CN103981178A - Cotton fiber length-associated major gene locus linked SSR (signal sequence receptor) molecular marker and application thereof - Google Patents
Cotton fiber length-associated major gene locus linked SSR (signal sequence receptor) molecular marker and application thereof Download PDFInfo
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Abstract
The invention relates to a cotton fiber length-associated major gene locus linked SSR (signal sequence receptor) molecular marker and an application thereof. The SSR molecular maker can be as follows: an SSR molecular marker linked with cotton fiber length-associated major gene locus qFL-19-1 and qFL-19-2 on a 19# chromosome of luyuan 343 of a cotton high-quality fiber product, wherein the qFL-19-1 and the qFL-19-2 are on a Chr.19# chromosome; an SSR molecular marker HAU1185 linked with qFL-19-1, an SSR molecular marker DPL0595, an SSR molecular marker GH109, an SSR molecular marker DPL0556 linked with qFL-19-2, and an SSR molecular marker CGR5582. The molecular marker disclosed by the invention has important application value for assisting polymerized cotton fiber quality improvement, even introgression excellent fiber major QTL (quantitative trait locus) candidate gene map-based cloning.
Description
Technical field
The present invention relates to the chain SSR molecule marker of major gene loci and application that a kind of cotton fiber length is relevant, be particularly related to SSR (Simple Sequence Repeats) mark upper with the Chr.19 of cotton high-quality filamentary material Shandong former 343 and that staple length is chain, belong to biotechnology applications technical field.
Background technology
Cotton is important cash crop, occupies very consequence in national economy.High yield, high-quality, disease-resistant be the important goal of cotton breeding always.Along with the development of textile technology, the requirement of fibrous quality is also being improved constantly.Nearly ten years, along with the development of molecular marking technique, perfect, and the development and utilization of a series of mapping softwares, breakthrough has been obtained in cotton genetic map construction and important character QTL location.For gene clone and the molecular mark of Main Agronomic Characters and fibrous quality proterties lay a solid foundation.
There are some researches show, lack excellent fiber gene resource in upland cotton gene pool, intravarietal crossing is difficult to realize effective fibrous quality improvement.Studies confirm that of a large amount of distant hybirdization both at home and abroad, the nearly edge cultivar of Gossypium inland basin cotton can be served as the GENE SOURCES of improvement upland cotton fiber quality as plucked instrument Bai Shi cotton (G.thurberi.), extremely cotton (G.armourianum) etc. as sea island cotton (Gossypium barbadense), nearly edge wild species, and create a collection of fibrous quality and had the distant hybirdization intermediate materials increasing substantially, owing to containing the excellent fiber hereditary component of external source, we these materials are referred to as to excellent fiber introgressive line or gradually ooze be).The genetic background of these materials is upland cotton, can be for the improvement of fibrous quality, but in actual breeding utilizes, often in the excellent successful while of fiber gene transformation, there is the genetic locus of negative impact to be also carried into (Linkage drag) to proterties such as output in a large number, this is also to cause cotton fiber quality Improvement advance major reason slowly, only relies on conventional breeding method to be difficult to realize the synchronous improvement of fibrous quality and output.
