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

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

A Puroindoline Mutigene Family Exhibits Sequence

*Pinb-2v2* CTTGTAGTGAGCACAACCTTT

*Pinb-2v3* GCAGAAAAAGCCATTGCACCT

*Pinb-2v4* CCTTTCTCTTGTAGTGAGCAC

**traits, grain yield components, and flag leaf size** 

GCA

A

*N-Pinb-D1b* 

wheat

wheat than in hard.

AACCA

Diversity in Wheat and is Associated with Yield-Related Traits 283

*Pinb-2v5* has 57.8%, 96.0%, 91.3%, 87.9% and 96.5% identity with *Pinb-D1a*, *Pinb-2v1*, *Pinb-*

*Pinb-2v1* GGTTCTCAAAACTGCCCAT ACTTGCAGTTGGAATCCAG 57 319

*Pinb-D1b* ATGAAGGCCCTCTTCCTCA CTCATGCTCACAGCCGCT 58 250

Table 1. PCR primers used in generating *Puroindoline b-2* variant gene sequences in durum

**3.3 Association** *of puroindoline b-B2* **variants with SKCS grain hardness, other grain** 

In order to investigate the influence of *puroindoline b-2* variants on kernel hardness independent of puroindoline a and b alleles, the average SKCS hardness index of two different *Puroindoline b-2* variant combinations, *Pinb-2v1/Pinb-2v2/Pinb-2v4* and *Pinb-2v1/Pinb-2v3/Pinb-2v4,* were compared by sub-setting the lines according to *Pin-D1*  haplotype. Puroindoline alleles are associated with dramatic effects on kernel hardness (reviewed in Morris, 2002; Morris and Behave, 2008; Behave and Morris, 2008a, b). Results indicated that in soft wheat cultivars with *Pina-D1a/Pinb-D1a,* the average of SKCS hardness index of cultivars with *Pinb-2v3* was 24.6, which was significantly higher than that of cultivars with *Pinb-2v2* (14.2). No significant difference was observed among cultivars with *Pinb-D1b*, possibly suggesting that the *Puroindoline b-2* variants had a larger impact in soft

No significant association between the puroindoline D1 alleles and the 9 agronomic traits relating to wheat yield were observed in the surveyed Chinese wheat cultivars of the Yellow and Huai Valleys, suggesting that the puroindoline genes have no apparent relationship with wheat grain yield. However, when the agronomic traits of cultivars with different *Puroindoline b-2* variant combinations were compared, the averages of grain number per spike, grain weight per spike, width of flag leaf and flag leaf area of cultivars with *Pinb-2v3* were significantly higher than those cultivars with *Pinb-2v2.* Differences in thousand kernel weight, grain length and spikelet number per spike were not different between these two *Pinb-2v2* and

ATGAAGGCCCTCTTCCTCA CTCATGCTCACAGCCGCC 58 250

GTATGGACGAACTTGCAGCTG

CATTAGTAGGGACGAACTTGC

GACGAACTTGCAGTTGGAATC

annealing Temp °С

<sup>A</sup>54 453

GAG 65 401

AGCTA 65 528

CAA 65 403

Fragment size (bp)

*2v2*, *Pinb-2v3* and *Pinb-2v4*, respectively, at the amino acid level (data not shown).

Gene Forward primer Reverse primer PCR

*Pinb-2vU* ATGAAGACCTTATTCCTCCTA TCASTAGTAATAGCCATTAKT

N7D-T7B, but absent in LND 7D(7A), CS N7A-T7B and CS N7A-T7D, indicating that *Pinb-2v4* is located on 7A of Langdon and Chinese Spring.

Therefore, it is concluded that *Pinb-2v1* is on 7D of Chinese Spring, *Pinb-2v2* is on 7B of Chinese Spring, *Pinb-2v3* is on 7B of Langdon and *Pinb-2v4* is on 7A of Langdon and Chinese Spring. The results of mapping *Pinb-2v* in Chinese Spring and its derived nullisomic-tetrosomic lines are consistent with the locations of the four *Puroindoline b-2* variants reported by Chen et al. (2010a).

