CN101880714A - Method for building cotton fiber transcription genetic linkage map by EST-SSR sign - Google Patents
Method for building cotton fiber transcription genetic linkage map by EST-SSR sign Download PDFInfo
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Abstract
The invention belongs to the technical field of cotton molecule breeding, particularly relating to a method for building a cotton fiber transcription genetic linkage map by EST-SSR signs. The method comprises the following steps: taking gossypium barbadense Pima 3-79 as a male parent and Gossypium hirsutum Emian 22 as a female parent, and hybridizing to obtain F1; planting F1, and causing F1 to be inbreeded to obtain F2; taking the F2 single plant as a starting material for the fiber transcription map to be plotted; extracting the RNA of the fiber of the field F2 single plant in 5 days after the single plant blooms, and carrying out reverse transcription of the RNA into cDNA to be served as a plotting group of the transcription map; utilizing the reported EST-SSR primer and the primer shown by a self-designed sequence table SEQ ID NO:1-90 to carry out the plotting group analysis to the F2 single plant by a denatured polyacrylamide gel electrophoretic analysis; and finally, analyzing data by MAPMAKER/ EXP.3.0 mapping software, and manufacturing and obtaining the transcription map. Compared with the existing method, the method of the invention is simple and practical, has accurate QTL positioning and is convenient for cloning genes relevant to cotton fiber development.
Description
Technical field
The invention belongs to cotton molecular breeding technical field.Be specifically related to utilize the EST-SSR mark to make up cotton fiber and transcribe the genetic linkage map spectral method.
Background technology
Molecular genetic linkage figure makes up in various plants.The cotton molecular linkage maps starts from 1994.Make up one from Reinish application limitations fragment length polymorphism (RFLP) mark in 1994 and comprised 705 pleomorphism sites, behind the cotton genetic linkage map of length overall 4675cM, many investigators (Rong, J., C.Abbey, et al, 2004; Nguyen, T., M.Giband, et al, 2004; He, D., Z.Lin, et al, 2007; Guo, W., C.Cai, et al, 2007; Zhang, Y., Z.Lin, et al, 2008) also utilize different marking methods to make up the genetic linkage map of many cottons.
But mark in the past all carries out on dna level, and its limitation is arranged: at first, genetic linkage map in the past adopts the mark of stochastic distribution, the QTL of location fiber trait, and different investigators obtain the result does not almost have anastomose property; Secondly, biological RNA information content is far longer than the information content of its DNA, can't study that part of genetic information of " being compressed " on dna level.So, make up the transcripting spectrum of cotton fiber on the cDNA level, the QTL of the fibrous quality that carries out on fiber cDNA level studies, and will increase verity and the practicality of QTL, help breeding man to find the gene of controlled target proterties faster and better, for molecular mark lays the foundation.
Make up the strategy that transcripting spectrum adopts cDNA-AFLP more at present.Fu Fengling etc. (2003) have introduced the roughly process of utilizing cDNA-AFLP technique construction genome transcripting spectrum; Fan Shuying etc. (2008) utilize the cDNA-AFLP technique construction one comprise 164 marks, form by 13 linkage groups, the collection of illustrative plates total length is the molecule transcripting spectrum of 1401.2cM; Ritter etc. (2008) utilize cDNA-AFLP to make up the transcripting spectrum of potato, and this figure contains 700 TDFs, total length 800cM.In addition, in cotton research, Pan Yuxin etc. (2007) utilize the cDNA-AFLP technology that cotton fiber development secondary wall thickening phase (20-25 days) expression of gene spectrum is analyzed, and have made up the transcripting spectrum of cotton fiber secondary wall thickening phase.
But adopt cDNA-AFLP construction of strategy transcripting spectrum that a lot of weak points are arranged: at first, the AFLP operating process is loaded down with trivial details, needs enzyme to cut and needs twice amplification; Secondly, very high to the cDNA specification of quality, and the RNA of some crop (for example cotton) extracts complexity, of low quality, the resulting double-stranded cDNA of reverse transcription is difficult to reach quality and the concentration that enzyme is cut; The 3rd, the expense height is not suitable for population analysis is mapped.
