**4. Discussion**

284 Gene Duplication

*Pinb-2v3* genotypes (Table 2). The results indicated that *Pinb-2v3* cultivars possessed superior grain yield traits compared to *Pinb-2v2*, possibly suggesting that the *Puroindoline b-2* gene could modulate wheat grain yield to some extent, and that the grain yield of cultivars with *Pinb-2v3* is slightly higher than that of cultivars with *Pinb-2v2*. Another likely possibility is that

Gsp-1 80 Pina-D1a 80 Pinb-D1a 80 Pinb-2v1 80 Pinb-2v2 80 Pinb-2v3 80 Pinb-2v4 80 Pinb-2v5 80

Gsp-1 157 Pina-D1a 124 Pinb-D1a 127 Pinb-2v1 127 Pinb-2v2 127 Pinb-2v3 127 Pinb-2v4 127 Pinb-2v5 127

Gsp-1 231 Pina-D1a 189 Pinb-D1a 195 Pinb-2v1 195 Pinb-2v2 195 Pinb-2v3 195 Pinb-2v4 195 Pinb-2v5 195

Gsp-1 311 Pina-D1a 266 Pinb-D1a 272 Pinb-2v1 272 Pinb-2v2 272 Pinb-2v3 272 Pinb-2v4 272 Pinb-2v5 272

Gsp-1 391 Pina-D1a 346 Pinb-D1a 352 Pinb-2v1 352 Pinb-2v2 352 Pinb-2v3 352 Pinb-2v4 352 Pinb-2v5 352

Gsp-1 470 Pina-D1a 422 Pinb-D1a 427 Pinb-2v1 429 Pinb-2v2 429 Pinb-2v3 429 Pinb-2v4 429 Pinb-2v5 429

Gsp-1 495 Pina-D1a 447 Pinb-D1a 447 Pinb-2v1 453 Pinb-2v2 453 Pinb-2v3 453 Pinb-2v4 453 Pinb-2v5 453

Fig. 1. DNA sequence alignment of *Puroindoline b-2* gene variants *Pinb-2v1*, *Pinb-2v2*, *Pinb-*

*2v3*, *Pinb-2v4*, *Pinb-2v5, Pinb-D1a, Pina-D1a* as well as *Gsp-1* from bread wheat.

C C C C C C C C

C C C C C C C C

C G C C C C G G

A C T T T T T T

C C C C C C C C

C C A C C C C C

C A A A A A A A

A G A A A A A A

G G G A A A A A

T T T T T T T T

G G G G G G G G

C C C C C C C C

A A A A A A A A

A A A A A A A A

C C C A A A A A

A C A C A A T T

T A T T T T T T

T G G G G G G G

G G G G G G G G

A G G A A A A A

T C C T C T T T

C C G T T T T T

C C C C C C C C

G T C C C T C C

A C G A A A A A

A C A A G G A A

A . . . . . . .

T . . . . . . .

T . . . . . . .