In recent years, molecular marking technique development rapidly, utilizes the auxiliary pyramiding breeding of molecule marker to improve new breeding method is provided for cotton fiber quality, and the QTL Position Research of cotton fiber quality has obtained greater advance.Yu etc. (1998) are at upland cotton and sea island cotton hybrid F
2in colony, utilize tri-kinds of molecular marking techniques of RFLP, RAPD and SSR the QTLs of 3 staple lengths to be detected; Shappley etc. (1998) detect the QTLs of 7 staple lengths in Lu Lu hybrid Population; Ulloa etc. (2000) utilize Lu Lu hybrid Population, with RFLP Marker Identification 2 QTLs that staple length is relevant; Kohel etc. (2001) utilize sea, land F
2crowd surveillance is to 3 staple length QTLs; Ren Lihua etc. (2002) are replacing the upland cotton substitution line F of sea island cotton dyad
2:3in family, find on No. 16 karyomit(e)s, there be QTLs2 of staple length; Wu Mao waits clearly (2003) with upland cotton and sea island cotton hybrid F
2colony is mapping population, 2 staple length QTLs detected; Paterson etc. (2003) utilize sea, land F
2and F
2:3colony has identified the QTLs of 6 staple lengths; Lin etc. (2005) utilize sea, land F
2colony adopts SRAP, SSR, tri-kinds of molecule markers of RAPD to build genetic linkage spectrum, the QTLs of 4 staple lengths detected, explains 17.14%～27.52% phenotypic variation; Shen etc. (2005) utilize three upland cotton combination F
2and F
2:3colony, detects the QTLs of 11 staple lengths; James etc. (2006) utilize recombinant inbred lines (Recombined Inbred Lines, RIL) QTLs of 10 staple lengths to be detected, can explain 5.7%～9.4% phenotypic variation; Wang etc. (2006) utilize SSR molecular marking technique, 48 QTLs detected in RIL colony, wherein, control 4 of staple length; Wang Juan etc. (2007) utilize Lu Lu F
2and F
2:3the QTL of crowd surveillance to 1 staple length, can explain 6.1% phenotypic variation.To locate colony used be elementary target group to cotton fiber quality QTL in the past, and have not been reported about the molecule marking research relevant with staple length on No. 19 karyomit(e)s of cotton.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, the chain SSR molecule marker of major gene loci and application that a kind of cotton fiber length is relevant are provided.The present invention derives from the molecule marker that Chr.19 is upper and cotton fiber length is chain of Shandong former 343 by screening, be applied to the assisted Selection in cotton fiber quality pyramiding breeding, improves polymerization efficiency.
Technical scheme of the present invention is as follows:
A detection primer of the chain SSR molecule marker DPL0556 of the major gene loci relevant to cotton fiber length, it is a pair of detecting primer, and upstream primer nucleotide sequence is as shown in SEQ ID NO.1, and downstream primer nucleotide sequence is as shown in SEQ ID NO.2.
A detection primer of the chain SSR molecule marker CGR5582 of the major gene loci relevant to cotton fiber length, it is a pair of detecting primer, and upstream primer nucleotide sequence is as shown in SEQ ID NO.3, and downstream primer nucleotide sequence is as shown in SEQ ID NO.4.
A detection primer of the chain SSR molecule marker HAU1185 of the major gene loci relevant to cotton fiber length, it is a pair of detecting primer, and upstream primer nucleotide sequence is as shown in SEQ ID NO.5, and downstream primer nucleotide sequence is as shown in SEQ ID NO.6.
A detection primer of the chain SSR molecule marker DPL0595 of the major gene loci relevant to cotton fiber length, it is a pair of detecting primer, and upstream primer nucleotide sequence is as shown in SEQ ID NO.7, and downstream primer nucleotide sequence is as shown in SEQ ID NO.8.
A detection primer of the chain SSR molecule marker GH109 of the major gene loci relevant to cotton fiber length, it is a pair of detecting primer, and upstream primer nucleotide sequence is as shown in SEQ ID NO.9, and downstream primer nucleotide sequence is as shown in SEQ ID NO.10.
With on No. 19 karyomit(e)s of cotton high-quality fiber strain Shandong former 343 with staple length the chain SSR molecule marker of relevant major gene loci qFL-19-1, qFL-19-2, qFL-19-1, qFL-19-2 are all positioned on Chr.19 karyomit(e), with qFL-19-1 chain be labeled as SSR molecule marker HAU1185, SSR molecule marker DPL0595, SSR molecule marker GH109, with qFL-19-2 chain be labeled as SSR molecule marker DPL0556, SSR molecule marker CGR5582.
