2018年第二周小麦文献推荐（1.14）

大家好！不知不觉今天是18年的第二周周末了。

昨天的推送收到很多反馈，胖丫让我感谢你们，她受益良多。有小伙伴问起Sentieon，昨天也没多说，今天多说两句，这个软件的功能和GATK功能类似，准确性上不输GATK，但是速度上要比它快N多倍。特别是当有大量样本需要做的时候，建议还是试一试Sentieon，毕竟时间就是金钱呢。这是一款商业软件，有兴趣的小伙伴可以邮件联系support@sentieon.com，可以获取试用版。也欢迎大家进群交流小麦方面的内容，可以加“wheatgenome”,老师或者博后也可以加“xiaoxuan8765”。

小麦组学研究在接来下2-3年一定是热点，除了中国春参考基因组，其它材料的基因组也会陆续发表。现在有各种组装技术加持，这些小麦族基因组不比参考基因组差，数据增多势必会促进我们的研究，但同时竞争也会更加激烈。作为传统的小麦研究者来讲，我们有材料，但是技术方面不成熟，而对于早期研究拟南芥、水稻等模式作物的研究者来讲，带着新技术新方法进入小麦也是顺其自然的过程。一个有技术，一个有材料或者数据，两者结合一定会有火花擦出来。回头看看最近2年的研究，这种合作方式越来越常见，最近黄三文小组和罗杰小组最近就联手在Cell上发表了一篇利用多组学数据研番茄果实的营养和风味的文章，这就是一个极好的合作共赢的例子。世界的发展也是这样的趋势，国家与国家之间联系越来越紧密，在这样一个信息和数据爆炸的时代，科研节奏会越来越快，专业的东西交给专业的人做，所以敞开胸怀合作一定不是坏选择。

又有点扯远了，下面让我们看看最近一周小麦方面又有哪些东西更新。

1 High throughput SNP discovery and genotyping in hexaploid wheat

Because of their abundance and their amenability to high-throughput genotyping techniques, Single Nucleotide Polymorphisms (SNPs) are powerful tools for efficient genetics and genomics studies, including characterization of genetic resources, genome-wide association studies and genomic selection. In wheat, most of the previous SNP discovery initiatives targeted the coding fraction, leaving almost 98% of the wheat genome largely unexploited. Here we report on the use of whole-genome resequencing data from eight wheat lines to mine for SNPs in the genic, the repetitive and non-repetitive intergenic fractions of the wheat genome. Eventually, we identified 3.3 million SNPs, 49% being located on the B-genome, 41% on the A-genome and 10% on the D-genome. We also describe the development of the TaBW280K high-throughput genotyping array containing 280,226 SNPs. Performance of this chip was examined by genotyping a set of 96 wheat accessions representing the worldwide diversity. Sixty-nine percent of the SNPs can be efficiently scored, half of them showing a diploid-like clustering. The TaBW280K was proven to be a very efficient tool for diversity analyses, as well as for breeding as it can discriminate between closely related elite varieties. Finally, the TaBW280K array was used to genotype a population derived from a cross between Chinese Spring and Renan, leading to the construction a dense genetic map comprising 83,721 markers. The results described here will provide the wheat community with powerful tools for both basic and applied research

2 Mapping of quantitative trait loci for grain yield and its components in a US popular winter wheat TAM 111 using 90K SNPs

