**2. Materials and methods**

### **2.1 Wheat germplasm, DNA extraction and PCR amplification**

A total of 109 bread wheat lines developed for the Yellow and Huai Valleys of China were planted at the Zhengzhou Scientific Research and Education Center of Henan Agricultural University during the 2009-10 cropping seasons according to local management practices. Analysis of agronomic traits and measurement of SKCS hardness as well as identification of puroindoline b-2 variants were performed. Each plot was comprised of four 200 cm-long rows with 23 cm between neighboring rows and 10 cm between neighboring plants. All surveyed cultivars grew well and no lodging was present in the trial. After harvest, all wheat samples were cleaned.

Durum wheat cultivar Langdon (LND), bread wheat cultivar Chinese Spring (CS), two disomic substitution lines of Langdon 7D(7A) and Langdon 7D(7B) with substituted 7D chromosomes of Chinese Spring and six nullisomic-tetrasomic lines of Chinese Spring involving group 7 chromosomes (CS N7A-T7B, CS N7A-T7D, CS N7B-T7A, CS N7B-T7D, CS N7D-T7A and CS N7D-T7B) were used in this study. DNA was extracted from two seeds each of the 109 bread wheat lines, genetic stocks, and 15 Chinese, CIMMYT and Italian durum wheat cultivars following the rapid extraction method of genomic DNA derived from Chen et al. (2006).

PCR primer sequences were designed using Primer Premier 5.0 software. Reactions were performed in 25 μL containing 100 ng of genomic DNA, 10 pmol of each primer, 250 μM of each dNTP, 1x Taq DNA polymerase reaction buffer containing 1.5 μM of MgCl2 and 0.5 unit of Taq DNA polymerase (Promega, Madison, WI). The cycling conditions were 94°C for 5 min followed by 35 cycles of 94°C for 50 s, 45°C to 65°C for 50 s (primer-specific annealing temperatures, see Table 1), 72°C for 1 min, followed by a final 10-min extension at 72°C. An aliquot (8 μL) of the PCR products was analyzed on 1.5% (w/v) agarose gels, stained with ethidium bromide, and visualized with UV light. Forty-eight clones from three independent PCR reactions using the universal primers on durum cv. Langdon were sequenced from both strands by SinoGenoMax Co., Ltd.

Multiple alignments of sequences and translations of nucleotide into amino acid sequence were performed by software DNAMAN Version 6.0. Sequence chromatograms were analyzed by Chromas Version 1.4.5 and FinchTV Version 1.4.0.