In the present invention, QTL name is the naming rule in paddy rice with reference to McCouch etc., form with q+ proterties (english abbreviation)+linkage group code name+QTL number represents (reference: McCouch SR, Cho YG, Yano M, et al.Report on QTL nomenclature, Rice Genet Newslett, 1997,14:11-13).
On No. 19 karyomit(e)s of Shandong former 343, a screening method for the chain SSR mark of major gene loci qFL-19-1, the qFL-19-2 relevant with cotton fiber length, comprises the steps:
(1) former 343 grind 22 hybridization selfing continuously afterwards as male parent and Shandong cotton taking Shandong, obtaining secondary gradually the oozing of high-quality is R497;
(1) utilize that high-quality is secondary gradually oozes that to be R497 grind No. 22 for male parent and Shandong cotton backcrosses, build F
2and F
2:3colony;
(2) gradually ooze so that high-quality is secondary that to be R497 grind 22 as male parent and transgenic cotton against pests Shandong cotton backcrosses and obtain F
1, obtain the secondary F of target group through selfing
2seed; The secondary F of target group
2plant selfing, results F
2:3seed, the plantation F of backcross progeny colony
2:3family;
(3) extract parent and F
2the genomic dna of colony;
(4) utilize the high staple length mark of SSR molecular marker screening cotton, obtain parent R497 and Shandong cotton is ground the polymorphism primer between No. 22, the F that utilizes polymorphism primer to extract step (3)
2the genomic dna of colony increases, and builds linkage map, in conjunction with utilizing F
2linkage map and F that colony builds
2:3family fibrous quality measurement result, carries out QTL location to fibrous quality proterties.
Preferred according to the present invention, in described method steps (4), utilize polymorphism primer to F
2the reaction system that colony increases is 10 μ l, specific as follows:
Ultrapure water (ddH
2o) 6.5 μ 1, F
2genomic dna 1 μ 1,10 × Buffer1 μ 1 of colony, the dNTP0.2 μ 1 that concentration is 10mM, concentration is upstream primer 0.6 μ 1 of 10 μ M, concentration is downstream primer 0.6 μ 1 of 10 μ M, TaqDNA polysaccharase 0.1 μ 1;
Amplified reaction program:
95 DEG C of denaturation 45s; 94 DEG C of sex change 30s, 52～57 DEG C of annealing 45s; 72 DEG C are extended 1min, circulate 33 times; 72 DEG C are extended 2min, preserve 4 DEG C to taking out; Utilize 8% native polyacrylamide gel electrophoresis, record result.
According to QTL positioning result, qFL-19-1 can detect under two environment, is positioned between No. 19 chromosomal HAU1185--GH109 mark zones, and synergy gene is former 343 from parent Shandong, and the phenotypic variation of explanation is 4.31%--5.54%.With qFL-19-1 chain be labeled as HAU1185, DPL0595, GH109; Another staple length main effect QTL, qFL-19-2 is positioned between the DPL0556--DPL0595 mark zone of Chr.19, synergy gene is former 343 from parent Shandong, the phenotypic variation of explanation is 10.77%, with the linked marker of qFL-19-2 be DPL0556, CGR5582.Only in an environment, can detect to there is environment specificity.
Beneficial effect
It is the derivative R497 of being (the staple length 35.47mm of Shandong former 343 that the present invention is gradually oozed with sea island cotton, fiber specific tenacity 39.30cN/Tex, mic value 4.01) be parent, grinding No. 22 with Shandong cotton backcrosses, build secondary mapping population, further dissect the molecule marker relevant to fibrous quality in Shandong former 343, obtain with on the Chr.19 karyomit(e) of cotton high-quality filamentary material Shandong former 343 with staple length relevant major gene qFL-19-1, the SSR mark that qFL-19-2 is chain, the auxiliary polymerization cotton fiber quality of molecule marker is improved and even gradually oozed excellent fiber main effect QTL candidate gene map based cloning and there is important using value.
Brief description of the drawings
Fig. 1 is the chain and staple length QTL site plan of the molecule marker of the Chr.19 of F2 colony of the present invention (R497 × Shandong cotton is ground No. 22).