Stable quantitative trait loci (QTL) are important for deployment in marker assisted selection in wheat (Triticum aestivum L.) and other crops. We reported QTL discovery in wheat using a population of 217 recombinant inbred lines and multiple statistical approach including multi-environment, multi-trait and epistatic interactions analysis. We detected nine consistent QTL linked to different traits on chromosomes 1A, 2A, 2B, 5A, 5B, 6A, 6B and 7A. Grain yield QTL were detected on chromosomes 2B.1 and 5B across three or four models of GenStat, MapQTL, and QTLNetwork while the QTL on chromosomes 5A.1, 6A.2, and 7A.1 were only significant with yield from one or two models. The phenotypic variation explained (PVE) by the QTL on 2B.1 ranged from 3.3-25.1% based on single and multi-environment models in GenStat and was pleiotropic or co-located with maturity (days to heading) and yield related traits (test weight, thousand kernel weight, harvest index). The QTL on 5B at 211 cM had PVE range of 1.8-9.3% and had no significant pleiotropic effects. Other consistent QTL detected in this study were linked to yield related traits and agronomic traits. The QTL on 1A was consistent for the number of spikes m-2 across environments and all the four analysis models with a PVE range of 5.8-8.6%. QTL for kernels spike-1 were found in chromosomes 1A, 2A.1, 2B.1, 6A.2, and 7A.1 with PVE ranged from 5.6-12.8% while QTL for thousand kernel weight were located on chromosomes 1A, 2B.1, 5A.1, 6A.2, 6B.1 and 7A.1 with PVEranged from 2.7-19.5%. Among the consistent QTL, five QTL had significant epistatic interactions (additive × additive) at least for one trait and none revealed significant additive × additive × environment interactions. Comparative analysis revealed that the region within the confidence interval of the QTL on 5B from 211.4-244.2 cM is also linked to genes for aspartate-semialdehyde dehydrogenase, splicing regulatory glutamine/lysine-rich protein 1 isoform X1, and UDP-glucose 6-dehydrogenase 1-like isoform X1. The stable QTL could be important for further validation, high throughput SNP development, and marker-assisted selection (MAS) in wheat.

3 Genotyping-by-Sequencing Derived High-Density Linkage Map and its Application to QTL Mapping of Flag Leaf Traits in Bread Wheat

Winter wheat parents ‘Harry’ (drought tolerant) and ‘Wesley’ (drought susceptible) were used to develop a recombinant inbred population with future goals of identifying genomic regions associated with drought tolerance. To precisely map genomic regions, high-density linkage maps are a prerequisite. In this study genotyping-by- sequencing (GBS) was used to construct the high-density linkage map. The map contained 3,641 markers distributed on 21 chromosomes and spanned 1,959 cM with an average distance of 1.8 cM between markers. The constructed linkage map revealed strong collinearity in marker order across 21 chromosomes with POPSEQ-v2.0, which was based on a high-density linkage map. The reliability of the linkage map for QTL mapping was demonstrated by co-localizing the genes to previously mapped genomic regions for two highly heritable traits, chaff color, and leaf cuticular wax. Applicability of linkage map for QTL mapping of three quantitative traits, flag leaf length, width, and area, identified 21 QTLs in four environments, and QTL expression varied across the environments. Two major stable QTLs, one each for flag leaf length (Qfll.hww-7A) and flag leaf width (Qflw.hww-5A) were identified. The map constructed will facilitate QTL and fine mapping of quantitative traits, map-based cloning, comparative mapping, and in marker-assisted wheat breeding endeavors.

4 A genome-wide association study of wheat yield and quality-related traits in southwest China

Wheat (Triticum aestivum L.) is one of the most productive and important crops and its yield potential and quality characteristics are tightly linked with the global food security. In this study, genome-wide association study (GWAS) was conducted for yield and quality-related traits. Based on the high-density wheat 90K Illumina iSelect SNP Array, 192 bread wheat lines from southwest China, including 25 synthetic hexaploid wheat lines, 80 landraces, and 87 cultivars were analyzed. Association analysis results indicated that there were 57, 27, 30, and 34 SNPs associated with plant height (PH), grain protein content (GPC), thousand kernel weight (TKW), and SDS sedimentation volume (SSV) have been detected, respectively. Then, integrating RNA-Seq with bioinformatics analysis, 246 candidate genes (102 for GPC, 52 for TKW, and 92 for SSV) were found. Further analysis indicated that one up-regulated and two down-regulated expression genes affect GPC. Additionally, two haplotypes significantly affecting PH were detected in a 2.2-Mb genome region encompassing a gene which encoded an ubiquitin-specific protease, TaUBP24. The functional markers of TaUBP24 have been developed, which could be used for marker-assisted selection to improve wheat quality and yield.

