CN1541514A - Molecular genetic linkage mapping method of cotton - Google Patents
Molecular genetic linkage mapping method of cotton Download PDFInfo
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Abstract
The present invention is linked inheritance mapping method utilizing SRAP molecular mark for cotton. The mapping population is hybridization between sea island cotton and upland cotton to generate F2 single plant. Applying SRAP molecular mark and PCR proliferation for detecting population polymorphism in inheritance mapping obtains 285 polymorphic bands in high yield. The 285 marks are used in constructing MAPMAKER linkage group, and 237 marks enter 39 linkage groups, each of which has 2-13 marks. All the linkage groups have the total length of 3030.7 cM, covers 65.4 % of the total cotton genome, and the average mark interval is 12.79 cM. The marks are distributed in the linkage groups relatively homogeneously. The specific molecule marking steps and electrophoresis parameters are also proposed.
Description
Technical field
The invention belongs to a kind of method of estimating the cotton linkage inheritance,, be specifically related to utilize sequence amplification polymorphism (SRAP) molecular labeling of be correlated with to carry out the invention of the method that the cotton molecular genetic linkage maps particularly about the method for cotton molecular genetic linkage mapping.
Background technology
Molecular genetic linkage figure makes up in various plants.The cotton molecular linkage maps starts from 1994, Reinish application restriction fragment length polymorphism (RFLP) mark has made up one and has comprised 705 pleomorphism sites, be distributed to the collection of illustrative plates of 41 linkage groups of different sizes, length overall 4675cM (Reinisch A J, Dong J M, Brubaker C L, StellyD M, Wendel J F, Paterson A H.A detailed RFLP map of cotton Gossypium hirsutum * Gossypiumbarbadense:chromosome organization and evolution in a disomic polyploid genome.Genetics, 1994,138:829-847).Shappley etc. utilize upland cotton * upland cotton F
2It is 865cM that colony's application restriction fragment length polymorphism (RFLP) mark has made up length overall, contain 120 marks, linkage map (the Shappley Z.W of covering gene group about 18.6%, Jenkins J.N., C.E.Meredith W R, McCarty, Jr J C.An RFLP linkage map ofUpland cotton, Gossypium hirsutum L.Theor Appl Genet, 1998,97:756～761).Application restriction fragment length polymorphism (RFLP) such as Jiang obtain containing 261 marks, 27 linkage groups, average headway 14.4cM, the linkage map of length overall 3767cM (Jiang CX, Wright R J, El-Zik K M, Paterson A H.Polyploid formation created uniqueavenues for response to selection in Gossypium (cotton) .Proc Natl Acad Sci, 1998,95:1419～1424).It is 700.7cM that Ulloa etc. have made up length overall, covering gene group about 15%, molecular labeling linkage map (the Ullia M that includes 81 restriction fragment length polymorphism (RFLP) pleomorphism site, Meredith Jr W R.Genetic linkage mapand QTL analysis of agronomic and fiber quality traits in an intraspecific population.The Journal ofCotton Science, 2000,4:161～170).A left side drives a well etc. and to utilize 152 F of nasal mucus continuous No. 3 (Bt) and Hubei Province chaste tree No. 1
2Individual plant obtains 67 pleomorphism sites (40 RFLP, 11 RAPD, 16 SSR sites), is distributed on 8 linkage groups, and collection of illustrative plates length is that (left side drives a well 1337.4cM, Sun Jizhong, Zhang Xianlong etc.Utilize RAPD, SSR and RFLP mark to make up upland cotton molecular labeling linkage map.Hua Zhong Agriculture University's journal, 2000,19 (3): 190～193).Two linkage groups and their new two collection of illustrative plates polymerizations mappings in addition that make up that Ulloa etc. utilize Shappley etc. (1998) and Ulloa and Meredith (2000) to make up, 47 linkage groups have been obtained, contain 284 sites, length overall 1502.6cM, but be marked at each linkage group skewness (UlloaM, Meredith W R Jr, Shappley Z W, Kahler A L.RFLP genetic linkage maops from F
2.3Populations and a joinmap of Gossypium hirsutum.Theor Appl Genet, 2002,104:200～208).ZhangJ cooperates the monoploid colony that produces 58 Gossypium hirsutum L. and Gossypium barbadense L. by half, being applied to linkage map makes up, 489 in 624 marks (510 SSR and 114 RAPD) enter 43 linkage groups, length overall 3314.5 cM (Zhang J, Guo W, Zhang T.Molecular linkage map of allotetraploid cotton (Gossypium hirsutum L. * Gossypium barbadense L.) with a haploid population.Theor ApplGenet, 2002,105:1166～1174).
