The overlap length of the two amplicons was 149 bp. Two fragments of this candidate gene were amplified by PCR in two separate PCR reactions, of which the volumes were 15 μL containing 30 ng DNA, 150 nmol L− 1 of each primer, 1 × Pfu polymerase reaction buffer, 1.5 or 2.0 mmol L− 1

MgCl2, 0.2 mmol L− 1 of each dNTP, and 0.5 U Pfu polymerase. After initial denaturation at 95 °C for 6 min, 34 cycles were conducted at 95 °C for 1 min, primer-specific annealing temperatures at 58 °C for 1 min, Z-VAD-FMK ic50 72 °C for 1 min, and a final extension step at 72 °C for 10 min. PCR products were then separated by polyacrylamide gel electrophoresis. The band of interest was cut out from the gel with a razor blade. The gel slice was soaked and crushed briefly in ddH2O, and the water was used as template for a second PCR. The second PCR products were directly sequenced by the Sunny Sequencing Service (Sunny, Shanghai, China). Amplicons of each accession

were sequenced with both forward and reverse PCR primers. Sequence reads were checked and assembled into contigs. The sequences of AF512540 and AY189969 were used as the reference sequences. The sequence reads were aligned using ClustalW2.1 [23] and manually corrected using BioEdit [24]. Sequence polymorphisms were deduced from sequence comparisons in gene-wise sequence alignments. Reference sequences were excluded from all subsequent analyses, and InDels were treated MG-132 mouse as single polymorphic sites. Nucleotide diversity (π), haplotype identification, haplotype diversity (Hd) and LD were determined with software DnaSP v5.10

[25]. Analyses of π and Hd were performed separately for each species as well as for full populations. Population structure was inferred from SSR data with Structure version 2.2 [26]. We used prior population information, predefining accessions as belonging to specific populations. Accessions were defined as 1) G. arboreum accessions, 2) G. barbadense accessions, and 3) G. hirsutum accessions. The optimum number of populations Oxalosuccinic acid (K) was selected after five independent runs with a burn-in of 500,000 iterations followed by 500,000 iterations testing for K = 2 to K = 10. Structure produced a Q matrix that lists the estimated membership coefficients for each accession in each cluster. The estimated Q matrices were used in the subsequent AM, by logistic regression, performed in TASSEL software [27]. SNPs or InDels at site frequencies of 0.05 or greater among the 92 accessions were evaluated using TASSEL. Mean phenotypic values were applied for the association analysis. One thousand permutations of the data were run to account for multiple testing, and a significant association was assigned if the P-value of the most significant polymorphism in a region was seen in < 5% of the permutations. We analyzed DNA polymorphisms in the Exp2 genomic region in 92 Gossypium accessions.