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Published in Crop Sci. 44:1808-1816 (2004).
© 2004 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Sequence Diversity of Puroindoline-a, Puroindoline-b, and the Grain Softness Protein Genes in Aegilops tauschii Coss

A. N. Massaa, C. F. Morrisb,* and B. S. Gillc

a Dep. of Crop & Soil Sciences, Washington State Univ., Pullman, WA 99164-6394
b USDA-ARS Western Wheat Quality Lab., E-202 Food Sci. & Human Nutrition East, Washington State Univ., Pullman, WA 99164-6394
c Dep. of Plant Pathology, Kansas State Univ., Manhattan, KS 66506-5502

* Corresponding author (morrisc{at}wsu.edu).


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aegilops tauschii Coss. constitutes a valuable resource of genetic variability for improving cultivated bread wheat, Triticum aestivum L. Nucleotide sequence diversity was examined at the Hardness (Ha) locus, which bears the Puroindoline-a (Pina-D1) and Puroindoline-b (Pinb-D1) genes, and the closely related Gsp-D1 gene. A distinct signature of polymorphism and selection was evident in each of the three genes across loci for the 50 accessions of Ae. tauschii. Pinb-D1 was characterized by a high level of nucleotide diversity ({pi} = 0.02966; {theta} = 0.0166) relative to a low level of variation in the neighboring linked Pina-D1 and Gsp-D1 genes. Despite the close genetic and physical linkage among these three loci, recombination and linkage disequilibrium analyses detected at least two recombination events, most likely at the interlocus level. Tests for neutrality showed that polymorphism at Pina-D1 and Gsp-D1 did not depart from a drift–mutation model. Genetic variation at the Pinb-D1 locus was higher than expected under neutrality suggesting that selection might operate to maintain polymorphism at this locus. The sequence variation resolved here will help elucidate the kind of variation that may be important for the function of these genes as well as to identify novel Ae. tauschii genotypes that may be used to broaden the range in wheat endosperm texture. Lastly, these results on sequence diversity, linkage disequilibrium, and recombination may help in the development of new strategies for breeding purposes.


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEXAPLOID WHEAT (T. aestivum) was formed some 7000 to 10 000 yr ago through one or more rare hybridization events between a wild tetraploid species (AABB) and a diploid Ae. tauschii (DD) (McFadden and Sears, 1946; Zohary and Hopf, 2000). Consequently, all modern wheat is limited by a bottleneck of evolution, and the specific genetic composition of those few individual Ae. tauschii plants of long ago (Ladizinsky, 1984; Talbert et al., 1998).

Substantial genetic variation exists within Ae. tauschii as compared with the D genome of wheat (Galili et al., 2000; Gill et al., 1986; Lagudah et al., 1991; Lelley et al., 2000). Estimates of RFLP diversity at RbcS have shown that wheat has, on average, approximately 30% of the diversity levels found in its diploid relatives of the genera Aegilops and Triticum, where the species with the D and the S genomes were the most polymorphic (Galili et al., 2000). Then, in continuing to characterize the genetic variability of Ae. tauschii, the assessment of nucleotide polymorphism for genes that play a role in endosperm texture of modern hexaploid wheat will be significant for understanding the kind of variation that may be important for the function of these genes.

Indolines are basic, cysteine-rich proteins characterized by a tryptophan-rich domain and occur in a variety of cereals of the Triticeae tribe (Blochet et al., 1993; Gautier et al., 1994). In hexaploid, wheat these proteins are present in two recognized isoforms, Puroindoline-a (Pina-D1) and Puroindoline-b (Pinb-D1) (from Greek puro = wheat, and indoline, for the indole ring of tryptophan). Genes encoding these two proteins have been mapped to the Hardness (Ha) locus on the short arm of chromosome 5D (Mattern et al., 1973). Puroindoline genes constitute the molecular genetic basis of grain endosperm texture and therefore represent the major genetic components that determine variation in wheat grain hardness (Morris, 2002). The wild-type allele (Ha) determines the soft endosperm phenotype, as that observed in T. aestivum ‘Chinese Spring’, while the hard phenotype (ha) is a result of mutations in either Puroindoline-a or -b gene (Giroux and Morris, 1997, 1998; Lillemo and Morris, 2000; Morris, 2002). Although puroindoline gene variation was recently investigated in diploid Aegilops and Triticum species (Gautier et al., 2000; Lillemo et al., 2002; Morris et al., 2001), little is known about the variability of these genes in Ae. tauschii.