For now, many molecular labeling linkage maps that cotton has made up, the dna marker of application mainly are that restriction fragment length polymorphism (RFLP), simple sequence repeat (SSR) and randomly amplified polymorphic DNA (RAPD).Restriction fragment length polymorphism (RFLP) mark has advantages such as codominance, information completely, repeatability and good stability, but the experimental implementation process of RFLP technology is complicated, is difficult for realizing automation, and to the requirement height of DNA, consumption is big, and the expense that large group is analyzed is big.Randomly amplified polymorphic DNA (RAPD) mark is few to the requirement of DNA, less demanding to DNA, and operation is simple, but the RAPD mark is subject to the experiment condition influence, poor repeatability, and productive rate is low.It is a kind of good mark that simple sequence repeats (SSR), but the SSR of cotton is limited, and developing SSR is very time-consuming very expensive.The molecular labeling linkage map density of cotton must increase mark and could satisfy the needs of using not enough, and existing mark must be used new mark to increasing the limited in one's ability of figure spectrum density.
Summary of the invention
The objective of the invention is to overcome the existing defective that cotton molecule linkage map makes up that is applied to, use the molecular labeling linkage map that SRAP makes up cotton, average each combination of primers can produce 3.75 polymorphic bandses, can produce 13 bands at most, have the productive rate height, increase the short characteristics of mark required time, can increase considerably the density of cotton molecular labeling linkage map.
The present invention is achieved through the following technical solutions:
A kind of method of estimating the cotton linkage inheritance, the particularly method of cotton molecular genetic linkage genetic mapping is characterized in that according to the following step:
1) be male parent with sea-island cotton strain Pima90, upland cotton kind Handan 208 is maternal, and the preparing hybrid combination is with the F that obtains
2Individual plant is as the starting material of molecular genetic linkage mapping, and described material is planted in the field;
2) from the material of described field, get described parent and F
2The light green blade of colony plant extracts its total DNA;
3) relevant amplification polymorphism (the Sequence-related amplified polymorphism SRAP) labeling method of application sequence, utilize polymerase chain reaction (PCR) (PCR) amplimer to detect the polymorphism of described colony and carry out genetic mapping, wherein said primer removes Li and Quiros (Li G, Quiros C F.Sequence-related amplified polymorphisim (SRAP), anew marker system based on a simole PCR reaction:its application to mapping and gene tagging inBrassica.Theor Appl Genet, 2001,103:455～461) outside Bao Dao the primer, designed primer in addition voluntarily, the numbering of this primer is from me6--m9, (upstream primer); Em7--e17 (downstream primer).The primer that the present invention designs voluntarily as shown in Figure 1;
4) dna fragmentation with step 3) amplification carries out polyacrylamide gel electrophoresis, and this method comprises being 30mA with 2000 volts of voltage prerunnings to electric current earlier, with 2500 volts of constant voltages electrophoresis 1.5～2h again, controls the offset plate temperature and be not higher than 50 ℃ in electrophoresis process behind the last sample;
5) gel film that step 4) is obtained carries out silver and dyes, and dyes the gel bottom during dyeing earlier, catches portion again after waiting to show band;
6) the shown banding pattern of attached gel sheet carries out genetic linkage evaluation and mapping.