Embodiment
Detailed description below by embodiment is further illustrated the present invention, but is not limitation of the present invention, only does example explanation.
Embodiment 1
The screening method that is present in the chain SSR mark of major gene loci qFL-19-1, qFL-19-2 relevant with cotton fiber length on No. 19 karyomit(e)s of Shandong former 343 of the present invention is as follows:
(1) gradually oozing with high-quality fiber is that Shandong former 343 and high yield, disease-resistant transgenic Insect Resistant Cotton kind Shandong cotton are ground No. 22 and hybridize as parent, F
1carry out strict selfing, obtain F
2seed, builds recombinant inbred lines with single seed descent, and each generation is all carried out strict selfing to F therebetween
8in generation, utilizing Shandong former 343 and Shandong cotton to grind between 22 has polymorphic SSR primer to scan F
8for the genotype of recombinant inbred lines, in conjunction with phenotypic character, pick out the R497 that carries the former 343 chromosome dyad sections in Shandong and fibrous quality excellence as the parent who builds secondary group.
2011 in Linqing testing station of Shandong Cotton Research Center taking high-quality secondary gradually ooze be R497 and transgenic cotton against pests Shandong cotton grind 22 as parent backcross obtain F
1, add for selfing winter in Hainan Island, obtains the secondary F of target group
2seed.2012, at Linqing, Shandong plantation F
2, the whole selfings of colony's individual plant, every strain harvesting time is collected cotton sample and is measured for fibrous quality.Results selfing bell is for succeeding year plantation F
2:3colony.2013, respectively at Linqing, Shandong and Changsha plantation F
2:3family, investigates fibrous quality data, and the qualitative data of comprehensive 2 years is carried out QTL and accurately located.
The secondary target group of plantation in (2) 2012 years, 2013, gathers cotton sample in the term of opening bolls and measures fibrous quality, F
2measure fibrous quality, F according to individual plant
2:3collect 50 bells and carry out fibrous quality mensuration as species test sample, obtain fibrous quality phenotypic data.Fibrous quality utilizes HVI900 (HVICC calibrated horizontal) to measure.
(3) F
2colony's (R497 × Shandong cotton is ground No. 22) individual plant DNA extraction adopts CTAB method (Paterson et al., 1993).
(4) screening of polymorphism SSR mark: utilize the Shandong cotton that filters out grind No. 22 and Shandong former 343 between have 334 pairs of polymorphic primers, screen secondary mapping population parent R497 and Shandong cotton and grind the DNA polymorphism of No. 22, obtain altogether 105 pairs of polymorphism primers.The primer of numbering taking NAU in these primers is Agricultural University Of Nanjing's exploitation, and totally 4 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_NAU.xls); Primer taking BNL numbering is developed as U.S.'s BNL, and totally 9 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_date/BNL_ PRIMER_marker_in fo.xls); The exploitation taking the primer of the numberings such as CGR, DPL, SHIN, DC as Monsanto Company, totally 59 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_MON.xls); The primer of numbering taking HAU is Hua Zhong Agriculture University's exploitation, and totally 24 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_HAU.xls); The primer of numbering taking JESPR, GH is the exploitation of the agro-industrial university of texas,U.S, and totally 4 to (primer source: 1. http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_JESPR.xls; 2. http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_GH.xls); Primer taking CIR numbering is developed as International Agriculture researchdevelopment center, and totally 3 to (primer source: 1. http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_CIR.xls; 2. Nguyen TB, Giband M, Brottier P, et al., Wide coverage of the tetraloid cotton genome using newly developed microsatellite markers.Theor Appl Genet, 2004,109:167-175); Primer taking TMB numbering is affixed one's name to exploitation as USDA farming research service, and totally 2 to (primer source: http://www.cottonmarker.org/cmd_downloads/ssr_project_date/CMD_ PRIMER_TMB.xls).
