DNA extraction, PCR amplification, and SSR genotyping were performed as previously described [5] and [30]. PCR amplification was performed on a PTC-200 Thermocycler (MJ Research/Bio-Rad, USA) with 5′ fluorescent end-labeled

primers and PCR products were visualized by silver staining after separation by 6% SDS-polyacrylamide gel electrophoresis. The products were used for genotypic analysis on a Mega BACETM 1000 (Amersham Biosciences, USA) and allele fragment sizes were obtained with software BioCalculator 2.0 (QIAGEN, Germany). A total of 14 phenotypic traits (nine qualitative and five quantitative Obeticholic Acid solubility dmso traits) were used for phenotypic diversity analysis. The proportions of different classes of nine qualitative phenotypic traits (seed coat color, cotyledon color, seed shape, growth habit, stem termination, pubescence color, flower color, leaf shape and hilum color) in the 159 accessions and a PIC   (polymorphic information content) value for each trait were calculated. Chi-square tests were used for detecting similarity of distribution with the accessions in the established MCC. Seed coat has five colors

including yellow, green, black, brown and di-color, designated as 1–5. Cotyledon has yellow and green colors, designated as 1 and 2. The codes for seed shape are 1–6 and refer to spherical, spherical flattened, ellipse, flat ellipse, long ellipse and reniform. Codes 1–4 of growth habit refer to erect, semi-erect, semi-rampant, and rampant, and codes 1–3 of stem termination refer to determinate, semi-determinate, and indeterminate. Codes INCB024360 clinical trial 1–2 of pubescence color and flower color refer to gray and tawny pubescence and to white and purple flower, respectively. The four leaf shapes (lanceolate, ovoid, ellipse and round) are designated

as 1–4 and six hilum colors (yellow, buff, brown, dark brown, blue, imperfect black and black) as 1–6. Mean value, standard deviation (SD  ) and coefficient of variation (CV  ) of five quantitative phenotypic traits (growth duration, 100-seed weight, plant height, protein content and fat content) were calculated using Microsoft Excel software. A large-sample Z  -test was used for detecting the similarity of distributions to those of accessions in the MCC. Numbers of observations, allele number, gene diversity, observed heterozygosity, and PIC  -value of molecular Smoothened markers were calculated with PowerMarker V3.25 [31].The PIC  -value was calculated as: PIC=1−∑i−1nPi2, where Pi is the frequency of the ith allele.The chi-square value was calculated as X2=∑i−1nAi−Ti2Tiwhere Ai is the frequency of the ith allele among soybean accessions in IACC and Ti is the frequency of the ith allele among soybean accessions in MCC. The Z  -value was calculated as: Z=X1¯−X2¯S12n1+S22n2Where X1¯/X2¯, S1/S2 and n1/n2 refer to mean, standard deviation, and sample size of soybean accessions in the IACC or MCC, respectively.