Genetic characterization of kernel polyphenol oxidases in wheat and related species

Polyphenol oxidase (PPO) activity causes undesirable darkening of raw Asian noodles and other wheat products. In this study we investigate the genetic origins and diversity of wheat kernel PPO. PPO was characterized via activity assays, antigenic staining, and Southern blots in Triticum aestivum, Triticum dicoccoides, Triticum durum, Triticum dicoccum, Triticum monococcum, Triticum urartu, Aegilops speltoides, and Aegilops tauschii. Among these species, PPO activity was well-correlated with antigenic staining intensity toward a wheat kernel-type PPO antibody. High PPO activity was observed in all three T. monococcum accessions (A^m genome), one Ae. speltoides accession, one T. durum accession, and two hexaploid wheat cultivars. Southern blots suggested the presence of two or more kernel-type PPO genes in diploid progenitors of the hexaploid A, B, and D genomes. Whole-kernel PPO activity was evaluated in disomic substitution lines derived from three T. dicoccoides accessions in the background of T. durum ‘Langdon’. PPO activity was primarily associated with chromosome 2A and to a much lower degree with chromosome 2B. DNA sequence comparisons showed that the intron associated with the high PPO allele on chromosome 2AL of hexaploid wheat had 94% nucleotide identity with the homeologous intron found in T. monococcum, a species with high kernel PPO activity. This implies that the ancestral PPO allele on the A genome is one of the high activity, and the low PPO allele found in hexaploid wheat represents a relatively recent genetic alteration. Results confirm the presence of multiple kernel-type PPO genes in the diploid and tetraploid progenitors and relatives of hexaploid wheat. However, it is likely that relatively few of the many kernel-type PPO genes present in wheat contribute substantially to kernel PPO activity. A single genetic locus on homeologous group 2 chromosomes may be the primary cause of high PPO activity in wheat kernels.     

Development and characterization of a Triticum aestivum-H. villosa T5VS•5DL translocation line with soft grain texture

The Hardness locus on the short arm of chromosome 5D is the main determinant of grain texture in bread wheat. The Pina and Pinb genes are tightly linked at this locus, and the soft kernel texture phenotype results when both genes are present and encode the wild-type puroindoline proteins PINA and PINB. In this study a compensating T5VS•5DL Triticum aestivum-Haynaldia villosa translocation line, NAU415, was characterized by chromosome C-banding, genomic in situ hybridization and molecular markers. Single Kernel Characterization System (SKCS) analysis and scanning electron microscopy indicated that NAU415 had soft endosperm although it lacked the wheat Pina-D1a and Pinb-D1a genes, suggesting the presence of functional Pin gene orthologs on chromosome 5VS. Using a PCR approach, Pina-related (designated Dina) and Pinb-related (Dinb) genes in H. villosa and NAU415 were identified and sequenced. The nucleotide and predicted amino acid sequences showed close similarities to the wild-type puroindolines of T. aestivum cv. Chinese Spring. The tryptophan-rich regions of both Dina and Dinb showed a sequence change from lysine-42 to arginine, a feature that may have an effect on grain texture. The potential of T5VS•5DL translocation line as a source of genes that may be used for modulation of endosperm texture and other valuable traits in wheat breeding is discussed.     

Conserved regulatory elements identified from a comparative puroindoline gene sequence survey of Triticum and Aegilops diploid taxa

Kernel texture (‘hardness’) is an important trait that determines end-use quality of wheat (Triticum aestivum L. and Triticum turgidum ssp. durum [Desf.] Husn.). Variation in texture is associated with the presence/absence or sequence polymorphism of two proteins, puroindoline a and puroindoline b. This work describes the flanking and coding region sequences of puroindoline genes from 25 accessions representing wild diploid taxa of the Triticeae related to the three genomes of T. aestivum. Analysis of variation at the nucleotide level included hard and soft T. aestivum wheat cultivars. Various degrees of insertions/deletions and point mutations were found, that did not affect the overall sequence structure identity. Nucleotide sequence comparisons and database searches facilitated the identification of the 5′ proximal regulating regions, revealing the presence of several putative control elements. An absolute conservation of some known regulatory elements for tissue specificity was observed, while different rates of conservation of reiterated motifs with possible enhancer functions, and the exclusive presence of some elements either in puroindoline a or puroindoline b were also found. A total of 24 new puroindoline alleles (unique sequences) were identified. Despite some primary structure variation, the main features of puroindolines, i.e. the signal peptide, the cysteine backbone, the tryptophan-rich domain, the hydrophobicity and basic identity of the proteins were all conserved.     