5 Characterization of Novel Gene Yr79 and Four Additional QTL for All-stage and High-temperature Adult-plant Resistance to Stripe Rust in Spring Wheat PI 182103

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. Exploring new resistance genes is essential for breeding resistant wheat cultivars. PI 182103, a spring wheat landrace originally from Pakistan, has shown a high level of resistance to stripe rust in fields for many years, but genes for resistance to stripe rust in the variety have not been studied. To map the resistance gene(s) in PI 182103, 185 recombinant inbred lines (RILs) were developed from a cross with Avocet Susceptible (AvS). The RIL population was genotyped with SSR and SNP markers and tested with races PST-100 and PST-114 at the seedling stage under controlled greenhouse conditions and at the adult-plant stage in fields at Pullman and Mt. Vernon, Washington under natural infection by the stripe rust pathogen in 2011, 2012, and 2013. A total of five quantitative trait loci (QTL) were detected. QyrPI182103.wgp-2AS and QyrPI182103.wgp-3AL were detected at the seedling stage, QyrPI182103.wgp-4DL was detected only in Mt. Vernon field tests, and QyrPI182103.wgp-5BS was detected in both seedling and field tests.QyrPI182103.wgp-7BL was identified as a high-temperature adult-plant (HTAP) resistance gene and detected in all field tests. Interactions among the QTL were mostly additive, but some negative interactions were detected. The 7BL QTL was mapped in chromosomal bin 7BL 0.40-0.45 and identified as a new gene, permanently designated as Yr79. SSR markers Xbarc72 and Xwmc335 flanking the Yr79 locus were highly polymorphic in various wheat genotypes, indicating that the molecular markers are useful for incorporating the new gene for potentially durable stripe rust resistance into new wheat cultivars.

6 Succession of Fungal and Oomycete Communities in Glyphosate-Killed Wheat Roots.

The successional dynamics of root-colonizing microbes are hypothesized to be critical to displacing fungal pathogens that can proliferate after the use of some herbicides. Applications of glyphosate in particular, which compromises the plant defense system by interfering with the production of aromatic amino acids, is thought to promote a buildup of root pathogens and can result in a ‘greenbridge’ between weeds or volunteers and crop hosts. By planting two to three weeks after spraying, growers can avoid most negative impacts of the greenbridge by allowing pathogen populations to decline, but with the added cost of delayed planting dates. However, the specific changes in microbial communities during this period of root death and the microbial taxa likely to be involved in displacing pathogens are poorly characterized. Using high-throughput sequencing, we characterized fungal and oomycete communities in roots after applications of herbicides with different modes of action (glyphosate or clethodim) and tracked their dynamics over three weeks in both naturally infested soil and soil inoculated with Rhizoctonia solani *AG-8. We found that many unexpected taxa were present at high relative abundance (eg. *Pythium volutum and Myrmecridium species) in live and dying wheat roots and may play an under-recognized role in greenbridge dynamics. Moreover, communities were highly dynamic over time and had herbicide-specific successional patterns, but became relatively stable by two weeks after herbicide application. Network analysis of communities over time revealed patterns of interactions among taxa that were both common and unique to each herbicide treatment and identified two primary groups of taxa with many positive associations within-groups but negative associations between-groups, suggesting that these groups are antagonistic to one another in dying roots and may play a role in displacing pathogen populations during greenbridge dynamics.

7 Characterization of Adult Plant Resistance to Leaf Rust and Stripe Rust in Indian Wheat Cultivar ‘New Pusa 876’

Growing resistant varieties is the most effective and economical method for controlling rust of wheat (Triticum aestivum L.). Resistance to leaf rust and stripe rust, caused by Puccinia triticina Erikss. and P. striiformis Westend. f. sp. tritici, respectively, was investigated in 148 F5recombinant inbred lines derived from a cross between ‘Avocet’ and ‘New Pusa 876’ (NP876). The parents and population were phenotyped for resistance in field trials for 3 and 2 yr for leaf rust and stripe rust, respectively, and genotyped with gene-linked molecular markers. The segregation analyses indicated that the adult plant resistance to leaf rust and stripe rust was conferred by five and four additive effect genes, respectively. Among them, the slow-rusting adult plant resistance gene Lr46/Yr29 reduced 14 and 16% of mean leaf rust and stripe rust severities, respectively, whereas a severity reduction of 26% occurred due to Lr67/Yr46 for both rusts. Both resistance genes were contributed by NP876. An additive effect between leaf rust resistance genes Lr46 and Lr67 was detected, with a reduction up to 11% when they were present together. The effect of combining Yr29 and Yr46 was additive but not significant, with a mean reduction of 5% in severity. New Pusa 876 can be used as a multiple rust resistance source to breed wheat varieties that may contribute towards durable resistance.