(5) the parent R497 and the Shandong cotton that utilize screening to obtain are ground the polymorphism primer between No. 22, to F
2colony increases, and builds linkage map.In conjunction with F
2colony and F
2:3family fibrous quality measurement result, carries out QTL location to fibrous quality proterties.PCR reaction system is 10 μ l, wherein ultrapure water (ddH
2o) 6.5 μ 1, template DNA 1 μ 1,10 × Buffer1 μ 1, concentration is the dNTP0.2 μ 1 of 10mM, concentration is forward primer (Forward primer) 0.6 μ 1 of 10 μ M, reverse primer (Reverse primer) 0.6 μ 1, Taq archaeal dna polymerase 0.1 μ 1.SSR amplified reaction program: 95 DEG C of 45s of preheating; 94 DEG C of 30s of sex change; 52～57 DEG C of 45s anneal; Extend 72 DEG C of 1min; Circulate 33 times; Extend 72 DEG C of 2min; Preserve 4 DEG C to taking out.Utilize 8% native polyacrylamide gel electrophoresis, silver dyes colour developing, records result.
The 5 pairs of characteristic primer sequences and amplified fragments size, as shown in table 1:
Table 1
| Mark | Forward primer sequence | Reverse primer sequence | Amplified fragments size (bp) |
| DPL0556 | CTCAAGGAGGCCTTTCACTAAATA | AGGATCTCTAGCATTCTGATTTGC | 324 |
| CGR5582 | GCTACTCCCGTAAGTGCTGC | CCACCCTGTTTATTTCAACCC | 254 |
| HAU1185 | ATTGTGATGAGCAGGGATCT | GCATGATGGATTGATCAGAA | 181 |
| DPL0595 | GTCTATCTTATCCCACGAGAACCA | CTACTCCCGTAAGTGCTGCTG | 255 |
| GH109 | CAAGAAGGAAATGGCTGAATTG | CAGACACCAGCTGTTGCC | 126 |
(6) use mapping software JoinMap4.0 to carry out mark linkage analysis, utilize Kosambi function, LOD=6.5 builds genetic linkage maps (referring to Fig. 1).
(7) method of QTL location: utilize software Windows QTL Cartographer2.5, in conjunction with utilizing F
2linkage map, F that colony builds
2colony and F
2:3family fibrous quality measurement result, carries out QTL location to fibrous quality proterties.Obtain two QTLss relevant with cotton fiber length that are positioned at karyomit(e) Chr.19, qFL-19-1 and qFL-19-2, as shown in Figure 1.QFL-19-1 is positioned between the HAU1185-GH109 mark zone of karyomit(e) Chr.19, peak value is positioned at 121.1cM place, the phenotypic variation of explaining is 4.31%-5.54%, synergy gene is from parent Shandong former 343, with this QTL site chain be labeled as HAU1185, DPL0595 and GH109, apart from this QTL site nearest be labeled as DPL0595, genetic distance is 3cM; QFL-19-2 is positioned between the DPL0556--DPL05582 mark zone of karyomit(e) Chr.19, and peak value is positioned at 91.4cM place, and the phenotypic variation of explanation is 10.77%, and synergy gene is from parent Shandong former 343.With this QTL chain be labeled as DPL0556 and CGR5582, apart from this QTL site nearest be labeled as CGR5582, genetic distance is 22.2cM.
Claims (2)
- With on No. 19 karyomit(e)s of cotton high-quality fiber strain Shandong former 343 with staple length relevant major gene loci qFL-19-1, qFL-19-2chain SSR molecule marker, qFL-19-1, qFL-19-2all be positioned on Chr.19 karyomit(e), with qFL-19-1the chain SSR molecule marker HAU1185 that is labeled as, SSR molecule marker DPL0595, SSR molecule marker GH109, with qFL-19-2chain SSR molecule marker DPL0556, the SSR molecule marker CGR5582 of being labeled as.