Isolation and characterization of EMS-induced Dy10 and Ax1 high molecular weight glutenin subunit deficient mutant lines of elite hexaploid wheat (Triticum aestivum L.) cv. Summit

The mixing properties of the dough are critical in the production of bread and other food products derived from wheat. The high molecular weight glutenin subunits (HMW-GS) are major determinants of wheat dough processing qualities. The different alleles of the HMW-GS genes in hexaploid wheat vary in their effect on dough quality. To determine the contribution of the individual HMW-GS alleles, lines deficient in HMW-GS proteins were generated by chemical mutagenesis in the elite bread wheat Triticum aestivum cv. Summit. In this report we describe the identification and characterization of Dy10 and Ax1 deficient lines. Examination of the effect of Dy10 and Ax1 deficiency on dough rheological properties by mixography showed shorter mixing time to reach peak resistance, and weaker and less extensible doughs relative to the wild type control. This is the first time that the role of Dy10 in vivo has been examined apart from the Dx5 + Dy10 allelic pair combination.     

粘果山羊草(Aegilops kotschyi) puroindoline b基因的克隆与表达分析

籽粒硬度是小麦加工品质的重要影响因素。puroindoline a（Pin a）和puroindoline b（Pin b）是控制小麦籽粒硬度的主效基因。根据已报导的小麦Pin b基因的保守序列,设计合成了一对特异性引物ForB1与RevB1,对粘果山羊草（Aegilops kotschyi,C^uM^k）的三个材料的基因组DNA和胚乳cDNA进行Pin b基因扩增、克隆、序列测定和表达分析,发现了4个新型Pin b等位基因,基因序列与六倍体小麦的同源基因存在较大的差异。与软粒小麦品种Capitole的Pinb—D1a相比较,其核苷酸同源性分别为93．3％、94．6％、94．6％、94．4％,氨基酸同源性分别为90．5％、93．2％、93．2％、92．6％。其ORF长447bp,编码148个氨基酸残基,都具有麦类作物Pinb基因特有的19个氨基酸的信号肽序列和WPTKWWK的色氨酸结构城。等位基因Pin b-11-1含有1个紧邻色氨酸结构城的突变位点（Val66Phe）。RT—PCR证实了Pin b基因在籽粒胚乳中的表达。Southern blot分析结果显示,三种材料均含有两个拷贝的Pin b基因。研究结果表明,粘果山羊草中包含与小麦差异较大的籽粒硬度控制基因,为栽培小麦的品质改良提供了丰富的基因资源。     

Reconciliation of D-genome puroindoline allele designations with current DNA sequence data

Kernel texture is an important trait in cereals, especially wheat (Triticum spp.). Throughout the Triticeae, the puroindoline genes act to soften kernel endosperm. Absence or mutation of either or both of the two puroindolines, ‘a’ and ‘b’, in Triticum aestivum results in harder grain texture. Apparently only one puroindoline haplotype was contributed by the Aegilops tauschii variety that contributed the D-genome during allopolyploidization. Yet, world collections of Ae. tauschii exhibit a range of puroindoline sequence polymorphisms. Consequently, these genes, through synthetic hexaploids (× Aegilotriticum) can enrich the wheat gene pool. Lastly, the puroindolines represent a useful tool for phylogenetic analyses. Here we review original sequence data published and/or available in public databases to reconcile the known gene sequence polymorphisms with a systematic approach to the designating of puroindoline gene and allele symbols in T. aestivum, Ae. tauschii, and × Aegilotriticum. This system follows the recommendations adopted by the International Wheat Genetics Symposium and described in the Catalogue of Gene Symbols for Wheat. Errors, discrepancies and ambiguities in the puroindolines are reviewed; a reconciliation of all existing data is outlined.     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

Apigenin di-C-glycosides (ACG) content and composition in grains of bread wheat (Triticum aestivum) and related species

Apigenin di-C-glycosides (ACGs) are present in the grain of bread wheat and other related cereals primarily as one or two sets of Wesseley-Moser isomers containing either arabinose and glucose (ACG1) or arabinose and galactose (ACG2) on the A ring of apigenin. These compounds may contribute to the yellow colour of wheat-based products made under alkaline conditions and in addition, have possible roles in a number of plant physiology processes and human health. The aims of this investigation were to survey genetic variation for ACG content and composition in hexaploid bread wheat (Triticum aestivum L.) and to examine ACGs in the putative progenitors of hexaploid wheat and available genetic stocks as a first step towards understanding the mechanisms involved in their biosynthesis and genetic control. Substantial variation in both grain ACG content and the ratio, ACG1/ACG2, were identified within bread wheat cultivars and related species. Genotype controlled the major portion of the variation. ACG content appeared to be a multigenic trait whereas variation in ACG1/ACG2 was associated with a limited number of chromosomes, in particular chromosomes 1B, 7B and 7D. The results suggest that it should be possible to manipulate both ACG content and composition traits through breeding.     

