| To increase the numbers of microsatellites available for use in constructing a genetic map,and facilitate the use of functional genomics to elucidate fiber development and breeding incotton, we sampled microsatellite sequences from expressed sequence tags (ESTs)transcribed during fiber development in the A-genome species Gossypium arboreum andAD-genome Gossypium hirsutum to evaluate their characterization, putative function, levelof polylnorphism and distribution in the At and Dt subgenomes of tetraploid cotton.From ESTs derived from G. arboreum fibers at 7～10 days post anthesis (dpa) fibercDNA library, 1,187 ESTs were found 605 containing simple sequence repeats (SSRs) and158 containing complex sequence repeats (CSRs); 763 EST-SSR primer pairs weredeveloped, and 687 (90%) amplified PCR products from allotetraploid cotton (G. hirsutumcv. TM-1 and G. barbadense cv. Hai7124). Among the 605 SSR-ESTs, hexanucleotides(36%) are the most abundant motif, followed by trinuleotides (31.3%), dinucleotides(20.4%), pentanucleotides (7.9%) and tetranucleotides(4.6%). AT/TA (12.6%) is the mostfrequent repeat in all the motif type. However, only 120 (17.4%) of 687 were found to bepolymorphic and segregating in our interspecific BC1 mapping population[(TM-1×Hai7124)×TM-1]. One hundred and thirty-five of 143 loci detected with these 120EST-SSRs were integrated into our backbone map including 511 SSR loci. The distributionof the EST-SSRs appeared to be non-random, since 84 loci were anchored to the At and 51to the Dt subgenome of aUotetraploid cotton based on linkage tests.From 13,505 ESTs developed from 7235 5～25 dpa fiber and Xuzhou142 0～5 dpa ovuleand 3～22 dpa fiber cDNA libraries, 5,811 were non-redundant ESTs and 966 contained oneor more SSRs. From them, 489 EST-SSR primer pairs were developed. Among theEST-SSRs, 59.1% are trinucleotides, followed by dinucleotides (30%), tetranucleotides(6.4%), hexanucleotides (2.7%), and pentanucleotides (1.8%). AT/TA (18.4%) is the mostfrequent repeat, followed by CTT/GAA (5.3%), AG/TC (5.1%), AGA/TCT (4.9%), AGT/TCA (4.5%), and AAG/TTC (4.5%). One hundred and thirty EST-SSR loci wereproduced from 114 informative EST-SSR primer pairs, which generated polymorphismbetween our two mapping parents. Of these, 129 were integrated on our allotetraploidcotton genetic map, including 66 on At subgenome and 63 on Dt subgenome.From 32,190 ESTs generated from -3～3dpa ovule of G. hirsutum cv. TM-1downloaded from Genbank dbEST, 12,463 non-redundant ESTs were developed by the softblastclust. And 454 EST-SSR primer paris were developed through the software SSRIT andPrimer3. Hexanucleotides and trinucleotides were observed at the highest frequencies,47.8% and 38.8%, respectively. And then di-, tetra-, pentanuleotides, is 7.5%, 3.2%, 3.2%,respectively. AGA/TCT (4.3%) is the most frequent repeat. AG/TC (2.6%) is the mostmotif in dinucleotides, AAAC/TTTG (1.5%) in tetranucleotides, CCCAA (0.4%) inpentanucleotides and CCACCT/GGTGGA (1.1%) in hexanucleotides. Eighty-four primerswere polymorphic between the parent TM-1 and Hai7124, and produced 90 loci.Eighty-four loci, including 8 distorted loci, were intergrated into the cotton genetic map,and 40 were distributed on At subgenome, 44 on Dt subgenome.There are 22 primer pairs produced polymorphic bands between TM-1 and Hai7124 from616 MUSS/MUSS EST-SSR primers. Twenty-two loci, including 3 distorted loci, wereintergrated into the genetic map, 11 and 11 loci distributed on the At and Dt subgenome,respectively.From a total 87,514 ESTs developed from G. arboreura and G. hirsutum, we obtained39,507 non-rududent ESTs, and 2,146 SSRs were found by the soft SSRIT with a SSR per13.05 kb. Hexanucleotides were the most abundant motif(36.8%) and AT/TA is found atthe highest frequency, 7.4%.Among these 386 loci in this study, there are 24 EST-SSRs producing duplicated locidistributed on the corresponding homoelogous chromosomes, and 16 EST-SSRs producingduplicated loci on the non-homoelogous chromosomes. The last genetic map contained1052 loci, and composed of 26 chromosomes with a genetic distance of 6321 cM (averageof 6.0 cM between loci). The chromosome A3, A11, D1 and D11 contain two linkagegroups, respectively. The number of loci on every chromosome is from 22 to 58, with agenetic distance from 144.5 to 383.5 cM. There are 43 distorted EST-SSR loci, including22 to TM-1 and 21 to Hai7124.All ESTs were categorized to 3 main classes, namely Molecular Function, BiologyProcess and Cell Component through the soft Blast2go online. Among the Cell Component class, 41% were classficated to cell, and 36% to organelle. Catalytic Activity and Bindingcontain 22% and 24%, respectively, in the Molecular Function. As to Biology Process, 38%were belonged to Physiological Process and 36% to Cellular Process.Some genes of carbohydrate metabolism, transcript factor and signal transduction weremapped. For instance, sucrose synthase were mapped on A6, E6 on A5 and D5, MYB60 onA3. And a gene coded salt tolerance protein was mapped on A8.Many kinds of molecular markers are being used in cotton genetic mapping, but there islittle report on SNP markers development of cotton genes. With the PCR-based method,FbL2A genes were isolated from TM-1 and Hai7124. Based on Single nucleotidepolymorphisms (SNPs) of the FbL2A gene sequences in TM-1 and Hai7124, the amplifiedproducts were digested by BstUI selected through NEBcutter software online. And the genewas mapped on D2 by Mapmaker v3.0 mapping software.FIF1 is predominantly expressed early in developing cotton fibers and identified be a keyregulator of cotton fiber development. In this paper, FIF1 genes from G. hirsuturn cv. TM-1and G. barbadense cv. Hai7124 were cloned on the basis of its published sequence in G.arboreum L. Based on the SNPs between FIF1 genes from two tetraploid cotton, SNAPand CAPs markers were developed, and the gene was mapped on A8. This result indicatesthat SNP marker development is feasible and effective in Gossypium.These EST-SSR and SNP markers can be used in genetic mapping, identification ofquantitative trait loci (QTLs), and comparative genomics studies of diploid and tetraploidcotton. |
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