Described in the present invention PCR reaction system is:
Dna profiling 60ng; Get each 1 of upstream and downstream primer in the described primer that relates at random, pairing is used, and each primer consumption is 30ng, and dNTPs is 200uM; 1 * Reaction Buffer; MgCl
2Be 1.5mM; Taq enzyme 1U, cumulative volume are 20ul, insufficient section ddH
2O replenishes.
Accompanying drawing and explanation thereof:
Fig. 1: be the SRAP molecular labeling primer that designs among the present invention;
Fig. 2: be that primer m3e14 of the present invention is at part F
2Amplification figure in the colony; Among the figure, M:100bp ladder; P
1: Handan 208; P
2: Pima90; Other is F
2The part individual plant; Arrow shows polymorphic bands.
Fig. 3: be cotton SRAP molecular labeling linkage map of the present invention;
The invention will be further described below in conjunction with specification and accompanying drawing:
1, for the examination material: take Pima90 (sea island cotton) as male parent, Handan 208 (upland cotton) is maternal, and the preparing hybrid combination obtains 129 F2Individual plant is as mapping population. The all material kind is in Hubei China province Wuhan City Hua Zhong Agriculture University experimental plot.
2, total DNA extracting method: from the material of described field, get described parent and F2The light green blade of colony plant extracts it Total DNA, concrete grammar was with reference to (Paterson A H, Curt L B, Wendel J F, the A rapid method such as Paterson in 1994 For extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP and PCR analysis.Plant Mol Bil Rep, 1993, the method for 11:112-127) delivering is extracted.
3, SRAP labeled analysis: (1) cotton SRAP labeled analysis: the PCR primer is except Li and Quiros (Li G, Quiros C F.Sequence-related amplified polymorphisim (SRAP), a new marker system based on a simole PCR reaction:its application to mapping and gene tagging in Brassica.Theor Appl Genet, 2001,103:455～461) outside the primer of delivering in the paper, designed, designed primer in addition, the numbering of this primer from Me6--m9, (upstream primer); Em7--e17 (downstream primer). Primer of the present invention is seen shown in the accompanying drawing 1. Afterwards with institute of the present invention It is synthetic that the primer of design is transferred to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd. The PCR reaction system is in the present invention: Dna profiling 60ng; Get at random each 1 of upstream and downstream primer in the described primer that relates to, pairing is used, each primer Consumption is that (consumption is with reference to the AFLP method: see document Vos P, Hogers R, Bleeker M, Reijans M, van de Lee for 30ng T, Homes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M.AFLP:a new technique for DNA Fingerprinting.Nucleic Acids Res, 1995,23:4407～4414); DNTPs is 200uM; 1 * Reaction Buffer; MgCl2Be 1.5mM; Taq enzyme 1U (available from MBI company), cumulative volume is 20ul, insufficient section ddH2O replenishes. The DNA cloning program is with reference to the method for Li and Quiros report, and amplified reaction carries out at PTC-100 PCR instrument. (2) electrophoresis: Electrophoresis apparatus is the EC-160 electrophoresis apparatus of U.S. life science company, and electrophoretic buffer is 0.5 * TBE. Add in the 20ul amplified production Enter 10ul Loading Buffer (98% deionized formamide, 10mM EDTA, 0.25% bromine powder orchid, 0.25% dimethylbenzene green grass or young crops), 95 ℃ sex change 5min, upper sample 5ul. Adopt 6%PAGE glue (7mol/L urea) to separate, 2000 volts of voltage prerunnings are to electric current Be 30mA, with 2500 volts of constant voltage electrophoresis 1.5～2h, guarantee in the electrophoresis process that the offset plate temperature is not higher than 50 ℃ behind the upper sample. (3) Silver dyes behind the electrophoresis, and silver staining method is with reference to method (Wu Guanyun, Pan Huazhen, the Wu Yu: biochemistry and molecular biosciences of Wu Guanyun etc. Learn experiment frequently-used data handbook, Beijing, Science Press, 1999). During dyeing, dye earlier the gel bottom, behind the band to be showed, again Catch section.