The another kind of method of the structure transcripting spectrum of having reported is cDNA-SRAP.CDNA-SRAP compares cDNA-AFLP certain advantage: the first, and cDNA-SRAP directly increases to cDNA, need not a series of steps such as enzyme is cut, connection; Secondly, this mark is not very strict to the requirement of cDNA quality, and is very suitable for the plant that this class of cotton is difficult to obtain high quality RNA; The 3rd, only need to get final product with cDNA first chain, need not to carry out the synthetic of second chain, not only simplified step but also saved cost; The 4th, most SRAP marks have comprised the exon in the encoder block, for the separating clone of gene provides very big convenience.The F that Li etc. (2003) have been hybridized with Cauliflower DH pillar and green vegetables flower
2Colony is a mapping population, utilizes the cDNA-SRAP mark to make up the transcripting spectrum that contains 247 marks.
Yet utilizing the cDNA-SRAP mark to make up transcripting spectrum is not the easiest approach, and its band is abundant inadequately, stability is not high.Along with constantly developing of ESTs database, the ESTs that rolls up provides sufficient rich in natural resources for the exploitation of EST-SSR, and the SSR mark has easy and simple to handle, advantages such as success ratio is high, non-false positive, codominance, so EST-SSR makes up the even more ideal method of transcripting spectrum.Also do not utilize at present EST-SSR to make up the report of transcripting spectrum.
ESTs in the cotton enriches constantly along with the expansion of Gossypium dbEST database, these ESTs sequence great majority are to come from the fiber that cotton is expressed, and these ESTs are used to develop the EST-SSR mark and are applied to (Saha.etal, 2004 in genetic mapping, the QTL Position Research; Chee P W et al, 2004; Han Z G, Guo W Z, Song X et al, 2004; Han Z G, Wang C B, Song X L et al, 2006; Park Y H et al, 2005; Taliercio E, Allen R D, Essenberg M et al, 2006; Zhang Yanxin etc., 2007).The EST-SSR mark can DNA amplification, and the cDNA that equally also can increase is because they itself come from cDNA.When with EST-SSR amplification cDNA, we come from transcriptional domain at resulting information, and amplified production is expressed, and the collection of illustrative plates that makes up with these marks is the expression genetic map.Because most of ESR-SSR come from cotton fiber, therefore, we can make up the genetic linkage maps of transcribing of cotton fiber with EST-SSR, and these EST-SSR marks are positioned on the transcripting spectrum, so that it is more accurate to carry out the QTL location in transcripting spectrum, use more.
Have not yet to see the relevant EST-SSR mark that utilizes and make up the report that cotton fiber is transcribed the genetic linkage map spectral method.
Summary of the invention
The objective of the invention is to the cotton linkage mapping is brought up to transcriptional level from dna level, be labeled as the basis with EST-SSR and set up the method that easy transcripting spectrum makes up, and set up the transcripting spectrum at cotton fiber development initial stage with this.The application of the gene studies of controlling fiber being grown from the transcriptional level.
The present invention is achieved through the following technical solutions:
Utilize easy EST-SSR to make up the method for cotton fiber transcripting spectrum, according to the following step:
1) with sea island cotton strain Pima 3-79 (Li Wu etc., the SRAP labeled analysis of sea island cotton genetic diversity, Acta Agronomica Sinica ACTAAGRONOMICA SINICA 2008,34 (5): 893-898) be male parent, upland cotton kind Hubei Province cotton 22 (ZhangYanxin etc., Characteristicsand analysis of simple sequence repeats in the cotton genome based on linkage map constructed from a BC
1Population between gossypium hirsutum and G.barbadense, Genome, 2008 (51): be maternal 534-546), hybridization obtains F1, makes the F1 selfing, obtains F
2,, it is planted in the field with the parent material of this F2 individual plant as the mapping of fiber transcripting spectrum;
2) extract described field material F
2The bloom RNA of back 5 days fiber of individual plant, and counter-rotating becomes cDNA;
3) adopt EST-SSR to F
2Mapping population is analyzed, and utilizes mapping software to make up the transcripting spectrum at cotton fiber development initial stage;
Wherein: EST-SSR comprises the called after HAU primer (seeing shown in the sequence table SEQ ID NO:1-90) that called after NAU, JESPR, STV, MUSS, MUCS, MGHES, BNL, CM, TMB, CIR, DPL, GH, MUSB series primer (primer sequence is seen http://www.cottonmarker.org/Downloads.shtml) and the applicant design voluntarily in the step 3).
4) with F
2Individual plant fiber cDNA is as the pcr template of EST-SSR, and with the polyacrylamide gel electrophoresis analysis of sex change.