### **3.2 Variation in** *puroindoline b-2* **variants and the discovery of a new** *puroindoline b-2* **variant**

Based on PCR amplification with four specific primers (Table 1), 35 of 36 durum wheat cultivars surveyed possessed variant combination *Pinb-2v3*/*Pinb-2v4*, whereas the durum wheat cultivar Norba, introduced from Italy, possessed combination *Pinb-2v2/Pinb-2v4.* None of the surveyed durum cultivars possessed the *Pinb-2v1* gene. Further sequencing of PCR products with specific primers showed that all of the sequences of *Pinb-2v4* were exactly the same as the *Pinb-2v4* sequence of bread wheat reported by Chen et al. (2010), whereas the coding region of *Pinb-2v3* in durum wheat cultivar Langdon had a single nucleotide change from G to T at the 6th position compared with the *Pinb-2v3* sequence reported by Wilkinson et al. (2008) and Chen et al. (2010). Furthermore, 18 durum wheat cultivars with the *Pinb-2v3* variant were sequenced and all of them uniformly contained a single nucleotide change from G to T at the 6th position. Sequencing of bread wheat cultivar Wichita showed the same change at the 6th position, compared with a previous *Pinb-2v3* sequence of Wichita reported by Wilkinson et al. (2008) and Chen et al. (2010). The discrepancy may have resulted from the previously used primers due to a single nucleotide change present in the forward primer sequence. However, after we modified the *Pinb-2v3* sequence from G to T at the 6th position of Wichita, a one base-pair change from T to C at the 311th position was found in the coding region of *Pinb-2v3* in 12 durum wheat cultivars, resulting in the deduced amino acid change from valine to alanine at position 104 in PINB-2v3. According to the gene nomenclature for puroindoline a and b, this *Pinb-2v3* allele in wheat cultivars containing the sequence of AM99733 and GQ496618 with the modification of G to T at the 6th position will be designated as *Pinb-2v3a*, and the *Pinb-2v3* allele with a nucleotide change from T to C at the 311th position in durum wheat cultivars will be designated as *Pinb-2v3b*.

Three repeated PCR amplifications of the durum cultivar Langdon were performed with the universal primer in order to reduce the influence of mismatching amplification of *Taq* polymerase. Cloning and sequencing results showed that 40 sub-clones belonged to either *Pinb-2v3* or *Pinb-2v4* whereas 7 other sub-clones did not belong to any known *Puroindoline b-2* variant. Further analysis indicated that a novel variant, designated as *Pinb-2v5* (HM245236), shared high homology and was most closely related to *Pinb-2v4* with only 5 SNPs (Fig. 1).

The overall alignment with *Pinb-D1a* with the five *Pinb-2* variant genes showed that *Pinb-2v5* has 74.3%, 95.8%, 94.0%, 92.3% and 98.8% identity with *Pinb-D1a*, *Pinb-2v1*, *Pinb-2v2*, *Pinb-2v3* and *Pinb-2v4*, respectively, at the DNA level. Moreover, all four puroindoline b variant sequences were at least 91.8% homologous amongst themselves. Full alignment of deduced amino acid sequences of *Pinb-D1a* and the five *Puroindoline b-2* variant genes indicated that

N7D-T7B, but absent in LND 7D(7A), CS N7A-T7B and CS N7A-T7D, indicating that *Pinb-*

Therefore, it is concluded that *Pinb-2v1* is on 7D of Chinese Spring, *Pinb-2v2* is on 7B of Chinese Spring, *Pinb-2v3* is on 7B of Langdon and *Pinb-2v4* is on 7A of Langdon and Chinese Spring. The results of mapping *Pinb-2v* in Chinese Spring and its derived nullisomic-tetrosomic lines are consistent with the locations of the four *Puroindoline b-2*

