Puroindolines: the molecular genetic basis of wheat grain hardness

The variation in grain hardness is the single most important trait that determines end-use quality of wheat. Grain texture classification is based primarily on either the resistance of kernels to crushing or the particle size distribution of ground grain or flour. Recently, the molecular genetic basis of grain hardness has become known, and it is the focus of this review. The puroindoline proteins a and b form the molecular basis of wheat grain hardness or texture. When both puroindolines are in their `functional' wild state, grain texture is soft. When either one of the puroindolines is absent or altered by mutation, then the result is hard texture. In the case of durum wheat which lacks puroindolines, the texture is very hard. Puroindolines represent the molecular-genetic basis of the Hardness locus on chromosome 5DS and the soft (Ha) and hard (ha) alleles present in hexaploid bread wheat varieties. To date, seven discrete hardness alleles have been described for wheat. All involve puroindoline a or b and have been designated Pina-D1b and Pinb-D1b through Pinb-D1g. A direct role of a related protein, grain softness protein (as currently defined), in wheat grain texture has yet to be demonstrated.     

Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars

Kernel hardness that is conditioned by puroindoline genes has a profound effect on milling, baking and end-use quality of bread wheat. In this study, 219 landraces and 166 historical cultivars from China and 12 introduced wheats were investigated for their kernel hardness and puroindoline alleles, using molecular and biochemical markers. The results indicated that frequencies of soft, mixed and hard genotypes were 42.7, 24.3, and 33.0%, respectively, in Chinese landraces and 45.2, 13.9, and 40.9% in historical cultivars. The frequencies of PINA null, Pinb-D1b and Pinb-D1p genotypes were 43.8, 12.3, and 39.7%, respectively, in hard wheat of landraces, while 48.5, 36.8, and 14.7%, respectively, in historical hard wheats. A new Pinb-D1 allele, designated Pinb-D1t, was identified in two landraces, Guangtouxianmai and Hongmai from the Guizhou province, with the characterization of a glycine to arginine substitution at position 47 in the coding region of Pinb gene. Surprisingly, a new Pina-D1 allele, designated Pina-D1m, was detected in the landrace Hongheshang, from the Jiangsu province, with the characterization of a proline to serine substitution at position 35 in the coding region of Pina gene; it was the first novel mutation found in bread wheat, resulting in a hard endosperm with PINA expression. Among the PINA null genotypes, an allele designed as Pina-D1l, was detected in five landraces with a cytosine deletion at position 265 in Pina locus; while another novel Pina-D1 allele, designed as Pina-D1n, was identified in six landraces, with the characterization of an amino acid change from tryptophan-43 to a ‘stop’ codon in the coding region of Pina gene. The study of puroindoline polymorphism in Chinese wheat germplasm could provide useful information for the further understanding of the molecular basis of kernel hardness in bread wheat.     

Prevalence of Puroindoline D1 and Puroindoline b-2 variants in U.S. Pacific Northwest wheat breeding germplasm pools, and their association with kernel texture

Kernel texture is a major factor influencing the classification and end use properties of wheat (Triticum aestivum L.), and is mainly controlled by the Puroindoline a (Pina) and Puroindoline b (Pinb) genes. Recently, a new puroindoline gene, Puroindoline b-2 (Pin b-2), was identified. In this study, 388 wheat cultivars and advanced breeding lines from the U.S. Pacific Northwest were investigated for frequencies of Puroindoline D1 alleles and Pinb-2 variants 2 and 3. Results indicated that Pinb–D1b (74.0%) was the predominant genotype among hard wheats (N = 196), the only other hard allele encountered was Pina-D1b (26.0%). Across all varieties, Pinb-2v3 was the predominant genotype (84.5%) compared with Pinb-2v2 (15.5%). However, among 240 winter wheat varieties (124 soft white, 15 club, 68 hard red and 33 hard white varieties), all carried Pinb-2v3. Among spring wheats, Pinb-2v2 and Pinb-2v3 frequencies were more variable (soft white 25.0:75.0, hard red 58.2:41.8 and hard white 40.0:60.0, respectively). Kernel texture variation was analyzed using 247 of the 388 wheat varieties grown in multi-location factorial trials in up to 7 crop years. The range of variety means among the four groups, soft winter, soft spring, hard winter and hard spring, was on the order of 15–25 single kernel characterization system (SKCS) Hardness Index. The least significant difference for each of these trials ranged from 2.8 to 5.6 SKCS Hardness Index. Observations lead to the conclusion that Pinb-2 variants do not exert a prominent effect on kernel texture, however, Pinb–2 variants do identify features of wheat germ plasm structure in the U.S. Pacific Northwest.     