4, data preparation and linkage analysis: the record of molecular labeling adopts Mapmaker software (Lincoln S, Daly M, Lander E S.Construction genetic maps with MAPMAKER/EXP 3.0.In:Whitehead Institute Technical Report, 2nd ed.Cambridge:Whitehead Institute, 1992) recording method. For the codominant marker, with " A " table The banding pattern that isozygotys that shows Handan 208 parents; The banding pattern that isozygotys with " B " expression Pima90 parent; " H " expression two parents' heterozygosis Banding pattern. For the dominant marker, Handan 208 parents are the banding pattern that isozygotys, and are designated as " A "; Pima90 parent's isozygoty banding pattern and heterozygosis Banding pattern is designated as " C ". Pima90 parent's the banding pattern that isozygotys is designated as " B "; Handan 208 parents' isozygoty banding pattern and heterozygosis banding pattern are designated as " D ". Missing data represents with "-".
Adopt the method for " combination of primers+labelled molecule amount " that mark is named, such as m1e1-550, " m1e1 " represents primer Combination me1 and em1, " 550 " represent the molecular weight of this mark; " K " of combination of primers back represents the molecular weight of this mark Be 1000bp.
Utilize MAPMAKER/EXP.3.0 (Lincoln S, Daly M, Lander E S.Construction genetic maps with MAPMAKER/EXP 3.0.In:Whitehead Institute Technical Report.2nd ed.Cambridge:Whitehead Institute, 1992) be mapping software, carry out linkage analysis (LOD=3.0, r=0.4) between mark with Group and Order order earlier, The mark that does not enter linkage group is linked on the corresponding linkage group with the Try order, is determined the optimal arrangement order with the Ripple order at last, Make up the cotton Linkage mapl. Utilize the Kasambi function that recombination fraction is converted to genetic distance (cM).
Effect of the present invention:
1, the cotton Linkage mapl that utilizes SRAP to make up is marked in the linkage map and is evenly distributed, and has overcome the company that has delivered Lock icon is remembered shortcoming pockety.
2, mark productive rate height, on average each combination of primers can produce 3.75 polymorphic bandses, can produce at most 13 polymorphism bars Band is all higher than the productive rate of other mark that is applied to cotton Genetic Linkage Map structure.
3, the electrophoretic process that adopts of the present invention, thus guarantee result's accuracy, repeatability and high-resolution.
Embodiment
Embodiment 1:
1, be male parent with sea-island cotton strain Pima90, upland cotton strain Handan 208 is maternal, and the preparing hybrid combination is with the F that obtains
2Individual plant is as the starting material of genetic linkage mapping, and described material is planted in the field;
2, from the material of described field, get described parent and F
2The light green blade of individual plant colony plant extracts its total DNA;
3, utilize the SRAP mark to make up cotton molecular labeling linkage map, obtain 39 linkage groups that contain 237 sites, length overall 3030.7cM covers 65.4% of whole genome, average headway 12.79cM between mark.This is a first cotton molecular genetic linkage collection of illustrative plates that makes up with SRAP.Concrete steps are as follows:
3.1 SRAP labeled analysis:
SRAP analyzes: the PCR primer removes Li and Quiros (Li G, Quiros C F.Sequence-related amplifiedpolymorphisim (SRAP), a new marker system based on a simole PCR reaction:its application tomapping and gene tagging in Brassica.Theor Appl Genet, 2001,103:455～461) outside the primer of delivering in the paper, (see shown in the accompanying drawing 1: upstream primer is numbered from me6--m9 to design primer voluntarily in addition; The downstream primer numbering is from em7--e17, and it is synthetic that the primer of the present invention's design is transferred to Shanghai biotechnology Services Co., Ltd.The PCR reaction system is: dna profiling 60ng; Get each 1 of upstream and downstream primer in the described primer that relates at random, pairing is used, and each primer consumption is 30ng; [(document is seen Vos P to consumption, Hogers R, Bleeker M with reference to the AFLP method of operating, Reijans M, van de Lee T, HomesM, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M.AFLP:a new technique for DNAfingerprinting.Nucleic Acids Res, 1995,23:4407～4414)].DNTPs is 200uM; 1 * ReactionBuffer; MgCl
