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.     

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。     

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.     

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。     

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.     

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.     

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.     

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.     

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.     

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%。     

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.     

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.     

小麦面粉及鲜面片色泽的影响因素

为明确小麦面粉及鲜面片色泽的影响因素,选取10个江苏沿淮及淮南地区大面积推广小麦品种进行2年3点种植,比较不同品种、不同地点和不同年份面粉及鲜面片色泽差异并分析其影响因素,探讨面粉及鲜面片色泽与品质性状间的相关性。结果表明,扬麦11、扬辐麦4号和宁麦14的面粉及鲜面片色泽较好,亮度高、黄度低。基因型是决定面粉白度、亮度(L~*值)和黄度(b~*值)的主要因素;环境因素亦影响面粉及鲜面片色泽。扬麦16、宁麦14和郑麦9023在不同年度及不同地点的色泽表现稳定。面粉与鲜面片放置24h内的色泽间呈显著正相关;面粉白度与籽粒硬度和出粉率呈极显著负相关;面粉及鲜面片L~*值与蛋白质含量、湿面筋含量、揉混仪参数和粉质仪参数呈显著或极显著负相关,与糊化特性参数呈显著或极显著正相关。综上所述,面粉色泽主要由基因型决定,鲜面片色泽与面粉色泽显著相关,二者与籽粒品质具有显著相关性。在小麦育种中,可通过面粉色泽指标筛选高白度及品质的品种。     

普通小麦籽粒硬度的分子标记研究

籽粒硬度是决定小麦磨粉品质和食品品质的重要性状，蛋白复合体Friabilin的两种主要肽Puroindolinea（PinA）和Puroindolineb（PinB）是决定籽粒硬度的关键。用单籽粒谷物特性仪（SKCS）、PCR技术和改进的SDS-PAGE方法分析了85份小麦品种的籽粒硬度和基因突变形式，结果表明，与软质小麦相比，硬质小麦品种在基因或蛋白质表达水平上发生了变化，有31份野生型的软质小麦Pina-Dla／Pinb-Dla和6份Pina-D1b／Pinb-D1a突变型，42份Pina-D1a／Pinb-D1b和6份Pina—D1a／Pinb-D1p突变型。     

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.     

安徽麦区软质小麦籽粒品质和终端制品品质评价

为明确安徽大田生产环境下软质小麦籽粒和终端产品品质表现,评价优质软麦品种的加工适用性,本研究选取该区当前推广种植的24个软质小麦品种,对其籽粒和面粉的主要品质性状及其制品南方馒头和曲奇饼干的品质进行差异性、相关性分析,并以美国软白麦近五年的主要品质性状平均值为理想指标进行灰色关联度比较。结果表明,供试材料的硬度、面粉色泽b^*、湿面筋含量、面团形成时间、稳定时间等籽粒品质性状变异系数较大,而容重、面粉L^*值和吸水率变异系数较小。南方馒头品质性状中,白度差异最小,比容差异最大;曲奇饼干品质性状中,感官评分变异系数较大,饼干直径均值和变异系数都较小。蛋白质含量、湿面筋含量、稳定时间均符合弱筋标准(GB/T 17320-2013)的样品数为0。相关分析表明,容重、降落值、面粉L^*、b^*、白度与大部分性状间相关性不显著;籽粒硬度与水SRC和乳酸SRC均呈显著正相关,与湿面筋含量和面粉a^*值均呈显著负相关。蛋白质含量与面粉a^*值等7个指标均呈显著正相关,与面粉b     

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.     

Effect of incorporation of an i-type low-molecular-weight glutenin subunit and a modified γ-gliadin in durum and in bread wheat doughs as measured by micro-mixographic analyses

The effects of incorporation of an i-type low-molecular-weight glutenin subunit (LMW-i) and of a modified γ-gliadin showing an additional cysteine residue, on 2 g Mixograph parameters of durum (biotypes 42 and 45 of the Italian cv. Lira) and bread wheat (Australian cv. Kukri) doughs were studied. In bread wheat flour incorporation of the modified γ-gliadin resulted in a significant decrease in dough strength (decreased mixing time and peak resistance), but at the same time it produced a slight increase in dough stability (decreased resistance to breakdown). The incorporation of the LMW-i type into bread wheat dough had minimal effects on dough mixing requirements. The incorporation of both LMW-i type and modified γ-gliadin in durum wheat doughs produced a significant decrease in the overall dough strength, especially in Lira 45 biotype doughs. Reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion high-performance liquid chromatography (SE-HPLC) and two-dimensional gels analyses of control and reconstituted semolina doughs showed that the two polypeptides were in the polymeric fraction. The effect of the incorporation of the two polypeptides in durum and bread wheat doughs showed remarkable differences and the reasons for this is discussed in terms of both intrinsic differences between wheat flour and durum semolina and in methodological approaches.