Grain softness proteins (GSPs) are closely related to puroindolines and all belong to the same group of proteins that includes the chloroform–methanol-soluble proteins and the nonspecific lipid transfer proteins (Gautier et al., 1994). Furthermore, the Grain Softness Protein-1 gene (Gsp-D1) is closely linked to puroindolines genes on chromosome 5DS of wheat (Jolly et al., 1996) and on chromosome 5Am of T. monococcum L. (Gsp-Am1), where Puroindoline-a is positioned between Puroindoline-b and Gsp-1 (Tranquilli et al., 1999; Turnbull et al., 2003). Despite the close relationship among these three genes, there is no clear evidence of the role of Gsp-1 on grain endosperm texture (Tranquilli et al., 2002).

This study examined the extent of nucleotide diversity of Puroindoline-a (Pina-D1), Puroindoline-b (Pinb-D1), and Grain Softness Protein-1 (Gsp-D1) genes in 50 Ae. tauschii accessions collected from their center of diversity. The primary goal was to provide a comprehensive, overall view of these loci with regard to nucleotide sequence diversity, neutrality–selection, linkage disequilibrium, and recombination at an important region of wheat. The ultimate goal is to gain better understanding of the amino acids that may be required for the endosperm "softening" effect of puroindolines, as well as identify allelic variants of Ae. tauschii that may be functionally significant in wheat grain texture.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Material
Seeds of Ae. tauschii were obtained from the Wheat Genetic Resources Center, Kansas State Univ., Manhattan, KS (Table 1). Accessions were those originally collected by Kihara et al. (1965) in the center of diversity and included the two morphological subspecies, i.e., Ae. tauschii subsp. tauschii and Ae. tauschii subsp. strangulata (Eig.) Tzvelev. Two seeds per accession were planted and seedlings were vernalized at 4°C for 5 wk. Plants were grown in the greenhouse to maturity.


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  		Table 1. Puroindoline-a (Pina-D1), Puroindoline-b (Pinb-D1), and Grain Softness Protein-1 (Gsp-D1) alleles in Aegilops tauschii accessions.

 
DNA Analysis
Genomic DNA was isolated from young leaves according to the method of Murray and Thompson (1980). Sequence analysis was performed on a single plant per accession because of the self-pollinating reproductive biology of Ae. tauschii and the highly homozygous populations of the accessions (Lubbers et al., 1991). Full-length Pina-D1 (447 bp), Pinb-D1 (447 bp), and Gsp-D1 (495 bp) genes were amplified with gene-specific primers (Table 2), which were designed with Primer3 software (Rozen and Skaletsky, 2000). 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, 0.5 unit of Taq DNA polymerase (Promega, Madison, WI), and 1.5 µM of MgCl2. The cycling conditions were 94°C for 4 min followed by 35 cycles of 94°C for 1 min, 58°C for 90 s, 72°C for 2 min. The extension was 10 min at 72°C. PCR products were analyzed on 1.5% (w/v) agarose gels, stained with ethidium bromide, and visualized by means of UV light. PCR products were purified from dNTPs and oligonucleotide primers with Exonuclease I and Shrimp Alkaline Phosphatase (ExoSAP-IT, UBS, Cleveland, OH). Sequencing reactions were performed with the Big Dye Terminator Version 3.1 Cycle Sequencing Kit and sequenced with an Applied Biosystems 3100 Genetic Analyzer (PerkinElmer Applied Biosystems Division, Foster City, CA). Singleton variants were confirmed by sequencing (both directions) PCR products from two plants to eliminate PCR artifacts.