2Be 1.5mM; Taq enzyme 1U (available from MBI company), cumulative volume is 20ul, insufficient section ddH
2O replenishes.The DNA cloning program is with reference to Li and Quiros (Li G, Quiros C F.Sequence-related amplified polymorphisim (SRAP), a new marker system based on a simole PCR reaction:its application to mapping and genetagging in Brassica.Theor Appl Genet, 2001,103:455～461) method, amplified reaction carries out on PTC-100 PCR instrument.(2) electrophoresis: electrophoresis apparatus is the EC-160 electrophoresis apparatus of U.S. life science company, and electrophoretic buffer is 0.5 * TBE.Add 10ul Loading Buffer (98% deionized formamide, 10mM EDTA, 0.25% bromine powder orchid, 0.25% dimethylbenzene green grass or young crops), 95 ℃ of sex change 5min, last sample 5ul in the 20ul amplified production.Adopt 6%PAGE glue (7mol/L urea) to separate, 2000 volts of voltage prerunning to electric currents are 30mA, with 2500 volts of constant voltage electrophoresis 1.5～2h, guarantee in the electrophoresis process that the offset plate temperature is not higher than 50 ℃ behind the last sample.(3) silver dyes behind the electrophoresis, and silver staining method is with reference to method (Wu Guanyun, Pan Huazhen, the Wu Yu of Wu Guanyun etc.Biochemistry and Molecular Biology experiment frequently-used data handbook.Beijing: Science Press, 1999).
3.2 data preparation and linkage analysis:
The record of molecular labeling adopts Mapmaker software records method.For the codominant marker, with " A " expression Handan 208 parents' the banding pattern that isozygotys; The banding pattern that isozygotys with " B " expression Pima90 parent; " H " expression two parents' heterozygosis banding pattern.For the dominant marker, Handan 208 parents are the banding pattern that isozygotys, and are designated as " A "; Pima90 parent's isozygoty banding pattern and heterozygosis banding pattern are designated as " C ".Pima90 parent's the banding pattern that isozygotys is designated as " B "; Handan 208 parents' isozygoty banding pattern and heterozygosis banding pattern are designated as " D ".Missing data is represented with "-".
Adopt the method for " combination of primers+labelled molecule amount " that mark is named, as m1e1-550, " m1e1 " expression combination of primers me1 and em1, " 550 " represent the molecular weight of this mark; " K " of combination of primers back represents that the molecular weight of this mark is 1000bp.
Utilize the MAPMAKER/EXP.3.0 mapping software, order with Group and Order earlier and carry out linkage analysis (LOD=3.0 between mark, r=0.4), the mark that does not enter linkage group is linked on the corresponding linkage group with the Try order, determine the optimal arrangement order with the Ripple order at last, make up cotton molecular labeling linkage map.Utilize the Kasambi function that recombination fraction is converted to genetic distance (cM).
3.3 the screening of polymorphic dna mark between the parent:
Two parents are screened with 136 combination of primers, and each combination can produce 50～100 clear and legible bands.76 good combination of primers are carried out population analysis to choose polymorphism, obtain 285 polymorphic bandses altogether, and the polymorphic bands number of every combination does not wait from 1～13.Average each combination of primers produces 3.75 polymorphic bandses (seeing shown in the accompanying drawing 2)
3.4 cotton SRAP molecular linkage maps
285 polymorphism marks that obtain are made up genetic linkage map with MAPMAKER/EXP3.0.237 marks enter 39 linkage groups (LOD 〉=3.0), 48 independences, length overall 3030.7cM, 65.4% (see figure 3) of covering whole genome.Each linkage group has 2～13 marks, and the longest linkage group is 227.4cM, and the shortest linkage group is 5cM.Maximum spacing is 42.8cM between mark, and minimum spacing is 0.2cM, average headway 12.79cM between mark.