Wherein: the PCR reaction system is as follows:
CDNA template 25ng, 1x damping fluid, 1.5mmol L
-1MgCl
2, 0.3mmol L
-1DNTPs, 0.2 μ mol L
-1Primer, 0.5U Taq archaeal dna polymerase, insufficient section is with aseptic distilled water polishing.
PCR program: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ of extension 1min, 30 circulations; Last 72 ℃ are extended 10min.
5) data preparation and linkage analysis: for the codominant marker, with " A " expression cotton 22 parents' in Hubei Province the banding pattern that isozygotys; The banding pattern that isozygotys with " B " expression Pima3-79 parent; " H " expression two parents' heterozygosis banding pattern.For the dominant marker, cotton 22 parents in Hubei Province are the banding pattern that isozygotys, and are designated as " A "; Pima3-79 parent's isozygoty banding pattern and heterozygosis banding pattern are designated as " C ".Pima3-79 parent's the banding pattern that isozygotys is designated as " B "; Cotton 22 parents' in Hubei Province isozygoty banding pattern and heterozygosis banding pattern are designated as " D ".Missing data is represented with "-".
Utilize MAPMAKER/EXP.3.0 software (Lincoln S, Daly M, Lander E S.Construction genetic maps withMAPMAKER/EXP 3.0.In:Whitehead Institute Technical Report, 2nd ed.Cambridge:Whitehead Institute, 1992) be mapping software, earlier with Group and Order order carrying out linkage analysis between mark (LOD=2.5), the mark that does not enter linkage group is linked on the corresponding linkage group with the Try order, the last Group (LOD=2.0) that utilizes again that does not enter linkage group divides into groups, and utilizes the Try order to be connected to chain mark then.Make up the transcripting spectrum at cotton fiber development initial stage.Utilize the Kasambi function that recombination fraction is converted to genetic distance (cM).
The present invention utilizes the genetic linkage maps of transcribing of cotton fiber that EST-SSR makes up, and its method is fast and simple.Utilize method of the present invention can make up the transcripting spectrum of fiber and even other organs, transfer to the cDNA level for the QTL location by dna level and lay a good foundation.
Description of drawings
Fig. 1: techniqueflow chart of the present invention
Fig. 2: cotton blooms and grew the transcripting spectrum of fiber in back 5 days
Fig. 3: the amplified production of She Ji HAU1741 (corresponding primer is SEQ ID NO:29,30) primer is at 6% denaturing polyacrylamide gel electrophoresis figure voluntarily
Fig. 4: the amplified production of She Ji HAU1739 (corresponding primer is SEQ ID NO:25,26) primer is at 6% denaturing polyacrylamide gel electrophoresis figure voluntarily
Embodiment
Embodiment 1:
With sea island cotton Pima 3-79 be male parent (Li Wu etc., the SRAP labeled analysis of sea island cotton genetic diversity, Acta Agronomica Sinica ACTAAGRONOMICA SINICA 2008,34 (5): 893-898), upland cotton kind Hubei Province cotton 22 (ZhangYanxin etc.,
Characteristics and analysis of simple sequence repeats in the cotton genome based on linkage mapconstructed from a BC
1Population between gossypium hirsutum and G.barbadense, Genome, 2008 (51) is maternal, obtain F1, the F1 kind is planted in breeding base, Chinese Hainan, make the F1 selfing, obtain F2, the F2 kind is planted the experiment Tanaka in Wuchang, Wuhan City, Hubei Province Hua Zhong Agriculture University, with the parent material of this F2 individual plant as the mapping of fiber transcripting spectrum;
2, the cotton fiber with the back 5 days growth of blooming makes up transcripting spectrum.Concrete steps are as follows:
Material is prepared
Get the cotton fiber of growing 5 days.Every morning lists in the field, and the flowers are in blossom same day also is designated as " 0dpa ", will hang over the plate on date on the same day on the anthocaulus, and to the flowers are in blossom sampling 6 days the time, the back 5 days cotton boll of promptly blooming places liquid nitrogen standby with tweezers picking fiber the cotton boll strip off rapidly.
All F
2After the fiber that individual plant was grown 5 days is all got sample, extract the RNA of fiber according to (2005) reported method such as ZHU LongFu.Superscript with invitrogen
III RT transcriptase is cDNA with the RNA reverse transcription, and each reaction guarantees that the RNA consumption is at 0.3 μ g.