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  		Table 2. Gene-specific PCR primers for producing full-length sequences of the genes Puroindoline-a (Pina-D1), Puroindoline-b (Pinb-D1), and Grain Softness Protein-1 (Gsp-D1).

 
Data Analysis
All sequences were aligned by ClustalW algorithm (Thompson et al., 1997) and have been deposited in the GenBank database under accession numbers AY251946 to AY252095 and AY255771. DNA sequences of T. aestivum were also included for comparison. DNA analyses were conducted by DnaSP version 4.0 (Rozas and Rozas, 1999). Nucleotide diversity was estimated as theta ({theta}), the number of segregating (polymorphic) sites (Watterson, 1975), and pi ({pi}), the average number of nucleotide differences per site between two sequences (Nei, 1987). Tests of neutrality were performed using Tajima's D (1989), and Fu and Li's D* and F* (Fu and Li, 1993) statistics. To test for intra- and interlocus linkage disequilibrium (LD) and recombination, the three genes were concatenated into a single sequence and arranged in the same order in which the genes are arranged along the chromosome (Tranquilli et al., 1999; Turnbull et al., 2003). The degree of LD between segregating sites was estimated by the D' and r2 parameters as implemented by DnaSP v.4.0 (Rozas and Rozas, 1999) and TASSEL (Buckler, software at http://www.maizegenetics.net/bioinformatics/tasselindex.htm; verified 27 April 2004), whereas recombination events were calculated by the four-gamete test of Hudson and Kaplan (1985). Haplotype trees were created using the program TCS v.1.13 (Clement et al., 2000), which determines allele relationships within the limits of parsimony criterion.


   RESULTS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene-specific PCR primers (Table 2) amplified the 444-bp coding region of both puroindoline genes (Gautier et al., 1994) and the 492-bp coding sequence of the Gsp-D1 gene (Rahman et al., 1994; Turner et al., 1999) for all 50 accessions of Ae. tauschii. Sequences of Pina-D1 and Pinb-D1 genes were aligned with T. aestivum ‘Capitole’ (accessions X69913 and X69914, Pina-D1, and Pinb-D1, respectively), whereas Gsp-D1 gene was compared with the Gsp-A1 gene sequence reported earlier (Turner et al., 1999) and several Gsp-1 gene sequences from the NCBI dbEST. Pairwise comparisons within genes diverged only by single nucleotide polymorphisms (SNPs), i.e., no insertion–deletion (indel) polymorphisms were detected in the coding regions.

Nucleotide Sequence Polymorphism
Pinb-D1 displayed the highest level of SNPs among the three genes with 33 polymorphic sites in the 444-bp coding region (Table 3). Eighteen of these SNPs were synonymous and 15 were nonsynonymous. Genealogical analysis resolved four different haplotypes, thus only a low level of SNPs were segregating among accessions within each of the four allelic variants (Fig. 1A) . One of these haplotypes was 100% identical to the puroindoline soft ("wild-type") allele found in Chinese Spring and was assigned Pinb-D1a (Table 1, Fig. 1A). The remaining three haplotypes were designated Pinb-D1h, Pinb-D1i, and Pinb-D1j following the convention established for allelic designations of the Pinb-D1 gene in hexaploid wheat (McIntosh et al., 1998; Morris, 2002). Although three of the four alleles were at a similar frequency, Pinb-D1i was the most common and broadly distributed (Table 1; Fig. 1A). The four haplotypes encode three different polypeptides of 148 amino acids each (Fig. 2A) . The 10 cysteine residues and the tryptophan-rich domain were found to be conserved.