8 Temperature and Alternative Hosts Influence Aceria tosichella Infestation and Wheat Streak Mosaic Virus Infection

Wheat streak mosaic, caused by Wheat streak mosaic virus (WSMV; family Potyviridae), is the most important and common viral disease of wheat (Triticum aestivum L.) in the Great Plains of North America. WSMV is transmitted by the wheat curl mite (WCM; Aceria tosichella). We evaluated how mean daily temperatures, cumulative growing degree-days, day of the year, and surrounding alternative host identity affected WCM infestation and WSMV infection of wheat from late summer through early autumn in Montana, United States. Cumulative growing degree-days, warm mean daily temperatures (i.e., >10°C), and surrounding alternative hosts interacted to alter risk of WCM infestation and WSMV infection. Wheat surrounded by Bromus tectorum L. and preharvest volunteer wheat had WCM infestation and WSMV infection rates of 88% in years when the mean daily temperature was 15°C in October, compared with 23% when surrounded by bare ground, and <1% when the temperature was 0°C regardless of surrounding alternative host. Mean daily temperatures in the cereal-growing regions of Montana during autumn are marginally conducive to WCM population growth and movement. As the region continues to warm, the period of WCM movement will become longer, potentially increasing the frequency of WSMV outbreaks.

9 Wheat resistances to Fusarium root rot and head blight are both associated with deoxynivalenol and jasmonate related gene expression

Fusarium graminearum is a major pathogen of wheat causing Fusarium head blight (FHB). Its ability to colonize wheat via seedling root infection has been reported recently. Our previous study on Fusarium root rot (FRR) has disclosed histological characteristics of pathogenesis and pathogen defense that mirror processes of spike infection. Therefore, it would be interesting to understand whether genes relevant for FHB resistance are induced in roots. The concept of similar-acting defense mechanisms provides a basis for research at broad Fusarium resistance in crop plants. However, molecular defense responses involved in FRR as well as their relation to spike resistance are unknown. To test the hypothesis of a conserved defense response, a candidate gene expression study was conducted to test the activity of selected prominent FHB defense-related genes in seedling roots, adult plant roots, spikes and shoots. FRR was examined at seedling and adult plant stages to assess age-related pattern of disease and pathogen resistance. This study offers first evidence for a significant genetic overlap in root and spike defense responses, both in local and distant tissues. The results point to plant development-specific rather than organ-specific determinants of resistance, and suggest roots as an interesting model for studies on wheat-Fusarium interactions.

10 Three-Dimensional Analysis of Chloroplast Structures Associated with Virus Infection

Chloroplasts are multifunctional organelles whose morphology is affected by environmental stresses. Although the three-dimensional (3D) architecture of thylakoid membranes has been reported previously, a 3D visualization of chloroplast under stress has not been explored. In this work, we used a positive-strand RNA ((+)RNA) virus, barley stripe mosaic virus (BSMV) to observe chloroplast structural changes during infection by electron tomography. The analyses revealed remodeling of the chloroplast membranes, characterized by the clustering of outer membrane-invaginated spherules in inner membrane-derived packets. Diverse morphologies of cytoplasmic invaginations (CIs) were evident with spherules at the periphery and different sized openings connecting the CIs to the cytoplasm. Immunoelectron microscopy of these viral components verified that the aberrant membrane structures were sites for BSMV replication. The BSMV αa replication protein localized at the surface of the chloroplasts and played a prominent role in eliciting chloroplast membrane rearrangements. In sum, our results have revealed the 3D structure of the chloroplasts induced by BSMV infection. These findings contribute to our understanding of chloroplast morphological changes under stress conditions and during assembly of plant (+)RNA virus replication complexes.

