**3. Results**

280 Gene Duplication

puroindoline a and b on only the D-genome (contributed by *Ae. tauschii* during the allohexaploidization of this taxon), but lacks homoeologous loci on the A- and B-genomes. The expression of puroindoline genes is mainly associated with 'soft' and 'hard' grain texture of bread wheat, whereas durum wheat is 'very hard' due to the lack of puroindoline genes (Capparelli et al. 2003). Extensive genetic surveys, cytological analysis, and transformation experiments in wheat and rice (*Oryza sativa*) have demonstrated that puroindoline a and b act to create soft kernel texture (Bhave and Morris 2008a, b, Morris 2002, Morris and Bhave 2008). However, puroindolines have also been extensively studied regarding their multiple roles in the development and resistance of plants against biotic stress, and their profound influence on the breeding and processing quality of food crops (Luo et al. 2008; Giroux and Morris 1997, 1998; Ragupathy and Cloutier 2008; Chen et al. 2007a, b). Considerable allelic variation in puroindoline a and b have been identified in a great number of genotypes stemming from different geographies. Gene sequence polymorphism and allele designations have been recently reconciled and reviewed by

Although clearly exerting a major role in wheat kernel texture variation, puroindolines do not account for all of the variation observed in bread wheat. Several minor quantitative trait loci (QTLs) for kernel texture have been mapped on different chromosomes (Sourdille et al. 1996; Turner et al. 2004). Four additional regions located on chromosomes 2A, 2D, 5B, and 6D were shown to have single-factor effects on hardness, while three others located on

Three new puroindoline gene-like sequences in hexaploid wheat were reported by Wilkinson et al. (2008). Chen et al. (2010) renamed them *Pinb-2v1*, *Pinb-2v2* and *Pinb-2v3*, and reported the discovery of a fourth novel puroindoline variant in bread wheat, designated *Pinb-2v4*. Physical mapping results of Chen et al. (2010) were not fully consistent with the results reported by Wilkinson et al. (2008). Interestingly, *Pinb-2v1* and *Pinb-2v4* were present in all of the surveyed cultivars whereas *Pinb-2v2* and *Pinb-2v3* were reciprocally present in different wheat cultivars (Chen et al. 2010). This result indicates that *Pinb-2v2* and *Pinb-2v3* are likely allelic. In this study, we remapped the four known *Puroindoline b-2* variants using aneuploids of bread wheat and durum wheat in order to confirm their physical location on the chromosomes of wheat, and report the discovery of a novel *Pinb-2v3* allele and a new *Puroindoline b-2* variant, designated *Pinb-2v5*. Also, we investigated the association of *Puroindoline b-2* variants with yield-related traits. This study provides useful information for illustrating the molecular and genetic basis of kernel

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

Morris and Bhave (2008) and Bhave and Morris (2008a, b).

hardness and gene duplication events in wheat.

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

**2. Materials and methods** 

wheat samples were cleaned.

chromosomes 5A, 6D and 7A had interaction effects (Sourdille et al. 1996).