Claims (2)
1, a kind of method of estimating the cotton linkage inheritance, the particularly method of cotton molecular genetic linkage mapping is characterized in that according to the following step:
1) be male parent with sea-island cotton strain Pima90, upland cotton kind Handan 208 is maternal, and the preparing hybrid combination is with the F that obtains
2Individual plant is as the starting material of genetic linkage mapping, and described material is planted in the field;
2) from the material of described field, get described parent and F
2The light green blade of colony plant extracts its total DNA;
3) relevant amplification polymorphism (the Sequence-related amplified polymorphism SRAP) molecular labeling of application sequence, polymerase chain reaction (PCR) (PCR) amplimer detects the polymorphism of described colony and carries out genetic mapping, wherein said primer sequence as shown in Figure 1:
4) dna fragmentation with step 3) amplification carries out polyacrylamide gel electrophoresis, and this method comprises being 30mA with 2000 volts of voltage prerunnings to electric current earlier, with 2500 volts of constant voltages electrophoresis 1.5～2h again, controls the offset plate temperature and be not higher than 50 ℃ in electrophoresis process behind the last sample;
5) gel film that step 4) is obtained carries out silver and dyes, and dyes the gel bottom during dyeing earlier, catches portion again after waiting to show band;
6) the shown banding pattern of attached gel sheet carries out genetic linkage evaluation and mapping.
2, the method, particularly cotton molecular genetic linkage of evaluation cotton linkage inheritance according to claim 1 pass the method for mapping, and it is characterized in that: described PCR reaction system is:
Dna profiling 60ng; Get each 1 of upstream and downstream primer in the described primer that relates at random, pairing is used, and each primer consumption is 30ng, and dNTPs is 200uM; 1 * Reaction Buffer; MgCl
2Be 1.5mM; Taq enzyme 1U, cumulative volume are 20ul, insufficient section ddH
2O replenishes.
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Cited By (5)
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| CN101880714A (en) * | 2010-06-04 | 2010-11-10 | 华中农业大学 | Method for building cotton fiber transcription genetic linkage map by EST-SSR sign |
| CN102994499A (en) * | 2013-01-05 | 2013-03-27 | 四川农业大学 | Ecotypic SRAP (Sequence Related Amplified Polymorphism) specificity molecular marker primer for determining switchgrass and ecotypic detection method of switchgrass |
| CN103276100A (en) * | 2013-06-19 | 2013-09-04 | 江苏省农业科学院 | Creation method of cotton fiber length single QTL near-isogenic line |
| CN107099599A (en) * | 2017-05-26 | 2017-08-29 | 红河学院 | A kind of tsaoko Genetic Diversity of Germplasm analysis method marked based on SRAP |
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| CN101880714A (en) * | 2010-06-04 | 2010-11-10 | 华中农业大学 | Method for building cotton fiber transcription genetic linkage map by EST-SSR sign |
| CN102994499A (en) * | 2013-01-05 | 2013-03-27 | 四川农业大学 | Ecotypic SRAP (Sequence Related Amplified Polymorphism) specificity molecular marker primer for determining switchgrass and ecotypic detection method of switchgrass |
| CN103276100A (en) * | 2013-06-19 | 2013-09-04 | 江苏省农业科学院 | Creation method of cotton fiber length single QTL near-isogenic line |
| 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 |
| CN107099599A (en) * | 2017-05-26 | 2017-08-29 | 红河学院 | A kind of tsaoko Genetic Diversity of Germplasm analysis method marked based on SRAP |
| CN107099599B (en) * | 2017-05-26 | 2021-02-23 | 红河学院 | SRAP marker-based tsaoko germplasm resource genetic diversity analysis method |
| CN107365861A (en) * | 2017-08-28 | 2017-11-21 | 华中农业大学 | A kind of molecular labeling for differentiating dark-brown cotton |
| CN107365861B (en) * | 2017-08-28 | 2020-06-26 | 华中农业大学 | Molecular marker for identifying dark brown cotton |
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