**3.2 Variation in** *puroindoline b-2* **variants and the discovery of a new** *puroindoline b-2*

Based on PCR amplification with four specific primers (Table 1), 35 of 36 durum wheat cultivars surveyed possessed variant combination *Pinb-2v3*/*Pinb-2v4*, whereas the durum wheat cultivar Norba, introduced from Italy, possessed combination *Pinb-2v2/Pinb-2v4.* None of the surveyed durum cultivars possessed the *Pinb-2v1* gene. Further sequencing of PCR products with specific primers showed that all of the sequences of *Pinb-2v4* were exactly the same as the *Pinb-2v4* sequence of bread wheat reported by Chen et al. (2010), whereas the coding region of *Pinb-2v3* in durum wheat cultivar Langdon had a single nucleotide change from G to T at the 6th position compared with the *Pinb-2v3* sequence reported by Wilkinson et al. (2008) and Chen et al. (2010). Furthermore, 18 durum wheat cultivars with the *Pinb-2v3* variant were sequenced and all of them uniformly contained a single nucleotide change from G to T at the 6th position. Sequencing of bread wheat cultivar Wichita showed the same change at the 6th position, compared with a previous *Pinb-2v3* sequence of Wichita reported by Wilkinson et al. (2008) and Chen et al. (2010). The discrepancy may have resulted from the previously used primers due to a single nucleotide change present in the forward primer sequence. However, after we modified the *Pinb-2v3* sequence from G to T at the 6th position of Wichita, a one base-pair change from T to C at the 311th position was found in the coding region of *Pinb-2v3* in 12 durum wheat cultivars, resulting in the deduced amino acid change from valine to alanine at position 104 in PINB-2v3. According to the gene nomenclature for puroindoline a and b, this *Pinb-2v3* allele in wheat cultivars containing the sequence of AM99733 and GQ496618 with the modification of G to T at the 6th position will be designated as *Pinb-2v3a*, and the *Pinb-2v3* allele with a nucleotide change from T to C at the 311th position in durum wheat cultivars will be

Three repeated PCR amplifications of the durum cultivar Langdon were performed with the universal primer in order to reduce the influence of mismatching amplification of *Taq* polymerase. Cloning and sequencing results showed that 40 sub-clones belonged to either *Pinb-2v3* or *Pinb-2v4* whereas 7 other sub-clones did not belong to any known *Puroindoline b-2* variant. Further analysis indicated that a novel variant, designated as *Pinb-2v5* (HM245236), shared high homology and was most closely related to *Pinb-2v4* with only 5

The overall alignment with *Pinb-D1a* with the five *Pinb-2* variant genes showed that *Pinb-2v5* has 74.3%, 95.8%, 94.0%, 92.3% and 98.8% identity with *Pinb-D1a*, *Pinb-2v1*, *Pinb-2v2*, *Pinb-2v3* and *Pinb-2v4*, respectively, at the DNA level. Moreover, all four puroindoline b variant sequences were at least 91.8% homologous amongst themselves. Full alignment of deduced amino acid sequences of *Pinb-D1a* and the five *Puroindoline b-2* variant genes indicated that

*2v4* is located on 7A of Langdon and Chinese Spring.

variants reported by Chen et al. (2010a).

**variant** 

designated as *Pinb-2v3b*.

SNPs (Fig. 1).

Gene Forward primer Reverse primer PCR annealing Temp °С Fragment size (bp) *Pinb-2vU* ATGAAGACCTTATTCCTCCTA TCASTAGTAATAGCCATTAKT <sup>A</sup>54 453 *Pinb-2v1* GGTTCTCAAAACTGCCCAT ACTTGCAGTTGGAATCCAG 57 319 *Pinb-2v2* CTTGTAGTGAGCACAACCTTT GCA GTATGGACGAACTTGCAGCTG GAG 65 401 *Pinb-2v3* GCAGAAAAAGCCATTGCACCT A CATTAGTAGGGACGAACTTGC AGCTA 65 528 *Pinb-2v4* CCTTTCTCTTGTAGTGAGCAC AACCA GACGAACTTGCAGTTGGAATC CAA 65 403 *Pinb-D1b* ATGAAGGCCCTCTTCCTCA CTCATGCTCACAGCCGCT 58 250 *N-Pinb-D1b*  ATGAAGGCCCTCTTCCTCA CTCATGCTCACAGCCGCC 58 250

*Pinb-2v5* has 57.8%, 96.0%, 91.3%, 87.9% and 96.5% identity with *Pinb-D1a*, *Pinb-2v1*, *Pinb-2v2*, *Pinb-2v3* and *Pinb-2v4*, respectively, at the amino acid level (data not shown).

