The Pinb-2 genes in wheat comprise a multigene family with great sequence diversity and important variants

The puroindoline genes Pina-D1 and Pinb-D1 located at the Ha locus on chromosome 5D of common wheat are considered the most important genetic determinants of grain hardness. The recent identification of Pinb-2 genes on group 7 chromosomes emphasises the need for detailed analysis of the genetics of this important trait. This study focussed on the analysis of Pinb-2 genes from accessions of hexaploid, tetraploid and diploid wheat, to address key questions related to their diversity and possible roles. Extensive DNA sequence heterogeneity was identified in the form of single nucleotide polymorphisms (SNPs), leading to seventeen reproducible haplotypes, of which thirteen are new. The results confirmed the known groups Pinb-2v1 to Pinb-2v5, identified a new group Pinb-2v6, and showed that the Pinb-2 genes comprised a small multigene family, at least in some genomes. The putative proteins exhibited changes at the important tryptophan-rich domain as well as basic and hydrophobic residues. A new Pina-D1 allele (at Ha locus) was also identified, designated Pina-D1t, with a premature stop codon at the TRD. Additionally, peptides designed on PINB-2 proteins displayed activity against bacteria and phytopathogenic fungi. The data strongly support the Pinb-2 genes being functionally relevant to roles including influencing grain texture.     

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.     

Variation in Friabilin Composition as Determined by A-PAGE Fractionation and PCR Amplification, and its Relationship to Grain Hardness in Bread Wheat

A-PAGE fractionation of starch granule proteins from 63 bread wheat cultivars with contrasting grain texture characteristics revealed two prominent polypeptides and three minor ones, approximately 15 kDa in size. These proteins were found to be encoded by genes on the short arm of chromosome 5D. The two major friabilin components were assumed to correspond to puroindolines a and b (pinA and pinB), as suggested by PCR amplification of genes coding for pinA, glycine-type or serine-type pinB. Two electrophoretic patterns for pinA (presence vs absence) and three patterns for pinB were obtained by A-PAGE. In cultivars with pinA (allele Pina-D1a), pinB was found to be encoded by wild-type Pinb-D1a, serine-type Pinb-D1b or by the novel glycine-type b1 allele. Cultivars lacking pinA (allele Pina-D1b) were shown to contain eitherPinb-D1a or the novel b2 allele, both alleles coding for glycine-type pinB. The intensity of pinB in A-PAGE gels was found to be associated with grain hardness as determined by the SKCS method. Cultivars lacking pinA had the highest SKCS values, suggesting that both pinA and pinB may affect grain texture. In the presence of pinA, cultivars with wild-type allelePinb-D1a had soft grain texture, whereas those with alleles Pinb-D1b or b1 showed increased grain hardness. It is suggested that allele b1 affects the interaction of pinB with starch granules because of a sequence mutation different from the glycine-to-serine change.     

Puroindoline genotype,starch granule size distribution and milling quality of wheat

Genetically-diverse wheat samples from the Australian Winter Cereals Collection propagated in two environments were sequenced to identify puroindoline genotypes then the relationships between flour yield, genotype, starch granule size distribution and starch-bound puroindoline protein content were investigated. The Pina-D1a, Pinb-D1b genotype resulted in a higher average flour yield than either the Pina-D1b, Pinb-D1a or the Pina-D1a, Pinb-D1a but the ranges of flour yields for the three genotypes showed considerable overlap. For both hard wheat genotypes (Pina-D1a, Pinb-D1b or Pina-D1b, Pinb-D1a), a higher proportion of type A to type C starch granules was associated with higher flour yield and this relationship accounted for between 31% and 33% of the variation in flour yield. This result is consistent with previously reported findings for soft wheat. For the Pina-D1a, Pinb-D1b genotype, increased flour yield was also associated with a decrease in starch granule-bound puroindoline protein, which accounted for 31–35% of the variation in flour yield across the two environments. The combined effect of starch granule type and associated puroindoline content accounted for 68% of the variation in flour yield within the Pina-D1a, Pinb-D1b genotype.     