- 2. the major gene loci relevant with cotton fiber length on No. 19 karyomit(e)s of a Shandong former 343 qFL-19-1, qFL-19-2the screening method of chain SSR mark, comprises the steps:(1) former 343 grind 22 hybridization selfing continuously afterwards as male parent and Shandong cotton taking Shandong, obtaining secondary gradually the oozing of high-quality is R497;?(2) utilize that high-quality is secondary gradually oozes that to be R497 grind No. 22 for male parent and Shandong cotton backcrosses, build F 2and F 2:3colony;?(3) gradually ooze so that high-quality is secondary that to be R497 grind 22 as male parent and transgenic cotton against pests Shandong cotton backcrosses and obtain F 1, obtain the secondary F of target group through selfing 2seed; The secondary F of target group 2plant selfing, results F 2:3seed, the plantation F of backcross progeny colony 2:3family;?(4) extract parent and F 2the genomic dna of colony;(5) utilize the high staple length mark of SSR molecular marker screening cotton, obtain parent R497 and Shandong cotton is ground the polymorphism primer between No. 22, the F that utilizes polymorphism primer to extract step (4) 2the genomic dna of colony increases, and builds linkage map, in conjunction with utilizing F 2linkage map and F that colony builds 2:3family fibrous quality measurement result, carries out QTL location to fibrous quality proterties.3 .method as claimed in claim 2, is characterized in that, described step utilizes polymorphism primer to F in (5) 2colony increases, and reaction system is 10 μ l, specific as follows:DdH 20 6.5 μ 1, F 2genomic dna 1 μ 1,10 × Buffer1 μ 1 of colony, the dNTP0.2 μ 1 that concentration is 10mM, concentration is upstream primer 0.6 μ 1 of 10 μ M, concentration is downstream primer 0.6 μ 1 of 10 μ M, Taq DNA polysaccharase 0.1 μ 1;Amplified reaction program:95 DEG C of denaturation 45 s; 94 DEG C of sex change 30 s, 52～57 DEG C of annealing 45 s; 72 DEG C are extended 1 min, circulate 33 times; 72 DEG C are extended 2min.
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| CN105734139A (en) * | 2016-03-29 | 2016-07-06 | 江苏省农业科学院 | Molecular marker in linkage with cotton verticillium-wilt disease-resistant main-effect QTLvw2 loci and application thereof |
| CN108184652A (en) * | 2018-01-24 | 2018-06-22 | 山东棉花研究中心 | A kind of molecular breeding method using the mono- QTL segments substitution line improvement cotton fiber lengths of chr.19 |
| CN111793714A (en) * | 2020-08-06 | 2020-10-20 | 南通大学 | Yellow-brown cotton fiber length related QTL and application thereof |
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2014
- 2014-05-26 CN CN201410225898.2A patent/CN103981178A/en active Pending
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200053A (en) * | 2015-11-04 | 2015-12-30 | 中国农业科学院棉花研究所 | Molecular markers derived from sea island cotton Hai 1 and associated with fiber length and applications of molecular markers |
| CN105200053B (en) * | 2015-11-04 | 2018-08-21 | 中国农业科学院棉花研究所 | From extra large 1 molecular labeling related with fibre length of sea island cotton and its application |
| CN105734139A (en) * | 2016-03-29 | 2016-07-06 | 江苏省农业科学院 | Molecular marker in linkage with cotton verticillium-wilt disease-resistant main-effect QTLvw2 loci and application thereof |
| CN105734139B (en) * | 2016-03-29 | 2019-03-29 | 江苏省农业科学院 | Chain molecular labeling and its application with the site verticillium wilt resistance of cotton by same main effect QTL vw2 |
| CN108184652A (en) * | 2018-01-24 | 2018-06-22 | 山东棉花研究中心 | A kind of molecular breeding method using the mono- QTL segments substitution line improvement cotton fiber lengths of chr.19 |
| CN111793714A (en) * | 2020-08-06 | 2020-10-20 | 南通大学 | Yellow-brown cotton fiber length related QTL and application thereof |
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Application publication date: 20140813 |
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