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  		Table 3. Summary of DNA polymorphism and test statistics for selection in 50 Ae. tauschii accessions, with average number of nucleotide differences (n), estimates of number of segregating sites (s), number of haplotypes (h), SNP frequency, and nucleotide diversity ({theta}, {pi}){dagger}.

 


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  		Fig. 1. Gene genealogy of Pinb-D1 (A), Pina-D1 (B), and Gsp-D1(C) for 50 Aegilops tauschii accessions using the statistical parsimony reconstruction method, as implemented by TCS version 1.13. All branches represent a single mutational change. Haplotypes are indicated by the alleles. The haplotypes with the highest outgroup probability are displayed as squares. The size of squares and ovals correspond to the haplotype frequency. Small, unlabeled ovals represent haplotypes inferred from mutational changes, but absent in this data set.

 


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  		Fig. 2. Comparative analysis of the deduced amino acid sequences of Puroindoline-a, -b and Grain Softness Protein-1 from Ae. tauschii and the wild-type ‘a’ alleles of T. aestivum. A dot (.) shows identity with the first sequence. Underlined amino acids indicate synonymous substitutions relative to the wild-type. Double underlined amino acids indicate synonymous and nonsynonymous substitutions.

 
Single nucleotide polymorphisms at Pina-D1 and Gsp-D1 were considerably lower than those observed in Pinb-D1 (Table 3). Over the entire Pina-D1 coding region, only five sites were polymorphic with an average number of 1.1 nucleotide differences between sequences (Table 3). All SNPs were transitions, and only one was nonsynonymous (A/G), which changed the codon CGG (ARG) to CAG (GLN) at position 58 (Fig. 2B) resulting in a non-conservative replacement. Gene genealogy at Pina-D1 traced evolutionary relationships among six allelic lineages, one was 100% identical to the wild-type allele of Chinese Spring, and therefore assigned the Pina-D1a allele designation (Fig. 1B). Remarkably, 12 of the 17 Ae. tauschii accessions carrying this allele also shared the Puroindoline-b soft allele of hexaploid wheat (Table 1). The remaining five alleles were designated by Pina-D1c, Pina-D1d, Pina-D1e, Pina-D1f, and Pina-D1g according to the allelic designation of Pina-D1 gene in T. aestivum (McIntosh et al., 1998, Morris 2002). Pina-D1a, Pina-D1c, and Pina-D1d were highly represented among the 50 accessions, while Pina-D1f and Pina-D1g were the least frequent (Fig. 1B). The six haplotypes encode two different polypeptides (Fig. 2B). One of these corresponded to the wild-type allele of Pina-D1 in hexaploid wheat and the other contains the ARG-to-GLN mutation at position 58. This mutation was present in all of the remaining 32 accessions of Ae. tauschii.

Single nucleotide polymorphisms at the Gsp-D1 locus occurred at seven sites in the 492-bp coding region (Table 3). Four of seven SNPs were nonsynonymous, whereas only one was a non-conservative replacement (Fig. 2C). Multiple sequence alignments included 50 Gsp-D1 gene sequences from Ae. tauschii and one from T. aestivum ‘Yecora Rojo’, which was amplified by means of the gene-specific primers listed in Table 2. Sequences were also compared to those of T. aestivum that were from publicly available databases, including Gsp-A1 from ‘Rosella’ (Turner et al. (1999), accession AF177218), Gsp-1a from ‘Timgalen’ (Rahman et al. (1994), accession S72696), and Gsp-1 gene sequence from Chinese Spring (Ogihara and Murai, unpublished, GeneBank dbEST, accession BJ242937). The 50 accessions coalesced into seven distinct haplotypes (Fig. 1C). None of these allelic variants however, were 100% identical to those of T. aestivum. Therefore they were designated Gsp-D1b, Gsp-D1c, Gsp-D1d, Gsp-D1e, Gsp-D1f, Gsp-D1g, and Gsp-D1h (Table 1) following the prior convention (McIntosh et al., 1998; Morris, 2002). The Gsp-1 sequence obtained from Yecora Rojo was 100% identical to that of Chinese Spring (above) and Timgalen (above), which was mapped to 5DS (Jolly et al., 1996).