Influence of puroindoline alleles on milling performance and qualities of Chinese noodles,steamed bread and pan bread in spring wheats

Two trials with a total of 75 spring bread wheat cultivars and advanced lines, were used to evaluate single kernel characterization system hardness, puroindoline alleles, milling yield, flour ash content, flour colour, and end-use qualities for Chinese noodles, steamed bread and pan bread. The results indicated that all International Maize and Wheat Improvement Center hard wheat lines surveyed were Pina-D1b genotype, whereas Pinb-D1b was the most common allele in the remaining cultivars. Genotypes with Pinb-D1b possess significantly lower flour ash content and higher milling yield than those of genotypes with Pina-D1b. For steamed bread, mean scores for loaf volume, crumb colour, width, structure and total score of Pinb-D1b genotypes were significantly higher than those of genotypes with Pina-D1b and wild type. For Chinese fresh white noodles, means for noodle a*, colour score, viscoelasticity and total score with Pinb-D1b were significantly higher than those of Pina-D1b and wild type. Means of loaf volume, texture and total score for Pinb-D1b genotypes were significantly higher than those of Pina-D1b genotypes. This study further illustrated the superiority of the Pinb-D1b allele on milling and processing qualities for various end products and provides useful information for wheat quality improvement.     

Milling and Chinese raw white noodle qualities of common wheat near-isogenic lines differing in puroindoline b alleles

Understanding the effects of different alleles at the puroindoline b (Pinb) locus on processing quality will provide crucial information for quality improvement. Seven near-isogenic lines (NILs) planted at two locations in the 2008 cropping season were used to determine the effect of puroindoline b alleles on milling performance and Chinese raw white noodle (CRWN) quality. The Pina-D1b/Pinb-D1a genotype possessed significantly higher values in grain hardness, protein content and starch damage than other genotypes, whereas the Pina-D1a/Pinb-D1d genotype had the lowest grain hardness and starch damage, with higher break flour yield, and less reduction flour yield, higher flour colour L*, and lower flour colour b*, than other genotypes. Farinograph parameters, except for water absorption, were not significantly affected by variation of puroindoline b alleles. Pina-D1a/Pinb-D1e had the highest peak viscosity, whereas the lowest value was observed in a Pina-D1b/Pinb-D1a genotype. For CRWN quality, higher noodle viscoelasticity was obtained in the genotype Pina-D1a/Pinb-D1e and Pina-D1a/Pinb-D1g, whereas Pina-D1a/Pinb-D1d had a lower smoothness score. Genotypes with Pina-D1a/Pinb-D1e and Pina-D1a/Pinb-D1g produced the best total noodle score. It was concluded that genotype Pina-D1a/Pinb-D1d had better milling qualities, whereas Pina-D1a/Pinb-D1e and Pina-D1a/Pinb-D1g had slightly superior CRWN qualities in comparison with other genotypes.     

Pins基因等位变异对磨粉品质和新疆拉面加工品质的影响

为了探讨新疆冬小麦品种Pins基因等位变异对小麦磨粉品质和新疆拉面加工品质的影响,对109份新疆冬小麦品种的籽粒硬度及其Pins基因等位变异、磨粉品质和新疆拉面加工品质进行测定,初步分析了新疆冬小麦品种资源籽粒硬度Pins基因的分布规律以及不同 Pins基因等位变异对籽粒硬度、磨粉品质和新疆拉面加工品质的影响。结果表明,新疆冬小麦品种属硬质麦类型,Pins基因型以 Pina-D1a、 Pinb-D1b和 Pina-D1a/ Pinb-D1b为主, Pins突变类型及Pins突变基因型组合类型小麦的籽粒硬度均显著高于野生型, Pinb-D1a基因型小麦的籽粒硬度最低,L^*值和a^*值最高,b^*值最低; Pinb-D1ab基因型小麦的吸水率最高。不同Pins基因型组合中,野生型小麦的籽粒硬度、b^*值和吸水率最低; Pina-D1a/ Pinb-D1aa的出粉率最高, Pina-D1a/ Pinb-D1ab的灰分含量最低,吸水率最高。Pins基因及其基因型组合对新疆拉面加工品质无直接影响,主要通过对灰分、面粉色泽和吸水率等磨粉品质的作用对新疆拉面产生间接影响。优质新疆拉面品种中,Pinb基因突变对新疆拉面加工品质的影响大于Pina基因突变,育种中应优先选择Pinb 基因突变型材料,其中 Pina-D1a/ Pinb-D1b可以作为重点选择的基因型组合。     