Physical mapping of <Emphasis Type="Italic">puroindoline b</Emphasis>-<Emphasis Type="Italic">2</Emphasis> genes and molecular characterization of a novel variant in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L.)

The puroindoline genes (Pina and Pinb) are the functional components of the common or bread wheat (Triticum aestivum L.) grain hardness locus that are responsible for kernel texture. In this study, four puroindoline b-2 variants were physically mapped using nulli-tetrosomic lines of bread wheat cultivar Chinese Spring and substitution lines of durum wheat (Triticum turgidum L.) cultivar Langdon. Results indicated that Pinb-2v1 was on 7D of Chinese Spring, Pinb-2v2 on 7B of Chinese Spring, Pinb-2v3 on 7B of Chinese Spring and Langdon, and Pinb-2v4 on 7A of Chinese Spring and Langdon. A new puroindoline b-2 variant, designated Pinb-2v5, was identified at the puroindoline b-2 locus of durum wheat cultivar Langdon, with a difference of only five single nucelotide polymorphisms compared with Pinb-2v4. Sequencing results indicated that, in comparison with the Pinb-2v3 sequence (AM99733 and GQ496618 with one base-pair modification of G to T at 6th position, designated Pinb-2v3a) in bread wheat cultivar Witchta, the coding region of Pinb-2v3 in 12 durum wheat cultivars had a single nucleotide change from T to C at the 311th position, resulting in a corresponding amino acid change from valine to alanine at the 104th position. This new allele was designated Pinb-2v3b. The study of puroindoline b-2 gene polymorphism in CIMMYT and Italian durum wheat germplasm and discovery of a novel puroindoline b-2 variant could provide useful information for further understanding the molecular and genetic basis of kernel hardness and illustrating gene duplication events in wheat.     

Puroindoline A-gene expression is involved in association of puroindolines to starch

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars (Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.Communicated by H.F. Linskens     

Chromosome 5H of <Emphasis Type="Italic">Hordeum</Emphasis> species involved in reduction in grain hardness in wheat genetic background

Grain hardness is an important factor affecting end-use quality in wheat. Mutations of the puroindoline genes, which are located on chromosome 5DS, control a majority of grain texture variations. Hordoindoline genes, which are the puroindoline gene homologs in barley, are located on chromosome 5HS and are also responsible for grain texture variation. In this study, we used three types of wheat–barley species (Hordeum vulgare, H. vulgare ssp. spontaneum, and H. chilense) chromosome addition lines and studied the effect of chromosome 5H of these species on wheat grain characteristics. The 5H chromosome addition lines showed significantly lower grain hardness and higher grain weight than the corresponding wheat parents. The effect of enhancing grain softness was largest in the wheat–H. chilense line regardless of having an increase in grain weight similar to those in the wheat–H. vulgare and wheat–H. spontaneum lines. Our results indicated that chromosome 5H of the Hordeum species plays a role in enhancing grain softness and increasing grain weight in the wheat genetic background, and the extent of effect on grain hardness depends on the type of Hordeum species. Protein analysis of hordoindolines indicated that profiles of 2D-electrophoresis of hordoindolines were different among Hordeum species and hordoindolines in the addition lines appeared to be most abundant in wheat–H. chilense line. The differences in enhancing grain softness among the Hordeum species might be attributed to the quantity of hordoindolines expressed in the 5H chromosome addition lines. These results suggested that the barley hordoindolines located on chromosome 5HS play a role in reducing grain hardness in the wheat genetic background.     

Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein

Kernel hardness is one of the most important factors determining the milling and processing quality of bread wheat (Triticum aestivum L.). In the present study, 267 wheat cultivars and advanced lines from the Yellow and Huai Valley of China, CIMMYT, Russia and Ukraine were used for identification of SKCS (Single Kernel Characterization System) hardness and puroindoline alleles. Results indicated that Pinb-D1b is the most popular genotype in wheat cultivars from the Yellow and Huai Valley, Russia and Ukraine, whereas PINA null is a predominant genotype in wheat cultivars and advanced lines from CIMMYT. Molecular characterization of PINA-null alleles indicated that one Chinese landrace Chiyacao had the allele Pina-D1l with a single nucleotide C deletion at position 265 in Pina coding region based on sequencing results, and 35 of 39 PINA-null alleles belonged to Pina-D1b according to PCR amplification with the sequence-tagged site (STS) marker Pina-N developed previously. The remaining three cultivars (Jiangdongmen, Heshangtou and Hongquanmang from China) with PINA-null alleles were characterized at the DNA level by a primer walking strategy, and the results showed that all three cultivars with PINA-null alleles possessed a uniform 10,415-bp deletion from −5,117 bp to +5,298 bp (ATG codon references zero), designated as Pina-D1r. Correspondingly, an STS marker Pina-N2 with an expected fragment size of 436-bp spanning the 10,415-bp deletion was developed for detection of the Pina-D1r allele. This study provided a useful molecular marker for straightforward detection of one of the PINA-null alleles and would also be helpful to further understand the molecular and genetic basis of kernel hardness in bread wheat.     

Wheat puroindolines interact to form friabilin and control wheat grain hardness

Wheat grain is sold based upon several physiochemical characteristics, one of the most important being grain texture. Grain texture in wheat directly affects many end use qualities such as milling yield, break flour yield, and starch damage. The hardness (Ha) locus located on the short arm of chromosome 5D is known to control grain hardness in wheat. This locus contains the puroindoline A (pina) and puroindoline B (pinb) genes. All wheats to date that have mutations in pina or pinb are hard textured, while wheats possessing both the soft type pina-D1a and pinb-D1a sequences are soft. Furthermore, it has been shown that complementation of the pinb-D1b mutation in hard spring wheat can restore a soft phenotype. Here, our objective was to identify and characterize the effect the puroindoline genes have on grain texture independently and together. To accomplish this we transformed a hard red spring wheat possessing a pinb-D1b mutation with soft type pina and pinb, creating transgenic isolines that have added pina, pinb, or pina and pinb. Northern blot analysis of developing control and transgenic lines indicated that grain hardness differences were correlated with the timing of the expression of the native and transgenically added puroindoline genes. The addition of PINA decreased grain hardness less than the reduction seen with added PINB. Seeds from lines having more soft type PINB than PINA were the softest. Friabilin abundance was correlated with the presence of both soft type PINA and PINB and did not correlate well with total puroindoline abundance. The data indicates that PINA and PINB interact to form friabilin and together affect wheat grain texture.Communicated by J. Dvorak     

The determinants of grain texture in cereals

Kernel hardness is an important agronomic trait that influences end-product properties. In wheat cultivars, this trait is determined by thePuroindoline a (Pina) andPuroindoline b (Pinb) genes, located in theHardness locus (Ha) on chromosome 5DS of the D genome. Wild type alleles code puroindoline a (PINA) and puroindoline b (PINB) proteins, which form a 15-kDa friabilin present on the surface of water-washed starch granules. Both the proteins are accumulated in the starch endosperm cells and aleurone of the mature kernels.Puroindoline-like genes coding puroindoline-like proteins in the starch endosperm occur in some of the genomes of Triticeae and Aveneae cereals. Orthologs are present in barley, rye and oats. However, some genomes of these diploid and polyploid cereals, like that ofTriticum turgidum var.durum (AABB) lack thepuroindoline genes, having a very hard kernel texture. The two wild type alleles in opposition (dominant loci) control the soft pheno-type. Mutation either inPina orPinb or in both leads to a medium-hard or hard kernel texture. The most frequent types ofPin mutations are point mutations within the coding sequence resulting in the substitution of a single amino acid or a null allele. The latter is the result of a frame shift determined by base deletion or insertion or a one-point mutation to the stop codon. The lipid-binding properties of the puroindolines affect not only the dough quality but also the plants’ resistance to pathogens. Genetic modification of cereals withPuroindoline genes and/or their promoters enable more detailed functional analyses and the production of plants with the desired characteristics.     

Molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses

The hardness or texture of cereal grains is a primary determinant of their technological and processing quality. Among members of the Triticeae, most notably wheat, much of the variation in texture is controlled by a single locus comprised of the Puroindoline a, Puroindoline b and Grain Softness Protein-1 (Gsp-1) genes. Puroindolines confer the three major texture classes of soft and hard common wheat and the very hard durum wheat. The protein products of these genes interact with lipids and are associated with the surface of isolated starch (as a protein fraction known as ‘friabilin’). During the past ten years a great diversity of alleles of both Puroindoline genes have been discovered and significant advances made in understanding the relationship between the gene presence/absence, sequence polymorphism and texture of cereal grains. Efforts have also focussed on Puroindoline and Gsp-1 genes in diploid progenitors, other Triticeae grasses and synthetic wheats in order to understand the evolution of this gene family and find potentially useful variants. The puroindoline homologues in other cereals such as rye and barley are also receiving attention. This work summarises new developments in molecular genetics of puroindolines in wheat and related Triticeae grasses, and the related genes in other cereals.     

Starch-bound 2S proteins and kernel texture in einkorn, <Emphasis Type="Italic">Triticum</Emphasis> <Emphasis Type="Italic">monococcum</Emphasis> ssp <Emphasis Type="Italic">monococcum</Emphasis>

The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE. All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B), as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured common wheat, “monococcum” accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules, respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele Eti-A ^m 1a and “valine-type” ETI encoded by allele Eti-A ^m 1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low as −2.05 ± 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in “monococcum” wheat, as determined by visual assessments in counting chambers, was estimated at 764 mm²/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification of factors that cause the extra-soft texture of einkorn kernels.     

Expression of wild-type pinB sequence in transgenic wheat complements a hard phenotype

Wheat grain hardness is a major factor in the wheat end-product quality. Grain hardness in wheat affects such parameters as milling yield, starch damage and baking properties. A single locus determines whether wheat is hard or soft textured. This locus, termed Hardness ( Ha), resides on the short arm of chromosome 5D. Sequence alterations in the tryptophan-rich proteins puroindoline a and b (PINA and PINB) are inseparably linked to hard textured grain, but their role in endosperm texture has been controversial. Here, we show that the pinB-D1b alteration, common in hard textured wheats, can be complemented by the expression of wild-type pinB-D1a in transformed plants. Transgenic wheat seeds expressing wild-type pinB were soft in phenotype, having greatly increased friabilin levels, and greatly decreased kernel hardness and damaged starch. These results indicate that the pinB-D1b alteration is most likely the causative Ha mutation in the majority of hard wheats.     

Real Time RT-PCR and flow cytometry to investigate wheat kernel hardness: role of puroindoline genes and proteins

Developing seeds from Triticum aestivum (wheat) cultivars were collected after flowering and analysed for puroindoline a and b gene expression by Real Time RT-PCR. Mature seeds were investigated for the presence and the amount of starch-associated puroindoline a and b proteins by flow cytometry. Puroindoline a gene and protein were found to have a predominant role in controlling wheat kernel hardness.     

The Ha locus of wheat: identification of a polymorphic region for tracing grain hardness in crosses.