2.1 EST-SSR labeled analysis
(this series primer comprises called after NAU, JESPR, STV, MUSS, MUCS, MGHES, BNL, CM, TMB, CIR, DPL, GH, MUSB series primer (see by primer sequence to utilize the EST-SSR primer
Http:// www.cottonmarker.org/Downloads.shtml) and voluntarily the serial primer (described primer is seen shown in the sequence table SEQ ID NO:1-90) of the called after HAU of design is respectively to F
2Individual plant carries out the mapping population analysis.
The PCR reaction system is as follows: cDNA template 25ng, 1X damping fluid, 1.5mmol L
-1 MgCl
2, 0.3mmol L
-1DNTPs, 0.2 μ mol L
-1Primer, 0.5U TaqDNA polysaccharase, insufficient section is with aseptic distilled water polishing.
PCR program: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ of extension 1min, 30 circulations; Last 72 ℃ are extended 10min.
The method that electrophoresis, silver dye is with reference to the method for (2005) such as Lin.
2.2 data preparation and linkage analysis
The record of molecule marker adopts Mapmaker/EXR3.0 software (Lander and Botstein, 1989) recording method.For the codominant marker, with " A " expression cotton 22 parents' in Hubei Province the banding pattern that isozygotys; The banding pattern that isozygotys with " B " expression Pima3-79 parent; " H " expression two parents' heterozygosis banding pattern.For the dominant marker, cotton 22 parents in Hubei Province are the banding pattern that isozygotys, and are designated as " A "; Pima3-79 parent's isozygoty banding pattern and heterozygosis banding pattern are designated as " C ".Pima3-79 parent's the banding pattern that isozygotys is designated as " B "; Cotton 22 parents' in Hubei Province isozygoty banding pattern and heterozygosis banding pattern are designated as " D ".Missing data is represented with "-".
Utilize MAPMAKER/EXP.3.0 (Lander and Botstein, 1989) mapping software, earlier with Group (LOD=2.5) grouping, 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 molecule marker linkage map.Utilize the Kasambi function that recombination fraction is converted to genetic distance (cM).
2.3 the screening of polymorphic dna mark between the parent
With NAU, JESPR, STV, MUSS, MUCS, MGHES, BNL, CM, TMB, CIR, DPL, GH, MUSB series primer (primer sequence is seen http://www.cottonmarker.org/Downloads.shtml) and self-designed HAU primer (seeing sequence table SEQ ID NO:1-90) respectively to F
2Individual plant carries out the mapping population analysis) carry out population analysis, obtain 279 pleomorphism sites altogether.
2.4 the structure of cotton EST-SSR transcripting spectrum
279 polymorphism marks that obtain are made up the transcripting spectrum at cotton fiber development initial stage with MAPMAKER/EXP3.0.227 marks enter 41 linkage groups (LOD 〉=2.0), length overall 3030.7cM, 72% (see figure 3) of covering whole genome.Each linkage group has 2～18 marks, and the longest linkage group is 292.2cM, and the shortest linkage group is 9cM.Maximum spacing is 38.4cM between mark, and minimum spacing is 0.3cM, average headway 13.35cM between mark.
Reference:
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Sequence table
＜110〉Hua Zhong Agriculture University
＜120〉utilize the EST-SSR mark to make up the method that cotton fiber is transcribed genetic linkage maps
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<221>Primer
<222>(1)..(20)
<223>
<400>25
tgcagtctca?acacaacaaa 20
<210>26
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>26
gagttcgagg?accaaactgt 20
<210>27
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>27
cgccaatgtc?gattgtagta 20
<210>28
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>28
cttcttcatt?catggccttc 20
<210>29
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>29
atccaacctg?tttccacact 20
<210>30
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>30
aacggttggt?gaacagaaat 20
<210>31
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>31
aaacaaaagg?gcaaacaaag 20
<210>32
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>32
agccatatat?gacggtggat 20
<210>33
<211>21
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(21)
<223>
<400>33
agatacaatc?aggcaaacac?a 21
<210>34
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>34
gagagacggg?cacagatagt 20
<210>35
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>35
ggaagggacc?ccttaataga 20
<210>36
<211>21
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(21)
<223>
<400>36
cccccttcag?atatcttcat?t 21
<210>37
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>37
cagcccaaaa?cagctaaaat 20
<210>38
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>38
ttaagatgtg?aaaggcagca 20
<210>39
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>39
ctgctttcca?agtggaagtt 20
<210>40
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>40
agtgcaacgg?cttaggatac 20
<210>41
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>41
gtggatgaat?gaggcatctg 20
<210>42
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>42
aatcgccatt?gtttactgct 20
<210>43
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>43
gcatgtgagg?ttagctcctt 20
<210>44
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>44
agagcctttg?tgttccagtc 20
<210>45
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>45
tcttgcagac?tagcctcctt 20
<210>46
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>46
aggagcgggt?ccggatgtaa 20
<210>47
<211>24
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(24)