Identification of new puroindoline genotypes and their relationship to flour texture among wheat cultivars

Puroindoline genotypes (Pina and Pinb) and their encoded proteins related to grain hardness were studied in various common wheat cultivars from Australia, China, Japan, Korea and North America. Most of the hard wheats had the Pinb-D1b genotype with a glycine to serine mutation at position 46. In addition to the known Pina and Pinb genotypes, cultivars were found with Pina and Pinb double-null mutations (Pina-D1b/Pinb-D1h (t)) and a new Pinb frameshift mutation (designated Pinb-D1i (t)) within the region encoding a tryptophan-rich domain. This new Pinb frameshift mutation was found only in Chinese cultivars. Endosperm proteins encoded by Pina and Pinb in these cultivars were analysed by 2D-gel electrophoresis (IPG×SDS-PAGE). Cultivars with Pina and Pinb double-null mutations showed no PIN-a or PIN-b protein, and cultivars with Pinb-D1i (t) had no PIN-b protein. Surprisingly, cultivars with Pinb-D1b had severely reduced amounts of PIN-b and cultivars with Pinb-D1c showed no PIN-b proteins. Grain hardness among cultivars having mutated Pinb may be explained by the amount of PIN-b protein and not by the type of amino acid substitutions.     

Genetic and environmental factors affecting grain texture in common wheat

Thirteen wheat cultivars grown in six locations were compared for kernel weight, protein content and grain texture, as determined by the Single Kernel Characterization System (SKCS). Moreover, puroindolines a (Pin-A) and b (Pin-B) bound to starch were quantified by densitometric scanning of A-PAGE fractionations. All cultivars shared allele Pina-D1a coding for wild-type Pin-A, and differed from each other in allele composition at Pinb-D1 coding for Pin-B. Cultivars with Pinb-D1a exhibited soft grain and high amounts of Pin-A and Pin-B compared to cultivars with Pinb-D1b or Pinb-D1d. Significant genetic variation for grain hardness and Pin-A level was detected in soft cultivars. The ratio between Pin-A and Pin-B levels in soft cultivars was approximately 6:5, whereas it varied between 9:5 and 10:1 in hard cultivars. Protein content was significantly correlated with Pin-B content (r=0.34) and SKCS value (r=0.36) in soft wheats. Significant correlations (0.68 and 0.73 for soft and hard wheats, respectively) were observed between Pin-A and Pin-B levels. Grain hardness was not correlated with puroindoline levels and Pin-A/Pin-B ratio in both textural classes. By contrast, kernel weight was found to act as a major environmental factor affecting grain texture in both soft and hard wheats.     

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.     

The co-operative interaction of puroindolines in wheat grain texture may involve the hydrophobic domain

The puroindoline genes are causatively associated with wheat grain hardness, a commercially significant property. The proteins puroindoline (PIN) A and B are both required in their wild-type (WT) to impart soft grain texture, and absence of/mutations in either/both PIN(s) results in hard wheat. However, there is no biochemical clarity yet that explains this interdependence. This work critically analyses the roles of the tryptophan-rich domain (TRD), the little-known hydrophobic domain (HD), and certain other residues, in the physical associations of PINs. Site-directed mutagenesis-PCR was used to delete the TRD or HD and introduce an Arg39Gly substitution in PINA. The PINB-D1c mutant (Leu60Pro) was also investigated. The yeast two-hybrid system was used to assess the protein–protein interactions (PPI) of proteins. The TRD deletion or Arg39Gly substitution in PINA did not adversely affect its PPI, while deletion of HD resulted in a significant reduction. No effect on PPI was observed for Leu60Pro PINB. The results of this expression system strongly suggest that the HD is essential (but not sufficient) in higher-order associations of PINs. We propose a two-event model that explains the co-operative action of the PINs and why mutations outside the TRD may alter grain texture.     