The grain softness proteins or friabilins are known to be composed of three main components: puroindoline a, puroindoline b, and GSP-1. cDNAs for GSP-1 have previously been mapped to group-5 chromosomes and their location on chromosome 5D is closely linked to the grain hardness (Ha) locus of hexaploid wheat. A genomic DNA clone containing the GSP-1 gene (wGSP1-A1) from hexaploid wheat has been identified by fluorescent in situ hybridization as having originated from the distal end of the short arm of chromosome 5A. A genomic clone containing the gene (wGSP1-D1) was also isolated from Aegilops tauschii, the donor of the D genome to bread wheat. There are no introns in the GSP-1 genes, and there is high sequence identity between wGSP1-A1 and wGSP1-D1 up to 1 kb 5' and 300 bp 3' to wGSP1-D1. However, regions further upstream and downstream of wGSP1-D1 share no significant sequence identity to corresponding sequences in wGSP1-A1. These regions therefore identified potentially valuable sequences for tracing the Ha locus through assaying polymorphic DNA sequences. The sequence from 300 to 500 bp 3' to wGSP1-D1 (wGSP1-D13) was mapped to the Ha locus in a mapping population. wGSP1-D13 was also tightly linked to genes for puroindoline a and puroindoline b which have been previously mapped to be at the Ha locus. In addition wGSP1-D13 was used to detect RFLPs between near isogenic soft and hard Falcon lines and in a random selection of soft and hard wheats.     

Genetic and physical characterization of grain texture-related loci in diploid wheat

Endosperm texture, i.e. the hardness or softness of the grain, is an important quality criterion in cereals because it determines many grain end-use properties. Grain softness is the dominant trait and is mainly controlled by the Ha locus on the short arm of chromosome 5D in hexaploid bread wheat. Genes for puroindoline a (Pina-D1), puroindoline b (Pinb-D1), and grain softness related protein (Gsp-D1) have been shown to be linked to the Ha locus in different mapping populations and have been associated with the expression of grain softness. The study of the linkage relationships among these genes has been limited by the low level of polymorphism in the D genome of hexaploid Triticum aestivum. In the present study, a highly polymorphic Triticum monococcum mapping population was used to analyze linkage relationships among these three genes. Gsp-A ^m 1 and Pina-A ^m 1 were found to be completely linked and lie 0.14 cM distal to Pinb-A ^m 1 in the distal region of the short arm of chromosome 5A^m. The tight genetic linkage among these three genes was paralleled by their physical proximity within a single 105-kb clone isolated from a T. monococcum bacterial artificial chromosome (BAC) library. A restriction map of this BAC clone showed that Pina-A ^m 1 is located between Pinb-A ^m 1 and Gsp-A ^m 1. Partial sequences of the T. monococcum genes showed a high degree of similarity with their T. aestivum counterparts (≥ 94%). Marker-assisted selection strategies based on the tight linkage among Ha-related genes are discussed. Received: 27 June 1999 / Accepted: 18 August 1999     

A microarray analysis of wheat grain hardness

Grain hardness is an important quality characteristic of wheat grain, and considerable research effort has focused on characterising the genetic and biochemical basis underlying the hardness phenotype. Previous research has shown that the predominant difference between hard and soft seeds is linked to the puroindoline (PIN) proteins. In this study the near-isogenic lines of Heron and Falcon, which differ only in the grain hardness character, were compared using a cDNA microarray consisting of approximately 5,000 unique cDNA clones that were isolated from wheat and barley endosperm tissue. Our analysis showed that major differences in gene expression were evident for puroindoline-a (Pina), with a minor but not consistent change in the expression of puroindoline-b (Pinb). These observations were confirmed using a 16,000 unique cDNA microarray in a comparison of hard wheats with either the Pina null or Pinb mutation.     