<223>
<400>47
tcacataaca?ttgcagcttg?aggt 24
<210>48
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>48
tttgacccag?agtcacgcgg 20
<210>49
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>49
gccctcctgc?tcctctgtct 20
<210>50
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>50
actgaccaat?ggccctccca 20
<210>51
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>51
aaacctcact?cccagccagt 20
<210>52
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>52
taagaagggc?tggggctcgg 20
<210>53
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>53
agcttcaccc?tcaccagagt 20
<210>54
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>54
aacaccatcg?ccctcgaacc 20
<210>55
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>55
ccgctaccat?ctccatcgcc 20
<210>56
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>56
tcggctgttc?cactgatgcc 20
<210>57
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>57
gtagcaactg?aggccacccc 20
<210>58
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>58
gcacgaggct?gcgtttcatc 20
<210>59
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>59
tccttgcgct?gaagccacat 20
<210>60
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>60
acgagccgaa?cccggatcta 20
<210>61
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>61
tgggagctgc?catctctggt 20
<210>62
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>62
gagctgccgg?ctgcattttc 20
<210>63
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>63
tacgaaccag?ctttgggggc 20
<210>64
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>64
ccgtcgcaat?caatgcccct 20
<210>65
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>65
ctcacggggg?cttgacgaaa 20
<210>66
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>66
gttgtttgaa?cacgggcggt 20
<210>67
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>67
tcaggcgatt?ttccccagca 20
<210>68
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>68
cgacgagctt?cacccacaga 20
<210>69
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>69
gcagctcgac?gacccttgaa 20
<210>70
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>70
tggagcctcc?ttcaatggcg 20
<210>71
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>71
gcagcagctg?aacaactccc 20
<210>72
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>72
ggctttacag?cggggagacc 20
<210>73
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>73
gctcacggcc?ggaatcttga 20
<210>74
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>74
agatcccaag?caaaacccga 20
<210>75
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>75
atcatggggc?gagagtcgga 20
<210>76
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>76
gatggatgcc?ccctgcaaca 20
<210>77
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>77
ggcaatggcg?agtgttagcc 20
<210>78
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>78
taccgagttt?ctctgcggcg 20
<210>79
<211>22
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(22)
<223>
<400>79
tctcaccgat?aaacagtttc?ca 22
<210>80
<211>25
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(25)
<223>
<400>80
tgcacatcat?caatcctatc?atatc 25
<210>81
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>81
gggcttgtga?cgtgtgttgc 20
<210>82
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>82
gatagctccg?cgtgatgcct 20
<210>83
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>83
tgctacgcaa?aaccactcca 20
<210>84
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>84
gaggccagtt?ggggcagttt 20
<210>85
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>85
gccctcgtgc?tcctctgtct 20
<210>86
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>86
gaggccagtt?ggggcagttt 20
<210>87
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>87
gccctcgtgc?tcctctgtct 20
<210>88
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>88
acagctcctc?gttatctcca 20
<210>89
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>89
tctttcttaa?cgccgaagac 20
<210>90
<211>20
<212>DNA
＜213〉cotton (Gossypium L)
<220>
<221>Primer
<222>(1)..(20)
<223>
<400>90
taccgagttt?ctctgcggcg 20
Claims (1)
1. make up the method for cotton fiber transcripting spectrum, it is characterized in that according to the following step:
1) be male parent with sea island cotton strain Pima 3-79, upland cotton kind Hubei Province cotton 22 is maternal, and hybridization obtains F1, plantation F
1, make the F1 selfing, obtain F
2, with this F
2Individual plant is as the parent material of fiber transcripting spectrum mapping;
2) extract described field F
2The bloom RNA of back 5 days fiber of individual plant, and reverse transcription is cDNA, as the mapping population of transcripting spectrum;
3) the EST-SSR primer of utilization report and the primer shown in the sequence table SEQ ID NO:1-90 are respectively to F
2Individual plant carries out the mapping population analysis, with the polyacrylamide gel electrophoresis analysis of sex change;
Wherein: the PCR reaction system is as follows:
CDNA template 25ng, 1x damping fluid, 1.5mmol L-1MgCl
2, 0.3mmol L-1 dNTPs, 0.2 μ mol L-1primer, 0.5U Taq archaeal dna polymerase, insufficient section is with aseptic distilled water polishing; PCR program: 94 ℃ of pre-sex change 2min; 94 ℃ of sex change 1min, 58 ℃ of renaturation 1min, 72 ℃ of extension 1min, 30 circulations; Last 72 ℃ are extended 10min;
4) the record banding pattern utilizes MAPMAKER/EXP.3.0 mapping software analytical data, makes to obtain transcripting spectrum.