Table 1. PCR primers used in generating *Puroindoline b-2* variant gene sequences in durum wheat

### **3.3 Association** *of puroindoline b-B2* **variants with SKCS grain hardness, other grain traits, grain yield components, and flag leaf size**

In order to investigate the influence of *puroindoline b-2* variants on kernel hardness independent of puroindoline a and b alleles, the average SKCS hardness index of two different *Puroindoline b-2* variant combinations, *Pinb-2v1/Pinb-2v2/Pinb-2v4* and *Pinb-2v1/Pinb-2v3/Pinb-2v4,* were compared by sub-setting the lines according to *Pin-D1*  haplotype. Puroindoline alleles are associated with dramatic effects on kernel hardness (reviewed in Morris, 2002; Morris and Behave, 2008; Behave and Morris, 2008a, b). Results indicated that in soft wheat cultivars with *Pina-D1a/Pinb-D1a,* the average of SKCS hardness index of cultivars with *Pinb-2v3* was 24.6, which was significantly higher than that of cultivars with *Pinb-2v2* (14.2). No significant difference was observed among cultivars with *Pinb-D1b*, possibly suggesting that the *Puroindoline b-2* variants had a larger impact in soft wheat than in hard.

No significant association between the puroindoline D1 alleles and the 9 agronomic traits relating to wheat yield were observed in the surveyed Chinese wheat cultivars of the Yellow and Huai Valleys, suggesting that the puroindoline genes have no apparent relationship with wheat grain yield. However, when the agronomic traits of cultivars with different *Puroindoline b-2* variant combinations were compared, the averages of grain number per spike, grain weight per spike, width of flag leaf and flag leaf area of cultivars with *Pinb-2v3* were significantly higher than those cultivars with *Pinb-2v2.* Differences in thousand kernel weight, grain length and spikelet number per spike were not different between these two *Pinb-2v2* and

A Puroindoline Mutigene Family Exhibits Sequence

Spikelet number per

Grain weight per spike

spike

(g)

traits

**4. Discussion** 

Diversity in Wheat and is Associated with Yield-Related Traits 285

No. of sample 2 23 19 48 21 71

SKCS Hardness 14.2a 24.6b 67.5a 60.7b - -

1000-kernel weight 59.2a 54.7b 50.9a 52.2b 51.7a 52.9a Grain length (mm) 6.85a 6.8a 6.61a 6.73a 6.63a 6.66a Grain width (mm) 3.65a 3.57a 3.47a 3.55a 3.49a 3.50a

Grain number per spike 42.1a 45.18b 40.7a 43.7b 40.86a 44.17b

Length of flag leaf (cm) 16.9a 17.4a 15.7a 17.9b 15.77a 17.70b

Width of flag leaf (cm) 1.85a 1.96a 1.85a 1.92a 1.85a 1.93a Area of flag leaf (cm2) 23.6a 25.6b 21.7a 25.7b 21.89a 25.69b

Table 2. Comparison of two *Puroindoline b-B2* variants on grain hardness and yield-related

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*

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

Different letters indicate significant difference at 5% probability level

combination was the predominant haplotype.

Wild type *Pinb-D1b* Total

*Pinb-2v2 Pinb-2v3 Pinb-2v2 Pinb-2v3 Pinb-2v2 Pinb-2v3* 

20.1a 20.83a 20.09a 20.59a 20.10a 20.67a

2.11a 2.27a 1.95a 2.17b 1.96a 2.21b

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

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.


Different letters indicate significant difference at 5% probability level

Table 2. Comparison of two *Puroindoline b-B2* variants on grain hardness and yield-related traits