Physical mapping and a new variant of Puroindoline b-2 genes in wheat

Puroindoline a and b proteins soften the endosperm of wheat kernels. When the underlying puroindoline genes are altered by mutation or are deleted, kernels become harder. Thus, puroindoline a and b (Pina and Pinb) play an important role in wheat quality and utilization. Recently, additional Pinb genes have been reported. In the present report, we provide corroborating coding and additional 5′ and 3′ flanking sequence for three Pinb variants: Pinb-2v1, Pinb-2v2, and Pinb-2v3. Additionally, a new Pinb variant, Pinb-2v4, is reported. All four variants were physically mapped using Chinese Spring (CS) diteolosomics, nullisomic–tetrasomics, and CS-Cheyenne disomic substitution lines. Results place Pinb-2v1 on 7DL, Pinb-2v2 on 7BL, Pinb-2v3 on 7B, and Pinb-2v4 on 7AL. Pinb-2v1 and Pinb-2v4 were present in all cvs. examined: CS, Cheyenne, Recital, Wichita and Winsome. Pinb-2v2 was present in CS and Recital; Pinb-2v3 was present in Cheyenne, Wichita, and Winsome. These results are not wholly consistent with prior research and additional studies will be required to reconcile discrepancies. The discovery of Pinb-2v4 and the mapping of all four variants will contribute to a better understanding of gene duplication events in wheat and their bearing on wheat kernel texture and grain utilization.     

Identification of genetic factors controlling kernel hardness and related traits in a recombinant inbred population derived from a soft × ‘extra-soft’ wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cross

Kernel hardness or texture, used to classify wheat (Triticum aestivum L.) into soft and hard classes, is a major determinant of milling and baking quality. Wheat genotypes in the soft class that are termed ‘extra-soft’ (with kernel hardness in the lower end of the spectrum) have been associated with superior end-use quality. In order to better understand the relationship between kernel hardness, milling yield, and various agronomic traits, we performed quantitative trait mapping using a recombinant inbred line population derived from a cross between a common soft wheat line and a genotype classified as an ‘extra-soft’ line. A total of 47 significant quantitative trait loci (QTL) (LOD ≥ 3.0) were identified for nine traits with the number of QTL affecting each trait ranging from three to nine. The percentage of phenotypic variance explained by these QTL ranged from 3.7 to 50.3%. Six QTL associated with kernel hardness and break flour yield were detected on chromosomes 1BS, 4BS, 5BS, 2DS, 4DS, and 5DL. The two most important QTL were mapped onto orthologous regions on chromosomes 4DS (Xbarc1118–Rht-D1) and 4BS (Xwmc617–Rht-B1). These results indicated that the ‘extra-soft’ characteristic was not controlled by the Hardness (Ha) locus on chromosome 5DS. QTL for eight agronomic traits occupied two genomic regions near semi-dwarf genes Rht-D1 on chromosome 4DS and Rht-B1 on chromosome 4BS. The clustering of these QTL is either due to the pleiotropic effects of single genes or tight linkage of genes controlling these various traits.     

Molecular Characterization of Puroindolines and their Encoding Genes in Aegilops Ventricosa

Puroindolines, the tryptophan-rich proteins controlling grain hardness in wheat, appeared as two pairs of 13 kDa polypeptides in the Acid-PAGE (A-PAGE) and two-dimensional A-PAGE×SDS-PAGE patterns of starch-granule proteins from wild allotetraploid wheat Aegilops ventricosa Tausch. (2n = 4x = 28, genomes D^vD^vN^vN^v). Puroindoline pair a1 + a2 reacted strongly with an antiserum specific for puroindoline-a from common wheat (Triticum aestivum L.), whereas puroindoline pair b1 + b2 exhibited A-PAGE relative mobilities similar to that of puroindoline-b in Aegilops tauschii (Coss.), the D-genome donor to both common wheat and Ae. ventricosa. Puroindolines a2 and b1 were found to be encoded by alleles Pina-D1a and Pinb-D1h on chromosome 5D^v, respectively, whereas puroindolines a1 and b2 were assumed to be under the genetic control of chromosome 5N^v. Puroindoline a1 encoded by the novel Pina-N1a allele exhibited a high level of amino acid variation with respect to puroindoline-a. On the other hand, the tryptophan-rich region of puroindoline b2 encoded by allele Pinb-N1a showed a sequence change from lysine-42 to arginine, with no effect on the amount of protein b2 accumulated on the starch granules. A partial duplication of the pin-B gene (Pinb-relic) was identified about 1100 bp downstream from Pinb-D1 on chromosome 5D^v. The present findings are the first evidence of a tetraploid wheat species in which four puroindoline genes are expressed. The potential of Ae. ventricosa as a source of genes that may be used to modulate endosperm texture and other valuable traits in cultivated wheat species is discussed.