C C C C C C C C

T T T T T T T T

G G G G G G G G

C C C C C C C C

A A A A A A A A

A A A A A A A A

C C G G G G G G

A A T T T T T T

T T T T T T T T

C C C T C C C C

C C C G G G G G

. . . T T T T T

C C . C C C C C

T G C G G G G G

C C T C C T C C

A A G C T T C C

A A A G G G G G

T T T T T T T T

C C C C C C C C

C C C C C C C C

A A A A A A A A

A A A A A A A A

G G G G G G G G

G G G G G G G G

C C C G C C C C

G G A A A A A A

A A G A A A A A

T T G C C C C C

C C C C C C C C

T T T T T T T T

A C C T T G T T

A G G G G G G G

G G G G G G G G

T T T T T T T T

G G G G G G G G

G G G G G G G G

C C C C C C C C

T A T A G A A A

T T T T T T T T

C C C C C C C C

A T T T T T T T

A T T T T T T T

G C G T T T T T

G G G G G G G G

G G G G G G G G

C A C T C C T T

C T A T T T T T

T T T T T T T T

T T T T T T T T

C C T C C C C C

A A G A A A A A

A G G G G G G G

C C C C C C C C

A G G A A A A A

A T A A A A A A

G G G G G G G G

T T G G G G G G

T T T T T T T T

T T G G G G G G

G G A A A A A A

T T G A A G A A

G C C C C C C C

A A A A A A A A

G A T A A A A A

G G G G G G G G

A A A A A A A A

G G G A G G G G

G C G G G G G G

T T T C C T C C

C C T C C C C C

C C C C C C C C

G T G T T T T T

A T G A A A A A

A G G G G G G G

A G A A A A A A

C G G C C C C C

C G A C C C C C

A A A A A A A A

G G G G A G G G

T T T T T T T T

G G G G G G G G

T T C T T T T T

T T T T T T T T

G G G G G G G G

T C C C C C C C

A A A C C C C C

A G A A A T A A

G T G G G G G G

C C C C C C C C

A G A A A A A A

A G G A A A A A

T C C C C C C C

T T T T T T T T

G C G A A A A A

A G A C C C C T

G G G G G G G G

G C C A C C C C

C C C C C C C C

A A A C C C C C

G G G G G G G G

G A G G G G G G

A A A A A A A A

T T T T T T T T

T T T T T T T T

A A A A A A A A

T C C T T C T T

G C G G G G G G

T T T T T T T T

T G G G G G G G

A C A A A A A A

T T T T T T T T

G A G G G G G G

G G G G G G G G

A A A A A A A A

T T G G G G G G

C C C C C C T C

G G G G G G G G

G A A G G A G G

T T T T T T T T

G G G G G G G G

T C T C C C C C

G T T T T T T T

T C T T T T T T

. A C G G G G G

. A A G G G G G

. C C C C C C C

G G A T G G G G

A A A G G T G G

T T T T T T T T

G G G G G G G G

A A A A A A A A

A A A A A A A A

G G G G G G G G

G G G G G G G G

A A A G G G G G

T T T T T T T T

A T T T T T T T

T T T T T T T T

G C T T T T T T

C C C T T C T T

C C C C C C C C

G G A G G G A A

C G G G G G G G

A A A A A A A A

A A A C C C C C

T T T T T T T T

G G G G G G G G

G C T C C C C C

G C C C C C C C

T C C C C C C C

T T G A A A A A

G G C T C C C C

C T A C C C C C

G A G G G G G G

A G G G G G G G

T . . . . . . .

A . . . . . . .

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G . . . . . . .

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G C C A A A A A

A A A A A A A A

G G G G G G G G

T C C T T T T T

G G G G G G G G

A A A A A A A A

G G G G G G G G

C C C C C C C C

A A A A A A A A

C C C C C C C C

T C A A A A A A

G G A A A A A A

C C C C C C C C

T C C C C C C C

A T T T T T A A

T T T T T T T T

T T C T T T T T

G G G G G G G G

C C C C C C C C

G G G G A G G G

C C C C C C C C

A A A A A A A A

A A A A A A A A

T T T T T T T T

A A A A A G A A

T T C C C C C C

G A T A A A A A

C G C A A A A A

A C A A A A A A

G G G G G G G G

A A A A A A A A

A A A G G G G G

G G G G G G G G

T T T T T T T T

T T T T T T T T

C G G G G G G G

C T G G G G G G

C T C T C T C C

A G G G G G G G

G G G G G G G A

C C C C C C C C

C A T A A A A A

C G G G G G G G

T T T T T T T T

these *Pinb-2* variants are identifying germplasm pools or founder effects.

A A A A A A A A

A T G C C C C C

G . . . . . . .

C G G A A A A A

A A G G G G G G

G A G G G G G G

C C . G C C G G

A G . C A C C C

T G . T T T T T

C C . A A A A A

A A . . . . . .