Although none of the Gsp-D1 alleles from Ae. tauschii were 100% identical to the sequence of hexaploid wheat, the Gsp-D1b allele was closely related to the sequence of the cultivars Yecora Rojo, Chinese Spring, and Timgalen. Gsp-D1b, which we mapped to chromosome 5DS using the Chinese Spring deletion line 5DS-2 (Endo and Gill, 1996), was 99.8% identical to the sequence of Gsp-1a and differed by one nonsynonymous nucleotide substitution, which changed the codon UCG (SER) to CCG (PRO). Interestingly, the group of Ae. tauschii accessions bearing the Gsp-D1b allele also shared the wild-type allele of both puroindoline genes. This group of accessions, which was classified as Ae. tauschii subsp. tauschii, was collected from natural populations over a vast geographic region including Armenia, Azerbaijan, Iran, Turkmenistan, and Turkey (Table 1). The seven Gsp-D1 haplotypes encoded four different polypeptides of 164 amino acids each (Fig. 2C). Comparison of deduced sequences revealed a total of four polymorphic sites with an average of 1.6 amino acid differences between any two sequences. Results also showed that the 10 cysteine residues in the deduced Grain Softness Protein were conserved.

Tests of Neutrality
Levels of nucleotide diversity as estimated by {theta} (Watterson, 1975) and {pi} (Nei, 1987) were evaluated for deviation from an equilibrium neutral model. These two estimates are expected to give similar values under a drift–mutation balance; otherwise, any form of natural selection may explain the maintenance of genetic variation. Two distinct signatures of polymorphism and selection were observed in this dataset. Pina-D1 and Gsp-D1 varied in a similar fashion, with nucleotide diversity estimates approximately eight-fold lower than that observed in Pinb-D1 (Table 3). Based on Tajima's (1989) and Fu and Li's (1993) tests statistics, the levels and patterns of polymorphism at these two loci were consistent with a neutral equilibrium (Table 3). Conversely, nucleotide diversity at Pinb-D1 was significantly higher than expected under neutrality (Table 3), with significantly positive values in all tests statistics. This finding as well as the observation of intermediate frequency variants at Pinb-D1 (Fig. 1A) is strong evidence that polymorphism at this locus is maintained by natural selection.

A sliding window analysis of DNA polymorphism and divergence between Ae. tauschii and an outgroup species, Triticum urartu Thüm. ex Gandilyan (sequences AJ302094, AJ302095, AJ302103, AJ302104, and unpublished data), was also performed across all gene arrangements (Fig. 3) . At the Pinb-D1 region (nucleotide positions 1–444, Fig. 3), the sliding window of the overall nucleotide site showed a similar to, or even higher divergence between Ae. tauschii alleles relative to the divergence between species. In contrast, nucleotide divergence at the Pina-D1 and Gsp-D1 regions, nucleotide positions 445 to 888 and 889 to 1380, respectively (Fig. 3), was lower between Ae. tauschii alleles compare to the divergence between species.



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  		Fig. 3. Sliding window analysis of overall polymorphic sites across loci. Abscissa indicates nucleotide position. Ordinate indicates nucleotide diversity between Ae. tauschii alleles (Pi) and divergence between Ae. tauschii and T. urartu (K) as estimated by the Jukes and Cantor (1969) correction.