青海小麦籽粒硬度等位变异研究

为了解小麦品种籽粒硬度的遗传多样性,利用单粒谷物硬度测定、PCR扩增和核苷酸测序技术,分析了66份青海小麦品种籽粒硬度主效基因的等位变异。结果表明,青海小麦以硬质类型为主,比例达到47.0%,混合麦比例为19.7%,软质麦比例为33.3%。硬度基因有5种组合类型:野生型、Pina-D1a/PinbD1b、Pina-D1a/Pinb-D1c、Pina-D1a/Pinb-D1x和Pina-D1b/Pinb-D1a。野生型小麦类型比例最高,占59.09%,SKCS硬度指数平均为44.12,变化范围为12.75~84.89。突变类型的品种籽粒均为硬质。因此,在青海硬质小麦可以通过突变类型的分子标记进行选育,软质小麦选育需在利用硬度基因分子标记筛选的基础上进一步考察籽粒硬度性状的表现型。     

长江中下游麦区小麦品种籽粒硬度及puroindoline基因等位变异的分子检测

为了明确长江中下游麦区小麦籽粒硬度及puroindoline基因型的分布,以该麦区105份小麦育成品种为材料,利用单籽粒硬度仪(SKCS)测定其籽粒硬度,利用分子标记检测和基因序列分析鉴定puroindoline基因的等位变异。结果表明,在长江中下游麦区历年育成的小麦品种中软质麦比例较高,占52.4%,硬质麦和混合麦分别占38.1%和9.5%;硬质麦和混合麦中存在Pinb-D1b、Pina-D1b和Pinb-D1p三种变异类型,突变频率分别为29.5%、10.5%和3.8%。     

Pastamaking and breadmaking quality of soft-textured durum wheat lines

The effects of grain texture on pastamaking and breadmaking quality were studied in three F₈ soft-textured durum wheat lines (SDLs) containing wild-type alleles Pina-D1a and Pinb-D1a as compared with their hard durum sister lines (HDLs). SDLs homozygous for a small 5DS segment, less than 14.4 cM in size, accumulated puroindolines A (Pin-A) and B (Pin-B) and showed SKCS values (19.9-23.6) significantly lower than those (72.6-76.8) of their hard-textured counterparts lacking Pin-A and Pin-B. In addition, SDLs exhibited approximately 24% higher flour extraction rates compared with HDLs. Reducing the kernel hardness decreased farinograph water absorption, dough tenacity (P) and, accordingly, alveograph P/L ratio, but increased farinograph stability, mixing tolerance and dough extensibility (L). Spaghetti cooking quality, as determined by the sensory judgment of firmness, stickiness and bulkiness, was unaffected by the kernel hardness, whereas the loaf volume exhibited a 10% increase associated with kernel softening. Flour and semolina, but not spaghetti, from SDLs showed a substantial reduction in yellowness (b^*) and brownness (100 − L^*) likely due to their finer particle size compared with HDLs. Alleles Pina-D1a and Pinb-D1a may offer new perspectives for breeding dual purpose (pasta and bread) durum wheat varieties.     

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.     