C C C A C C C A

T T T T T T T T

A A A A A A A A

G T G A A A A A

C T T T T T T T

G G G G G G G G

G G G G G G G G

A C C C C C C C

T T T T T T T T

A A A A A A A A

T T T T T T T T

T T T T T T T T

A A A A A A A A

C C C C C C C C

T T T T T T T T

T G G A A A A A

G G G G C C G G

T T T T T T T T

G G G G G G G G

A A A A A A A A

T T T C C T C C

C C G C A A C C

T G A A A A A A

T G G G G G G G

A G G A A A A A

A C T T T T T T

A A A A A A A A

G A T A A A A A

C G T C C C C C

C C C C G C C C

A A A A A A A A

G A A A A A A A

A A A A A A A A

A G C C C C C C

C T A A A A A A

G G A A A A A A

G A C A A A A A

T T T T T T T T

G A T T T T T T

C C C C C C C C

A A A A A A A A

G A G G G G G G

A G A A A A A A

C A G G G G G G

G A G G G G G G

G G G G G G G G

C C C C C C C C

C C C C C C C C

A A C A A A A A

A A A T T T T T

G G G G G G G G

A A A A A A A A

G A G T T T T T

C C C G G G G G

C C C T T T T T

T T T T T T T T

A A A A G G A A

C T T T T T T T

G G A A A A A A

A C G G G G G G

C C C C T C C C

G A A A A A A A

T C C A A A A A

C C C A A A A A

G G A A G G G G

C C C A A A A A

G A A A A A A A

T A A G G G G G

T T T T T T T T

G G G G G G G G

C C T C C C C C

C C C C C C C C

G G G G G G G G

T C C A A A T T

T T T T T T T T

G G G G G G G G

C C T T T T T T

A A G G G G G G

A A A A A A A A

G C T G G G G G

G A T G G G G G

C T C C C C C C

T C T C C C C C

A A A A A A A A

T T T T T T T T

A C C T T T T T

T C C T T T T T

G A G G G G G G

G G G G G G G G

A G C A A A A A

C G G G G G G G

A G A A G G A A

T T T T T T T T

C C C C C C C C

T A A G G T G G

C C C C C C C C

T C C T C C C C

T T T A A A A A

G G G G G C G G

G G G G A G G G

T C C C C C C C

T G C T T T T T

C T C T T T T T

T T A T T T T T

T G C T T T T T

T G A G G G G G

C T A A A A A A

C . . . . . . .

T . . . . . . .

C . . . . . . .

G G A A A A A A

G G A A A A A A

A A T T T T T T

C A G G G G G G

T A G G G G G G

T T T T T T T T

G G G G G G G G

G G G G G G G G

G T A A A A A A

G G A A A A A A

G G G G G G G G

A A G G G G G G

A A G G G G G G

G G C C C T C C

A G G G G G G G

G G G C C C C C

A A C C C C C C

A G T T T T T T

C . . . . . . .

G . . . . . . .

A . . . . . . .

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A . . . . . . .

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C . . . . . . .

A A A A A A A A

G G G G G G G G

C A C A A A A A

C C G A A A A A

A A G G G G G G

. . C A A A A A

. . C C C C C C

. . G G G G G G

A A A A A A A A

A A A A A A A A

G G G G G G G G

G C C C C C C C

T T T T T T T T

A A A A G G G G

G A A G G G G G

A A G A A A A A

C T C C C C C C

T T T C T T T T

C C C C C C C C

T A T T T T T T

T T T T T T T T

G G G G G G G G

T C C C C C C C

A A A A A A A A

G G A A A A A A

G T T T T T T T

C A A A A A A A

T C C T T T T T

. . A G G G G G

. . A A A A A A

. . T G G G G G

G G G A A A A A

C A A A A A A A

G T A C C T C C

C G G G G G G G

A T T G G G G G

A T T T T T T T

G G G G G G G G

C C G G G G G A

T T C C T T C C

C G G G A G G G

C G G G G G G G

C . . . . . . .

A . . . . . . .

C . . . . . . .

C C A A A A A A

G G G G G G G G

C G G G G G G G

G G A G G G G G

G G G T T T T T

A G G T T T T T

T T T T T T T T

G G G G G G G G

G G G G G G G G

T T T T T T T T

T G T T T T T T

T C C C C C C C

T T T T T T T T

G C C C C C C C

G A A A A A A A

G A A A A A A A

T T T T T T T T

G G G G G G G G

A A A A A A A A

A A A A A A A A

G G G G G G G G

A G A A A A A A

C C C C C C C C

C C C C C C C C

T C T T T T T T

T T T T T T T T

C C A A A A A A

T T T T T T T T

T T T T T T T T

C C C C C C C C

C C C C C C C C

T T T T T T T T

C C C C C C C C

C A C C C C C C

T T T T T T T T

A A A A A A A A

G G G G G G G G

C G C C C C C C

T A T T T T T T

T C C C C C C C

T T T T T T T T

C G C C C C C C

C C C C C C C C

T T T T T T T T

T T T T T T T T

G G G T T T T T

C C C C C C C C

T T T T T T T T

C C C C C C C C

T T T T T T T T

G G T T T T T T

G G G G G G G G

> Even though physical mapping of *Puroindoline b-2* variants was conducted using aneuploids of Chinese Spring by Chen et al. (2010), we re-mapped the *Puroindoline b-2* variants in the durum wheat cultivar Langdon and bread wheat cultivar Chinese Spring. The re-mapping was performed using different stocks than those reported by Chen et al. (2010) due to the controversial difference between the results of Chen et al. (2010) and Wilkinson et al. (2008). According to this study and the previous report (Chen et al. 2010), *Pinb-2v1* is located on chromosome 7D and is present in all the bread wheat cultivars surveyed. Therefore, in this study, the absence of *Pinb-2v1* in durum wheat is expected. *Pinb-2v2* and *Pinb-2v3* are reciprocally present on chromosome 7B in all of the bread wheat cultivars surveyed. Only one durum cultivar possessed the *Pinb-2v2/Pinb-2v4* haplotype combination, whereas 35 durum cultivars possessed the *Pinb-2v3/Pinb-2v4* combination, suggesting that *Pinb-2v3* and *Pinb-2v2* were likely allelic. In this set of durum germplasm, the *Pinb-2v3/Pinb-2v4* combination was the predominant haplotype.

> Wilkinson et al. (2008) mapped *Puroindoline b-2* variant 2 to chromosome 7AL in three doubled haploid populations and amplified sequences of *Pinb-2v3* and *Pinb-2v1* from the genomic DNA of the durum wheat cultivar Ofanto. We did not find *Pinb-2v1* in any of the durum cultivars surveyed, including 48 sequencing results of cloned PCR amplicon in the

A Puroindoline Mutigene Family Exhibits Sequence

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durum wheat Langdon. The reason for this discrepancy is possibly due to primer sequence and specificity (or lack thereof) for the various variant genes used in the two studies.

According to Chen et al. (2010), *Pinb-2v1, Pinb-2v2, Pinb-2v3* and *Pinb-2v4* are located on chromosome 7DL, 7BL, 7B and 7AL in bread wheat, respectively. Coincidently, the strongest QTL effects controlling grain yield, especially for grain weight, were found on chromosomes 7AL and 7BL in the report of Quarrie et al. (2005), and a QTL associated with grain yield has also been identified on chromosome 7D in the study of Kuchel et al. (2007). More recently, several QTLs including QTLs on 7A and 7B associated with grain yield and yield components have been discovered in a recombinant inbred line population (McIntyre et al. 2009). However, further studies are required to determine if this is 'cause and effect' or simply linkage occurring.

Even though many QTLs controlling wheat grain yield and its components have been studied for many years, no specific gene with detailed sequence has been reported so far. Therefore, the possibility of the *Puroindoline b-2* gene possessing some function in modulating grain yield traits may provide useful information for MAS (Marker Assisted Selection). Future studies should define the function of the *Puroindoline b-2* genes by using populations with defined genetic background.