 
Linkage Disequilibrium and Recombination
Pairwise comparisons of nucleotide variants across loci (1380 bp) were tested for a nonrandom association. Only polymorphic sites at which alleles occurred in more than 5% of the accessions (41 sites) were included in the analysis. A total of 575 (70%) of the 820 possible pairwise comparisons were significant (P < 0.01) by the Fisher's exact test, and 486 (49%) remained significant after applying the Bonferroni procedure (Fig. 4 , lower left). Disequilibrium tests were partitioned into intra- and interlocus site comparisons. On average, 48% of within-locus test and 50% of between-locus tests were in significant LD. A much greater level of LD was observed in Pinb-D1 compared with either of the other two genes (Pina-D1 and Gsp-D1) and to comparisons among all three genes (Fig. 4). Most of the polymorphic sites at Pinb-D1, 438 of the 528 site comparisons (83%), exhibited a highly significant association, while only 1 out of 3 (33%) and 4 out of 15 site comparisons (27%) in Pina-D1 and Gsp-D1, respectively, yielded significant tests (Fig. 4).



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  		Fig. 4. Linkage disequilibrium estimates for Pina-D1, Pinb-D1, and Gsp-D1 from Aegilops tauschii. Estimates consider only the 41 polymorphic sites that were present in at least 5% of the accessions examined (nucleotide site numbers are listed down the left-hand side and across the top, and are arranged according to the concatenated three-gene model). The lower left of the black-square diagonal indicates the significance level of each pair-wise comparison as determined by the Fisher's exact test (color code appears in the lower right as "Lower P value"). The upper right of the black-square diagonal indicates the correlation (r2) between the 41 pairs of polymorphic sites (color code appears in the upper right as "Upper R->2"). At the bottom of the figure is a display of the polymorphic sites along the concatenated three genes.

 
The four-gamete test (Hudson and Kaplan, 1985) showed no evidence for intragenic recombination. However, at least two recombination events were detected across all gene arrangements (1380 bp), one between nucleotide positions 99 and 501, and another between positions 510 and 925. These nucleotide intervals include the inter-locus site between Pinb-D1 and Pina-D1, and the interlocus site between Pina-D1 and Gsp-D1 loci, respectively (Fig. 4).


   DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study examined the extent of nucleotide sequence variation in genes located at the hardness (Ha) locus on chromosome 5DS of Ae. tauschii. The assessment of genetic diversity at these closely located and closely linked loci provided insight into neutrality–selection models and patterns of linkage disequilibrium and recombination in an important region of the D genome of wheat. Results document distinct signature of polymorphism and selection in each of the three genes and across loci in Ae. tauschii. Pinb-D1 was characterized by a high level of nucleotide polymorphism and diversity relative to a low level of variation in the neighboring linked Pina-D1 and Gsp-D1 genes. The degree of polymorphism in Pinb-D1 is similar to that reported for the adh3 gene in wild barley (Lin et al., 2002) or the RPS5 locus in Arabidopsis (Tian et al., 2002), which was also found to be higher than the average reported values in other Arabidopsis genes. Enhanced polymorphism in combination with an excess of intermediate frequency variants and deep coalescence, like that observed at the Pinb-D1 locus, is seen as evidence of balancing selection. Under the balanced model hypothesis, polymorphisms can be maintained by selection over an extended period. Thus, polymorphism at Pinb-D1 might have been maintained in the most recent common ancestor of the Pooideae subfamily. Therefore, during speciation events, copies of the same gene lineage could have been inherited by two (or more) species, and some allelic variants in each species are genetically more closely related to allelic variants in other species than they are to those in the same species. Consistent with this selection model, a sliding window analysis at the Pinb-D1 region showed that divergence between alleles, within Ae. tauschii, was similar to, or even higher than divergence between Ae. tauschii and T. urartu. Likewise, Pinb-D1 alleles of Ae. tauschii, except Pinb-D1a, were more similar to those of T. monococcum (accessions AJ243994, AJ302102, AJ302101, and AJ242716) than they were to Pinb-D1a. Remarkably, the Pinb-D1h allele of Ae. tauschii was found to be 100% identical to that reported for T. monococcum, accession AJ249934 (Gautier et al., 2000). Further investigations including accessions of diploid Aegilops and Triticum species are underway and may help elucidate the stochastic models of sequence evolution at Pinb-D1.

