.5 μ l, concentration is the Taq enzyme 0.1 μ l of 5U/ μ l, template DNA 1.2 μ l, ddH 2O 6.2 μ l, the forward primer 0.5 μ l of 10 μ M, the reverse primer 0.5 μ l of 10 μ M; PCR thermal response program is: 95 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30s, 55 ℃ of annealing 45s, 72 ℃ are extended 45s, circulate 30 times, and last 72 ℃ are extended 2min, 4 ℃ of preservations; Increase at TC-512 and the imperial EDC-810 amplification of east victory instrument, pcr amplification product is electrophoresis in 8% polyacrylate hydrogel, and silver dyes colour developing, the record result.
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| CN102586426B (en) * | 2012-01-12 | 2014-05-28 | 中国农业科学院棉花研究所 | SSR (Simple Sequence Repeat) molecular marking method for identifying variety authenticity and/ or variety purity of high-quality transgenic hybrid cotton CCRI (Chinese cotton research institute) 70 |
| CN103255139B (en) * | 2013-05-07 | 2015-04-15 | 南京农业大学 | Major QTL (Quantitative Trait Locus) of cotton high-strength fiber and molecular marker and application thereof |
| CN104745702B (en) * | 2014-06-23 | 2017-07-04 | 山东棉花研究中心 | EST SSR label primers and application based on the exploitation of upland cotton transcript profile sequence |
| CN105925668B (en) * | 2016-04-12 | 2019-10-29 | 江苏省农业科学院 | Method for rapidly positioning of the cotton unit point mass gene in chromosome |
| CN108085406A (en) * | 2017-12-13 | 2018-05-29 | 山东棉花研究中心 | A kind of identification method in upland cotton precocity molecular breeding correlation SSR marker site |
| CN107980618B (en) * | 2018-01-24 | 2020-05-01 | 山东棉花研究中心 | Molecular breeding method for improving strength of cotton fibers by using chr.7 single QTL (quantitative trait locus) fragment replacement line |
| CN109554492B (en) * | 2018-09-07 | 2021-06-15 | 中国农业科学院棉花研究所 | SNP molecular marker of fiber strength major gene from Xinluzao 24 and Lumian 28 |
| CN110777197A (en) * | 2019-08-02 | 2020-02-11 | 中国农业科学院棉花研究所 | Major QTL method for rapidly identifying cotton-related traits through compound BSA-seq |
| CN111206076B (en) * | 2020-02-26 | 2021-12-03 | 上海晶准生物医药有限公司 | Copy number variation universal verification method based on fragment analysis technology |
| CN112349351B (en) * | 2020-11-10 | 2022-10-11 | 邯郸市农业科学院 | Method for breeding ultra-early-maturing cotton germplasm with assistance of molecular markers |
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