Analysis of Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm by Ecotilling and identification of a novel Pinb allele

Kernel hardness is mainly conditioned by allelic variations of Pina-D1 and Pinb-D1 genes located on the short arm of chromosome 5D. In this work, the Ecotilling approach was optimized to investigate Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm, and three Pina and eight Pinb alleles were found. Generally, more Pinb alleles were detected in the accessions coming from the regions that grow winter or a mixture of spring and winter wheats. This was particularly evident for the Southwestern winter wheat, Xinjiang winter–spring wheat and Yellow and Huai River Valley winter wheat regions. A novel variant (designated as Pinb-D1x) was discovered in one of the accessions from the Xinjiang winter–spring wheat region. Compared to wild type (WT) allele Pinb-D1a, two nucleotide substitutions occurred in the coding region of Pinb-D1x, one (at nucleotide position 257) resulting in the replacement of a WT cysteine residue by tyrosine and the other (at nucleotide position 382) creating a premature stop codon. The implications of our data to understanding the diversity of Pina and Pinb alleles in wheat and to future molecular breeding of wheat kernel hardness are discussed.     

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。     

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.     

Transformation of Pinb-D1x to soft wheat produces hard wheat kernel texture

Kernel hardness - a key quality trait of common wheat (Triticum aestivum L.) - is mainly conditioned by the Pina and Pinb genes. Mutation or deletion of Pina or Pinb increases kernel hardness, resulting in a hard wheat kernel texture. Here, Pinb-D1x gene was cloned from a hard wheat landrace Kashibaipi and transformed into a soft wheat cultivar Yangmai19 to assess its effect on kernel hardness and flour properties. PCR, RT-PCR and Western blot data confirmed the successful transformation and overexpression of Pinb-D1x gene in transgenic offsprings. The data of single kernel characterization system and scanning electron microscopy revealed that the introduction of Pinb-D1x in soft wheat increased the kernel hardness significantly and changed the internal structure of the kernel. Similarly, transgenic lines exhibited hard wheat like flour properties; flour whiteness and pasting temperature were significantly reduced in the transgenic lines, while the total protein content, damaged starch content, and compound parameter in the Mixograph tests (PT × TW value) showed a significant increase over the wildtype. The results showed that the transformation of the Pinb variants is a powerful strategy to alter the kernel hardness and flour properties in wheat breeding.     

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.     

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.     

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.     

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%。     

新疆小麦籽粒SOD活性及TaSOD-A1位点等位变异的分布

为了解新疆小麦品种(系)籽粒超氧化物歧化酶(SOD)的活性及TaSOD-A1位点等位变异的分布,用两个功能标记SODA1和SODA11对117份新疆小麦品种(系)的 TaSOD-A1位点(基因ID为TraesCS5A01G290800)进行等位变异检测,并结合SOD活性检测结果,分析 TaSOD-A1位点不同等位变异与SOD活性的相关性。结果表明,含有 TaSOD-A1a等位变异材料的籽粒SOD活性显著高于含有 TaSOD-A1b等位变异材料的籽粒,二者占比分别为50.4%和49.6%;新疆冬小麦品种(系)中, TaSOD-A1a等位变异的分布频率高低依次为引进品种(系)>自育品种(系)>地方品种;新疆春小麦品种(系)中,只有3份材料含有 TaSOD-A1a等位变异,早期品种(系)中未发现含有 TaSOD-A1a等位变异的材料。新疆冬小麦品种(系)的籽粒SOD活性平均值显著高于新疆春小麦品种(系),且新疆冬小麦引进品种(系)中含有 TaSOD-A1a等位变异材料的籽粒SOD活性平均值也显著高于含有 TaSOD-A1b等位变异材料的籽粒SOD活性。     

White salted noodle characteristics from transgenic isolines of wheat over expressing puroindolines

The closely linked genes puroindoline a (Pina) and puroindoline b (Pinb) control most of the variation in wheat (Triticum aestivum) grain texture. Mutations in either Pina or Pinb result in hard grain with wild type forms of both genes giving soft grain. Asian noodles are prepared from both hard and soft classes of wheat. Our objective was to examine color and texture characteristics of white salted noodles processed from flours of transgenic isolines of Hi-Line hard red spring wheat over expressing Pina-D1a, Pinb-D1a or both and a control giving a range in grain texture from very soft to hard. White salted noodles were prepared and color and texture characteristics were measured. The three softer textured transgenic isolines showed greater change in L^* with time than Hi-Line. The noodles were more adhesive (more negative value), firmer, and chewier as the grain texture became successively softer when cooked at 5 min. These texture differences were not as apparent when noodles were cooked for an optimum time. Starch pasting properties did not explain the noodle textural differences. A possible explanation for the noodle texture differences may be related to starch damage which ranged from 2.2% for HGAB to 6.7% for Hi-Line, flour particle size differences and subsequent water absorption differences among the four genotypes. Over expression of puroindolines did not enhance quality of white salted noodles when prepared under these conditions.     

新疆小麦品种 Glu A3和 Glu B3位点等位变异的分布

为给新疆小麦品质育种提供理论依据,利用 GluA3、 GluB3位点上的17个STS标记检测了185份新疆冬、春小麦品种 GluA3和 GluB3位点的等位变异。结果表明,新疆小麦品种以 GluA3c、 GluB3a和 GluB3j亚基为主,其分布频率分别为64.86%、22.70%和17.84%。新疆冬、春小麦品种在 GluA3位点上均以 GluA3c亚基为主,分布频率分别为63.30%和67.11%;在 GluB3位点上,新疆冬、春小麦品种分别以 GluB3j和 GluB3a为主,分布频率分别为22.02%和26.32%。新疆冬、春小麦农家品种亚基类型较少,冬小麦农家品种仅有5种类型（以 GluA3c和 GluB3i为主）,春小麦农家品种有10种类型（以 GluA3c和 GluB3d为主）。引进品种和自育品种亚基类型丰富,冬小麦引进品种以 GluA3c和 GluB3i为主,分布频率为12.84%和6.42%;春小麦引进品种以 GluA3c和 GluB3j为主,分布频率为17.11%和6.58%。冬小麦自育品种以 GluA3c和 GluB3j亚基类型为主,分布频率为45.87%和18.35%;春小麦自育品种以 GluA3c和 GluB3a亚基类型为主,分布频率为36.84%和18.42%。     

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。     

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.     

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.     

Puroindolines co-localize to the starch granule surface and increase seed bound polar lipid content

Endosperm texture in wheat is controlled by the Pina and Pinb genes that comprise the Hardness (Ha) locus. Studies have shown that soft and hard varieties differ in the amount of starch bound polar lipids but have not addressed whether PINs are directly involved and whether the presence of one particular PIN affects seed polar lipid levels and cellular localization. Here, we determined the effect of overexpressing PINA or PINB on seed bound polar lipids and PIN localization. F₃ recombinants homozygous for either a Pina or Pinb null Ha locus with or lacking a transgenically added Pina or Pinb were analyzed for grain hardness, PIN abundance, and seed bound polar lipid levels. Overexpressed PINs resulted in reduced hardness, increased starch bound PINs, and increased seed bound polar lipids. Addition of PINA to the PINA nulls or PINB to the PINB nulls resulted in higher bound polar lipid levels than the addition of the alternative PIN. Both PINs localized to the starch surface in the presence or absence of the other protein. Our results indicate that PIN overexpression results in reduced endosperm texture and increased seed bound polar lipids and that PINs independently localize to the surface of starch granules.     

Identification of new Pina null mutations among Asian common wheat cultivars

Wheat grain hardness is one of the most important phenotypes related to milling, baking and noodle making. Either a mutation of the Puroindoline-a (Pina) gene or Puroindoline-b (Pinb) gene results in hard grain texture. A deletion mutation of Pina (Pina-D1b) is widely distributed among common wheat cultivars. Although North/South American and Australian cultivars and their descendants have a 15-kbp deletion in common, two new types of deletion mutation were found among Asian wheat cultivars. A 4.4-kbp deletion was found in one Korean and two Chinese wheat cultivars beginning at position +371 within the Pina coding region. The other, a 10.4-kbp deletion, was found in three Chinese and nine Japanese wheat cultivars, including five Japanese landraces, beginning at position −5112. It caused the deletion of the full-length Pina gene. These findings suggest that Asian wheat cultivars are genetically distinct from those in other regions. The 4.4-kbp and 10.4-kbp deletion mutants were designated as Pina-D1r and Pina-D1s, respectively.     

新疆小麦品种春化和光周期主要基因的组成分析

为了明确新疆冬春麦区小麦春化和光周期基因的分布特点,利用STS标记对185份品种(系)的重要春化基因Vrn-A1、Vrn-B1、Vrn-D1、Vrn-B3和光周期基因Ppd-D1位点的等位变异组成进行了检测和分析。结果表明,在新疆小麦品种中,春化和光周期基因位点显性等位变异分布频率不同。含有春化显性等位变异Vrn-A1的品种47个,占供试品种(系)的25.4%;Vrn-B1为43个,占23.3%;Vrn-D1为38个,占20.5%;Vrn-B3位点不存在显性等位变异。春化显性等位变异Vrn-A1、Vrn-B1和Vrn-D1在冬、春性小麦内的分布比例也不同。在春性小麦品种(系)中,显性等位变异Vrn-A1出现的频率较高(55.3%);其次为Vrn-B1,占50.6%;Vrn-D1占44.7%。在冬性小麦中,仅有显性等位变异Vrn-B1出现,占2.0%。在光周期基因Ppd-D1位点,80.0%的品种(系)携带光不敏感显性等位变异Ppd-D1a;其中在春性和冬性小麦品种(系)中,Ppd-D1a出现的频率分别为83.5%和77.0%。新疆小麦品种(系)中,存在11种春化和光周期基因显性等位变异组合。     

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。