**1. Introduction**

278 Gene Duplication

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prothrombin activator, multactivase, from the venom of Echis multisquamatus."

Kernel texture (grain hardness) is a leading quality characteristic of bread wheat (*Triticum aestivum* L.) as it dramatically influences the milling and processing properties, and consequently determines the classification and marketing of grain (Bhave and Morris 2008a, b). The word puroindoline is derived from the Greek word "*puros"* meaning wheat and "*indoline"* describing the indole ring of tryptophan (Gautier et al. 1994). Puroindolines, composed of puroindoline a and b, are amphipathic proteins of ca. 13,000 Da, and share homology with grain softness protein (GSP), purothionins, lipid transfer proteins, and other members of the prolamin super-family of proteins (Shewry and Halford 2002). Puroindoline proteins possess a characteristic tryptophan-rich domain and cysteine backbone; isoforms occur in the starchy endosperm of the Triticeae. Their secondary structure, determined by infrared and Raman spectroscopies, is comprised of approximately 30% *α*-helices, 30% *β*sheets, and 40% unordered structure at pH 7.4 in solution (Bihan et al. 1996). Puroindoline genes are present throughout the Triticeae tribe of the Poaceae (Gramineae), including wheat (*Triticum* sp.), rye (*Secale* sp.), barley (*Hordeum* sp.), and the wild relatives of wheat (*Aegilops* sp. and *Triticum* sp.). In *Triticum aestivum*, puroindolines exist as two expressed genes, Puroindoline a and Puroindoline b, and are located on the distal end of the short arm of chromosome 5 (5DS). An exception to this general situation lies with the tetraploid (AABB) wheats (*T. turgidum*), which include cultivated durum (ssp. *durum*). Apparently during the allotetraploidization formation of T. *diccocoides*, the wild ancestor of cultivated durum, both the A- and B-genome Puroindoline loci were eliminated due to transposable element insertion and two large deletions in the *Hardness* loci caused by illegitimate DNA recombination (Chantret et al. 2005). Consequently, hexaploid wheat, *T. aestivum*, possesses

A Puroindoline Mutigene Family Exhibits Sequence

from Chen et al. (2006).

**3. Results** 

both strands by SinoGenoMax Co., Ltd.

**3.1 Physical mapping of** *Pinb-2* **variants** 

analyzed by Chromas Version 1.4.5 and FinchTV Version 1.4.0.

**2.2 Measurement of agronomic traits and hardness index** 

were used to classify the genotypes tested into soft, mixed, and hard types.

Diversity in Wheat and is Associated with Yield-Related Traits 281

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

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

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

Before harvesting, ten plants of each genotype were randomly selected in each plot to determine spikelet number per plant, flag leaf length and flag leaf width, as well as flag leaf area calculated from the product of leaf length and maximum leaf width × 0.75. Grains per spike, grain weight per plant and grain weight per spike were determined after harvesting. Kernel hardness, grain diameter and thousand-kernel weight were measured on 300-kernel samples of each genotype using the Perten Single Kernel Characterization System (SKCS) 4100, following the manufacturer's operation procedure (Perten Instruments North America Inc., Springfield, IL). Mean, standard deviation, and distribution of SKCS hardness data

Physical mapping results with variant-specific primers (Table 1) showed that *Pinb-2v1* was present in LND 7D(7A), LND 7D(7B), Chinese Spring, CS N7A-T7B, CS N7A-T7D, CS N7B-T7A and CS N7B-T7D, but absent in wild-type Langdon, CS N7D-T7A and CS N7D-T7B, indicating that *Pinb-2v1* is located on 7D of Chinese Spring. *Pinb-2v2* was present in wildtype Chinese Spring, CS N7A-T7B, CS N7A-T7D, CS N7D-T7A and CS N7D-T7B, but absent in wild-type Langdon, LND 7D(7A), LND 7D(7B), CS N7B-T7A and CS N7B-T7D, indicating that *Pinb-2v2* is located on 7B of Chinese Spring. *Pinb-2v3* was present in wild-type Langdon, LND 7D(7A), but absent in LND 7D(7B), wild-type Chinese Spring and all of its six nullisomic-tetrosomic lines, indicating that *Pinb-2v3* is located on 7B of Langdon. *Pinb-2v4* was present in Langdon, LND 7D(7B), CS N7B-T7A, CS N7B-T7D, CS N7D-T7A and CS

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

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

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 hardness and gene duplication events in wheat.