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| CN2010101960310A CN101880714B (en) | 2010-06-04 | 2010-06-04 | Method for building cotton fiber transcription genetic linkage map by EST-SSR sign |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101960310A CN101880714B (en) | 2010-06-04 | 2010-06-04 | Method for building cotton fiber transcription genetic linkage map by EST-SSR sign |
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| CN101880714A true CN101880714A (en) | 2010-11-10 |
| CN101880714B CN101880714B (en) | 2012-08-22 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103497949A (en) * | 2013-10-15 | 2014-01-08 | 中国农业科学院棉花研究所 | Molecular markers from sea island cotton Hai 1 and related to cotton fiber strength and application of molecular markers |
| CN104017896A (en) * | 2014-06-23 | 2014-09-03 | 山东棉花研究中心 | EST-SSR marker primers developed on basis of upland cotton transcriptome sequence and application |
| CN104694661A (en) * | 2014-06-23 | 2015-06-10 | 山东棉花研究中心 | EST-SSR labeled primer and application based on upland cotton transcriptome sequence development |
| CN114196780A (en) * | 2021-12-31 | 2022-03-18 | 石河子大学 | Construction method of Xinjiang island cotton capillary electrophoresis DNA fingerprint spectrum and cotton variety identification method |
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|---|---|---|---|---|
| CN1541514A (en) * | 2003-04-29 | 2004-11-03 | 华中农业大学 | Molecular genetic linkage mapping method of cotton |
| CN101020924A (en) * | 2006-12-30 | 2007-08-22 | 华中农业大学 | Prepn process and application of sea island cotton EST SSR marker |
| CN101613761A (en) * | 2009-08-12 | 2009-12-30 | 中国农业科学院棉花研究所 | The SSR mark chain with the cotton fiber strength major gene |
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|---|---|---|---|---|
| CN1541514A (en) * | 2003-04-29 | 2004-11-03 | 华中农业大学 | Molecular genetic linkage mapping method of cotton |
| CN101020924A (en) * | 2006-12-30 | 2007-08-22 | 华中农业大学 | Prepn process and application of sea island cotton EST SSR marker |
| CN101613761A (en) * | 2009-08-12 | 2009-12-30 | 中国农业科学院棉花研究所 | The SSR mark chain with the cotton fiber strength major gene |
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| 《科学通报》 20070830 张艳欣 海岛棉EST-SSR 引物的开发与应用研究 第52卷, 第5期 2 * |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103497949A (en) * | 2013-10-15 | 2014-01-08 | 中国农业科学院棉花研究所 | Molecular markers from sea island cotton Hai 1 and related to cotton fiber strength and application of molecular markers |
| CN103497949B (en) * | 2013-10-15 | 2015-08-05 | 中国农业科学院棉花研究所 | The molecule marker relevant with cotton fiber strength from sea island cotton sea 1 and application thereof |
| CN104017896A (en) * | 2014-06-23 | 2014-09-03 | 山东棉花研究中心 | EST-SSR marker primers developed on basis of upland cotton transcriptome sequence and application |
| CN104694661A (en) * | 2014-06-23 | 2015-06-10 | 山东棉花研究中心 | EST-SSR labeled primer and application based on upland cotton transcriptome sequence development |
| CN104745701A (en) * | 2014-06-23 | 2015-07-01 | 山东棉花研究中心 | Primers of marker EST-SSR developed based on gossypium hirsutum transcriptome sequence and application thereof |
| CN114196780A (en) * | 2021-12-31 | 2022-03-18 | 石河子大学 | Construction method of Xinjiang island cotton capillary electrophoresis DNA fingerprint spectrum and cotton variety identification method |
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| CN101880714B (en) | 2012-08-22 |
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