11 No Time to Waste: Transcriptome Study Reveals that Drought Tolerance in Barley May Be Attributed to Stressed-Like Expression Patterns that Exist before the Occurrence of Stress

Plant survival in adverse environmental conditions requires a substantial change in the metabolism, which is reflected by the extensive transcriptome rebuilding upon the occurrence of the stress. Therefore, transcriptomic studies offer an insight into the mechanisms of plant stress responses. Here, we present the results of global gene expression profiling of roots and leaves of two barley genotypes with contrasting ability to cope with drought stress. Our analysis suggests that drought tolerance results from a certain level of transcription of stress-influenced genes that is present even before the onset of drought. Genes that predispose the plant to better drought survival play a role in the regulatory network of gene expression, including several transcription factors, translation regulators and structural components of ribosomes. An important group of genes is involved in signaling mechanisms, with significant contribution of hormone signaling pathways and an interplay between ABA, auxin, ethylene and brassinosteroid homeostasis. Signal transduction in a drought tolerant genotype may be more efficient through the expression of genes required for environmental sensing that are active already during normal water availability and are related to actin filaments and LIM domain proteins, which may function as osmotic biosensors. Better survival of drought may also be attributed to more effective processes of energy generation and more efficient chloroplasts biogenesis. Interestingly, our data suggest that several genes involved in a photosynthesis process are required for the establishment of effective drought response not only in leaves, but also in roots of barley. Thus, we propose a hypothesis that root plastids may turn into the anti-oxidative centers protecting root macromolecules from oxidative damage during drought stress. Specific genes and their potential role in building up a drought-tolerant barley phenotype is extensively discussed with special emphasis on processes that take place in barley roots. When possible, the interconnections between particular factors are emphasized to draw a broader picture of the molecular mechanisms of drought tolerance in barley.

12 How exogenous nitric oxide regulates nitrogen assimilation in wheat seedlings under different nitrogen sources and levels

Nitrogen (N) is one of the most important nutrients for plants and nitric oxide (NO) as a signaling plant growth regulator involved in nitrogen assimilation. Understanding the influence of exogenous NO on nitrogen metabolism at the gene expression and enzyme activity levels under different sources of nitrogen is vitally important for increasing nitrogen use efficiency (NUE). This study investigated the expression of key genes and enzymes in relation to nitrogen assimilation in two Australian wheat cultivars, a popular high NUE cv. Spitfire and a normal NUE cv. Westonia, under different combinations of nitrogen and sodium nitroprusside (SNP) as the NO donor. Application of NO increased the gene expressions and activities of nitrogen assimilation pathway enzymes in both cultivars at low levels of nitrogen. At high nitrogen supplies, the expressions and activities of N assimilation genes increased in response to exogenous NO only in cv. Spitfire but not in cv. Westonia. Exogenous NO caused an increase in leaf NO content at low N supplies in both cultivars, while under high nitrogen treatments, cv. Spitfire showed an increase under ammonium nitrate (NH4NO3) treatment but cv. Westonia was not affected. N assimilation gene expression and enzyme activity showed a clear relationship between exogenous NO, N concentration and N forms in primary plant nitrogen assimilation. Results reveal the possible role of NO and different nitrogen sources on nitrogen assimilation in Triticum aestivum plants.

13 Reference Quality Genome Assemblies of Three Parastagonospora nodorum Isolates Differing in Virulence on Wheat

Parastagonospora nodorum, the causal agent of Septoria nodorum blotch of wheat, has emerged as a model necrotrophic fungal organism for the study of host-microbe interactions. To date, three necrotrophic effectors have been identified and characterized from this pathogen, including SnToxA, SnTox1, and SnTox3. Necrotrophic effector identification was greatly aided by the development of a draft genome of Australian isolate SN15 via Sanger sequencing, yet remained largely fragmented. This research presents the development of near-finished genomes of *P. nodorum *isolates Sn4, Sn2000, and Sn79-1087 using long-read sequencing technology. RNAseq analysis of isolate Sn4 consisting of eight time-points covering various developmental and infection stages mediated the annotation of 13,379 genes. Analysis of these genomes revealed large-scale polymorphism between the three isolates, including the complete absence of contig 23 from isolate Sn79-1087 and a region of genome expansion on contig 10 in isolates Sn4 and Sn2000. Additionally, these genomes exhibit the hallmark characteristics of a 'two-speed' genome, being partitioned into two distinct GC-equilibrated and AT-rich compartments. Interestingly, isolate Sn79-1087 contains a lower proportion of AT-rich segments, indicating a potential lack of evolutionary hot spots. These newly sequenced genomes, consisting of telomere to telomere assemblies of nearly all 23 *P. nodorum *chromosomes provides a robust foundation for the further examination of effector biology and genome evolution.