Identification,mapping and evolutionary course of wheat lipoxygenase-1 genes located on the A genome

Durum wheat (Triticum turgidum) is the cereal of preference for semolina and pasta production. Bright yellow color, which is desirable for pasta making, depends on the amount of carotenoid pigments present in the grain. Lipoxygenases (LPXs) account for most of the carotenoid degradation activity. Although B genome Lpx genes have been extensively described, little information about the A genome has been reported. Here, we demonstrate that the Lpx-A1 locus is represented by a single gene in the diploid Triticum urartu, the tetraploid T. turgidum and the hexaploid Triticum aestivum wheats in contrast to the multiple copies reported in the B genome. The Lpx-A1_like pseudogene previously identified in T. turgidum genome A was also identified in the T. aestivum cv Chinese Spring wheat, whereas T. urartu possesses a complete copy, suggesting that pseudogenization occurred after the formation of the tetraploid and then passes to the hexaploid wheat. The nucleotide sequence of T. urartu Lpx is more closely related to genome B Lpx-1 than to Lpx genes of genome A from T. turgidum and T. aestivum, probably due to the deletions and insertions that occur. Thus, the present paper extends our knowledge of lipoxygenase gene organization and evolution in the wheat A genome.     

Genetic and Environmental Effects on Puroindoline-a and Puroindoline-b Content and their Relationship to Technological Properties in French Bread Wheats

Grain hardness (GHa) in hexaploid wheat (Triticum aestivum L.) is a major factor of end use quality. The variation of texture has been related to the Hardness locus, closely linked with the puroindoline-a (PIN-a) and puroindoline-b (PIN-b) genes. In order to study the role of puroindoline content in texture variation, the quantity of puroindolines was determined. Puroindoline-a (PIN-a) and puroindoline-b (PIN-b) content was determined and a total of 11 bread making parameters were obtained from 40 bread wheat cultivars grown in four experimental locations. The 11 parameters were significantly influenced by the genotype whereas location did not significantly affect PIN-a or PIN-b content and loaf volume. PIN-b and grain hardness displayed the highest heritability coefficients (both 0.88). PIN-a and PIN-b content were not correlated with grain protein content (Prot) and grain hardness in hard and soft wheat types. In soft samples PIN-(a+b) content was negatively correlated, with loaf volume in two locations. Multiple regression analyses, carried separately for soft and hard types, revealed that PIN-b content explained variation of dough strength (W) and loaf volume, however their influence was mostly significant in soft types. For each location, from 22 to 91% of the phenotypic variation of strength and loaf volume was explained by combining up to three flour traits. Protein content, PIN-b and the average score of high molecular weight glutenin subunits (HMW-GS) were frequently introduced by multiple regression (without GHa) as explanatory variables of strength and loaf volume. These results strengthened the significant role of PIN-b in breadmaking (loaf volume), and indicated that biochemical factors other than puroindolines are involved in the grain hardness variation.     

Content and relative composition of some phytochemicals in diploid,tetraploid and hexaploid Triticum species with potential nutraceutical properties

Seven cultivated Triticum species or subspecies (Triticum monococcum, Triticum turgidum ssp dicoccum, T. turgidum ssp. durum, T. turgidum ssp turanicum, Triticum timopheevii, Triticum aestivum and Triticum zhukovskyi) were compared for their contents of bioactive compounds, mainly 5-n-alkylresorcinols (ARs). Multivariate analysis of variance and principal component analysis were used to evaluate the differences in the phytochemical profiles and to establish the relationships among variables. Significant differences were observed for both total phenol (TP) and AR content. The highest AR level (377 μg/g) was observed in Triticum dicoccum, which also exhibited the highest variability for these compounds (298–436 μg/g). By contrast, the lowest AR content (286 μg/g) was found in Triticum durum. C21:0 was the main homologue chain in all the samples, its value ranging between 54.5% in T. durum and 41.2% in T. aestivum. The T. dicoccum and T. monococcum wheats had relatively low amounts of TP, whereas Triticum turanicum (215 mg/kg), T. timopheevii (250 mg/kg) and T. zhukovskyi (286 mg/kg) had approximately 3-fold higher TP levels. These results suggest that there are opportunities for breeding wheat varieties with superior health benefits and for promoting the use of ancient Triticum crops as novel sources of healthy food.     

Discriminant analysis of selected yield components and fatty acid composition of chosen Triticum monococcum, Triticum dicoccum and Triticum spelta accessions

This study analyses the variability of key yield components, the content of protein and crude fat in grain and the fatty acid composition of 50 spring accessions of Triticum monococcum, Triticum dicoccum and Triticum spelta of various origins. The average protein content of the grain of T. monococcum was 20.8%, of T. dicoccum 19.7%, and of spelt 17.0%. The crude fat content of T. monococcum grain (2.7%) was significantly higher compared with T. spelta (2.4%) and T. dicoccum (2.3%). In crude fat, fatty acids C18:2, C18:1 and C16 predominated. T. spelta was characterised by the highest concentrations of C18:2 and C16 (55.89% and 18.77% respectively), while T. monococcum had the highest content of C18:1 (26.35%). The structure of analysed fatty acids proved to be highly desirable in this species. A discriminant analysis performed separately for five variables: protein and fat content and three biometrical characters and separately for fatty acid composition enabled three Triticum species to be distinguished. These species also differed significantly with respect to the C18:1/C16 ratio which was equal to 1.78, 1.06, 1.47 and 0.99 in T. monococcum, T. dicoccum, T. spelta and Triticum aestivum respectively.     

Puroindoline grain hardness alleles in CIMMYT bread wheat germplasm

Grain hardness is an important quality parameter of bread wheat (Triticum aestivum L.) with importance for wheat classification and end use properties, and is controlled by the genes puroindoline a (Pina) and puroindoline b (Pinb). The presence of known hardness alleles was studied in a representative sample of 373 bread wheat lines from the breeding program at CIMMYT. The PINA-null mutation (Pina-D1b) was the most frequent hardness allele and present in 283 of the 328 lines with hard endosperm. All other hard wheat had the glycine to serine mutation in PINB (Pinb-D1b). A study of historically important CIMMYT bread wheat lines showed that Pina-D1b has been the dominating hardness allele since the inception of the wheat breeding program in Mexico. New puroindoline alleles have recently been introduced through the extensive use of synthetic hexaploid wheat, and the textural effects of various Aegilops tauschii-derived Pina and Pinb alleles were studied in 92 breeding lines derived from various crosses with synthetic wheat. Progeny lines with Pina-D1j/Pinb-D1i were on average 10 SKCS hardness units softer than those carrying the allelic combination Pina-D1c/Pinb-D1h. Further investigation is needed to validate the potential of such minor allelic differences for the improvement of soft wheat quality.     

Molecular and Biochemical Characterisation of HMW Glutenin Subunits fromT. tauschiiand the D Genome of Hexaploid Wheat

Electrophoretic (urea SDS–PAGE) and chromatographic (RP–HPLC) analysis was performed on 8 allelic variants of HMW glutenin subunits derived fromTriticum tauschiiand from the D genome of a hexaploid wheat species (Triticum macha) and hexaploid landraces. These subunits had been previously identified using SDS–PAGE. The characterisation revealed that subunits Dy10^tand Dy12^tfromT. tauschiicould be differentiated from their bread wheat counterparts using both urea SDS–PAGE and RP–HPLC. In the latter case, theT. tauschiiy-type subunits were clearly more hydrophobic than the Dy type subunits of bread wheat. The characterisation also suggested that subunit Dx5^t, derived from two separateT. tauschiiaccessions, did not contain the extra cysteine residue characteristic of Dx5 from bread wheat. RFLP analysis of the genes encoding the HMW glutenin subunits of interest suggested that the absence of Dx-type HMW glutenins in two hexaploid landraces was due to lack of expression of their encoding genes. The relationship betweenHindIII DNA fragment size and protein subunit size, as measured by electrophoretic mobility, is examined and discussed. Finally, the solubility properties of a HMW protein designated T1 (derived fromT. tauschiiaccession AUS 18913) suggested that it was not a HMW glutenin subunit as was previously thought. Further studies are needed to clarify the identity of this subunit.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

Agropyron elongatum HMW-glutenins have a potential to improve wheat end-product quality through targeted chromosome introgression

After screening of 177 disomic addition lines (DALs) of wheat (Triticum aestivum) containing a pair of chromosomes from different alien species, we found that the chromosome 1E addition line of Agropyron elongatum, that is known to be a potential genetic resource for drought and salinity tolerance, showed potential for improvement of bread-making quality of wheat. This was indicated by increased SDS sedimentation, specific sedimentation, mixograph peak time and SE-HPLC analysis of polymeric proteins. This addition line spontaneously gave rise to a substitution line for chromosome 1D in subsequent generations that showed weak dough strength. Analysis of the x-type HMW-glutenin subunit sequence of Ag. elongatum from DAL1E indicated that it closely resembled the x-type sequence of the A and B genomes of wheat, and the y-type was intermediate between x- and y-type HMW-glutenin subunit genes. From these observations, it was inferred that 1E-encoded seed storage proteins have considerable potential for improvement of wheat end-product quality if transferred to specific chromosomes such as 1A of Chinese Spring (CS) wheat, which has a negative overall effect on bread-making quality.     

Spectroscopic analysis of diversity in the spatial distribution of arabinoxylan structures in endosperm cell walls of cereal species in the HEALTHGRAIN diversity collection

Two varieties each of spelt (Triticum aestivum var. spelta), durum wheat (Triticum turgidum var. durum), rye (Secale cereale), barley (Hordeum vulgare), oats (Avena sativa), einkorn (Triticum monococcum var. monococcum) and emmer (Triticum turgidum var. dicoccum) (all members of the Pooideae sub-family of grasses) were selected according to variation in their contents of soluble and/or total arabinoxylan (AX) determined during the HEALTHGRAIN diversity screen, together with one genotype of the related “model” grass species Brachypodium distachyon. The spatial distribution of low substituted (LS-AX) and highly substituted arabinoxylan (HS-AX) was determined using FT-IR spectroscopic mapping of transverse thin cross-sections consisting of cell walls only. Variation in cell wall AX composition was observed between the cereals, and compared with that observed for wheat (Triticum aestivum var. aestivum). One line of each cereal type was analysed in more detail using ¹H NMR spectroscopy. The results of the two analyses were consistent, showing variation in the composition and structure of the endosperm cell wall AX that is consistent with the genetic relationships of the cereals studied.     

A new puroindoline b mutation present in Chinese winter wheat cultivar Jingdong 11

Kernel hardness is one of the most important characteristics in determining utilization and marketing of bread wheat. Genes coding for puroindoline a and b (PINA and PINB) were located at the Ha locus and designated as Pina-D1 and Pinb-D1, respectively. The coding sequence of the Pinb gene in a Chinese winter wheat cultivar Jingdong 11 (Triticum aestivum L.) was amplified with polymerase chain reaction (PCR), and the obtained 447-bp fragment sequenced from two strands, and compared with the eight known Pinb alleles. The results showed that Jingdong 11 possessed a new Pinb allele not reported previously, and was designated as Pinb-D1q. It is characterized by a single base T to G substitution, which results in a tryptophan to leucine substitution (TGG to TTG) at position 44 and is most likely the cause of hard grain texture in Jingdong 11. The characterization of Pinb-D1 alleles would be helpful in manipulating grain hardness of bread wheat in breeding programs.     

Variation in polar lipid composition among near-isogenic wheat lines possessing different puroindoline haplotypes

The exact mechanism underlying wheat (Triticum aestivum L.) kernel hardness is unknown. Similar to puroindoline proteins, polar lipids are present on the surface of starch granules. The objective of this research was to determine the specific polar lipid species present on the surface of wheat starch from near-isogenic wheat lines that have different puroindoline haplotypes and endosperm hardness. Four near-isogenic wheat lines were used in this study, all derived from the soft cultivar Alpowa. Direct infusion tandem mass spectrometry was used to identify the lipid species in whole-meal, flour and starch samples. Endosperm hardness had no significant effect on the polar lipid contents in wheat whole-meal, a slight influence on the polar lipid contents of the flour fractions and a significant influence on the polar lipid composition of the polar lipids located on the surface of wheat starch. The greatest quantities of polar lipids on the starch-surface occurred when both puroindoline proteins were present in their wild-type form. Starch-surface polar lipid content dramatically decreased when one of the puroindoline proteins was null or if pin-B was in the mutated form. The least amount of polar lipids was present when pin-B was in its mutated form and pin-A was in its wild-type form.