Segregation analysis indicates that Puroindoline b-2 variants 2 and 3 are allelic in Triticum aestivum and that a revision to Puroindoline b-2 gene symbolization is indicated

Genetic and kernel texture relationships between Puroindoline b-2 variants 2 and 3 have not been fully established in wheat (Triticum aestivum L.). Here, 480 F₂ plants, derived from three hard spring wheat populations were used to test the segregation of Puroindoline b-2 (Pinb-2) variants 2 and 3. Chi-square analysis indicated that Pinb-2 variants 2 and 3 in all three F₂ populations segregated as a single bi-allelic locus, with segregation ratios fitting a 1:2:1 ratio. Using 448 of the 480 plants derived from these three F₂ populations, the average SKCS hardness index of plants homozygous for Pinb-2 variant 2 vs. those homozygous for variant 3 was not significantly different (67.5 vs. 67.9). Results indicated that plants with Pina-D1b/Pinb-D1a were on average 10.0 Single Kernel Characterization System (SKCS) hardness index units harder than those carrying the Pina-D1a/Pinb-D1b haplotype. In conclusion, Pinb-2 variants 2 and 3 are allelic and exert little effect on kernel texture in hard-kernel T. aestivum germplasm. Further, the designation of Pinb-2v2 and Pinb-2v3 should be changed to Pinb-B2a and Pinb-B2b, respectively. We propose that Pinb-2 variants 1 and 4 of Chinese Spring be designated Pinb-D2a and Pinb-A2a, respectively.     

Association analysis of Puroindoline-D1 and Puroindoline b-2 loci with 13 quality traits in European winter wheat (Triticum aestivum L.)

Grain hardness, a major determinant influencing end-use quality of common wheat, is mainly controlled by Puroindoline a-D1 (Pina-D1) and Puroindoline b-D1 (Pinb-D1) genes. Recently, additional puroindoline genes, designated Puroindoline b-2 (Pinb-2), were described. This study examined frequencies of Pin-D1 alleles and Pinb-2 variants in 94 West European wheat genotypes and assessed their association with 13 quality traits considering population and family structure. The survey was completed by analyzing the Grain softness protein-1 gene. Results indicated sequence variation only for Pinb-D1 and Pinb-B2 genes. Pinb-D1b was the predominant hard allele. Pinb-B2v3-1 was the most common Pinb-2 variant, followed by a newly discovered variant Pinb-B2v3-5. Association mapping carried out in the whole sample population showed that Pinb-D1 alleles were associated with 11 quality traits, whereas Pinb-B2 variants were only associated with semolina extraction. Considering only the panel of hard wheat genotypes, variation for flour ash content, sedimentation value, gluten index and loaf volume was found to be associated with Pinb-D1 mutations suggesting that different Pinb-D1 mutations might have particular effects on quality traits. Our study indicated that Pinb-D1d was associated with inferior sedimentation value, gluten index and loaf volume, for which reason this mutation should be disregarded in breeding for quality wheat.     

江苏淮北地区小麦品种资源籽粒硬度基因等位变异的KASP检测

为了解江苏淮北地区小麦品种资源的籽粒硬度概况及硬度基因型分布规律,以74份近年来江苏淮北地区所育品种(系)和38份来自黄淮其他麦区的常用亲本为材料,采用单籽粒谷物硬度测试仪、KASP标记检测技术和基因扩增及测序技术对其SKCS硬度值及硬度基因型进行鉴定。硬度检测结果表明,供试小麦品种(系)硬度变化范围较大,但硬质麦的比例最大,为70.5%。与常用亲本相比,江苏淮北地区育成品种中软质麦比例较高,为34.3%,但在高代品系中软质麦比例下降到20.5%。基因型检测结果表明,在Puroindoline-D1位点,供试品种(系)中共检测到4种基因型,即野生型(Pina-D1a/Pinb-D1a)、Pina-D1b、Pinb-D1b和Pinb-D1p,其频率依次为25.0%、2.7%、67.9%和4.5%。其中,野生型和Pinb-D1p主要分布在江苏淮北地区。不同硬度基因型的硬度值也存在差异,其中以Pina-D1b基因型的硬度值最高,野生型(Pina-D1a/Pinb-D1a)硬度值最低,Pinb-D1b和Pinb-D1p两硬质类型的籽粒硬度没有显著性差异。在Pinb-2位点,供试品种(系)中共检测到25份材料为Pinb-B2b基因型,包含21份硬质麦、2份混合麦和2份软质麦,其平均硬度值为63.8。