14 Exploration of Mechanisms for Internal Deterioration of Wheat Seeds in Postharvest Storage and Nitrogen Atmosphere Control for Properties Protection

Wheat (Triticum aestivum L.) seeds were stored in simulated conditions of four regions for 300 d. Changes of biochemical properties and electron microscope pictures demonstrated that unfavorable storage conditions caused serious internal deterioration and promoted the accumulation of unhealthy products in wheat seeds. Two hypotheses were proposed to explain the internal deterioration during storage. This work confirmed that the unfavorable storage conditions damaged enzymatic scavenging systems. As a result, without an effective scavenging system, serious internal deterioration occurred in wheat seeds. Atmospheric composition was adjusted to evaluate the potential of applying nitrogen atmosphere control in wheat storage. The results showed that 98% nitrogen gas in atmosphere effectively protected scavenging systems in wheat seeds and alleviate the internal deterioration.

15 Winter Wheat Yield Gaps and Patterns in China

Wheat (Triticum aestivum L.) yield stagnation has been reported in some regions of the world. China is the largest producer of wheat across the globe, but the pattern of its wheat yield stagnation remains poorly addressed. Here, our goal is to examine the temporal trends and spatial patterns of wheat yields along with possible causes based on a comprehensive assessment of winter wheat yield throughout China over the 31-yr period from 1980 to 2010. Combined with the Agricultural Production Systems Simulator (APSIM) wheat model, we assessed the winter wheat yield gaps and patterns in 1414 counties and at five physiogeographic regional scales across China to ascertain the driving factors of yield variations. Wheat yields increased in 53% of the 1414 counties, but the remaining counties experienced yields that never improved, stagnated, or collapsed from 1980 to 2010. The yield gap analysis showed that actual yields represented only 59% of the national average yield potential, indicating a substantial opportunity to improve winter wheat yields. Relatively larger yield gaps were observed in the northern China Plain (NC, 47%) and in southwestern China (SW, 45%). Although the yield gaps in these regions were accompanied by significantly progressive uptrends of actual yields, our results suggest that agronomic management could be further improved. Moreover, underperforming regions could potentially benefit from new investments and strategies to reliably increase actual yields and reverse trends in stagnation in winter wheat performance.

16 Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome

We constructed and analyzed the pangenome of Zymoseptoria tritici, a major pathogen of wheat that evolved host specialization by chromosomal rearrangements and gene deletions. We used single-molecule real-time sequencing and high-density genetic maps to assemble multiple genomes. We annotated the gene space based on transcriptomics data that covered the infection life cycle of each strain. Based on a total of five telomere-to-telomere genomes, we constructed a pangenome for the species and identified a core set of 9149 genes. However, an additional 6600 genes were exclusive to a subset of the isolates. The substantial accessory genome encoded on average fewer expressed genes but a larger fraction of the candidate effector genes that may interact with the host during infection. We expanded our analyses of the pangenome to a worldwide collection of 123 isolates of the same species. We confirmed that accessory genes were indeed more likely to show deletion polymorphisms and loss-of-function mutations compared to core genes.

17 Expressed Ay HMW glutenin subunit in Australian wheat cultivars indicates a positive effect on wheat quality

Out of the six HMW-GS genes, 1Ay is usually not expressed in bread wheat cultivars. In the current study, an active 1Ay gene has been integrated into two Australian wheat cultivars, Livingston and Bonnie Rock, through conventional backcross approach. Three sister lines at BC4F4 generation for each cross were obtained and underwent a series of quality testing. Results show that the active 1Ay subunit increased the amount total protein, Glutenin/Gliadin ratio and unextractable polymeric protein. The expressed 1Ay also resulted in up to 10% increase of gluten content, 5% increase of glutenin, and hence increased the HMW- to LMW-GS ratio without affecting the relative amount of other subunits. Milling yield and Flour swelling were decreased in the Livingston lines and remained mostly unchanged for Bonnie Rock. Alveograph result showed that Ay improved dough strength in Livingston and dough extensibility in Bonnie Rock. Zeleny sedimentation value was found to be higher in all three lines of Bonnie Rock but only in one of Livingston derivatives. The dough development time and peak resistance, determined on the micro Z-arm mixer were increased in most cases. Overall, the integration of Ay subunit showed significant positive effects in bread making quality.