Linkage disequilibrium and recombination analyses suggested that the genomic region bearing the Pina-D1, Pinb-D1, and Gsp-D1 genes has undergone genetic exchange, most probably at the inter-locus level. Evidence for recombination among these loci has also been reported in F2 plants of T. monococcum between Pina- Am1 and Pinb-Am1 (Tranquilli et al., 1999); although complete linkage was found between the Pina-Am1 and Gsp-Am1 loci (Tranquilli et al., 1999), or between Pinb- D1 and the Pina-D1 in hexaploid wheat (Giroux et al., 2000). It has been proposed that gene-rich chromosome regions like that observed in the Ha locus region (Tranquilli et al., 1999), are also rich in recombination (Gill et al., 1996). However, even though recombination may occur, most recombination events may be undetectable in highly self-fertilizing species like several Aegilops or Triticum taxa (Nordborg et al., 2002). Further DNA sequence data between these loci may help localize the sites of recombination as well as determine the extent of LD over the entire region.

Although the origin of the Ha locus is unknown, accessions from Iran showed the highest levels of haplotype diversity. Interestingly, Ae. tauschii accessions from Iran have also been reported to be a good source of genetic variation for insect and disease resistant genes (Cox et al., 1991; Gill et al., 1986). In the present study, all the allelic variants, except Pina-D1g and Gsp-D1h, were present in accessions collected in Iran. Yet, no geographic patterns of DNA polymorphism were found across all gene arrangements, and identical genotypes were widely dispersed over the broad geographic regions.

Aegilops tauschii contains genetic variability not found in hexaploid wheat. Novel Pina-D1, Pinb-D1, and Gsp-D1 haplotype sequences were detected in the analyses. With the exception of the wild-type allele of Pina-D1 and Pinb-D1 genes, as defined by convention as those found in Chinese Spring and present in all other soft wheat varieties (Morris, 2002), none of the other Pinb-D1 alleles detected in Ae. tauschii accessions have been found in modern hexaploid wheat. Conversely, none of the previously reported Pina-D1 and Pinb-D1 alleles, those that confer hard endosperm texture in modern hexaploid wheat varieties (Morris, 2002), were found in the Ae. tauschii accessions investigated in this study.

Among the 50 Ae. tauschii subsp. tauschii accessions, 13 were the most closely related to wheat. They carried the wild-type allele of both Puroindoline-a and -b genes, and the Gsp-D1b allele of the Grain Softness Protein-1 gene, which was found to differ by only one nucleotide compared to Chinese Spring. Although none of the Gsp-D1 alleles were 100% identical to that of hexaploid wheat, the presence of the wild-type allele of the puroindoline genes in a group of accessions of Ae. tauschii subsp. tauschii, which also possessed the highly similar Gsp-D1b allele favors the hypothesis that this group is the most closely related to the donor of the D genome of modern hexaploid wheat. These findings, however, do not support the assertion that subsp. strangulata is the major donor to the D genome of hexaploid wheat (Dvoràk et al., 1998; Lagudah et al., 1991; Lubbers et al., 1991).

Overall, these results on sequence diversity of Ae. tauschii open up new perspectives for studying the role of puroindolines in grain texture in wheat and other cereals, in addition to facilitating preliminary selection of Ae. tauschii germplasm for the genetic manipulation of this important trait. Complementary studies to evaluate the phenotypic effects of the genetic variation of these genes have been initiated.


   ACKNOWLEDGMENTS
 
We thank Garrison King for helpful assistance in plant culture and Shelle Freston for technical assistance in DNA isolation.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mention of trademark or proprietary products does not constitute a guarantee or warranty of a product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable. This article is in the public domain and not copyrightable.

Received for publication August 19, 2003.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome