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[...]we have successfully mutated waxy gene to generate glutinous wheat with lower amylose content. PPO catalyses phenols oxidation into dark-coloured products, a feature often undesirable for wheat end-use products. [...]developing wheat cultivars with low PPO activity has always been an important goal in wheat breeding. [...]new allelic variations of the target genes (pinb, waxy, ppo and psy) were created in Fielder through Agrobacterium-delivered CRISPR/Cas9 system. Furthermore, many of the mutants had segregated out the CRISPR/Cas9 transgene. [...]we had successfully obtained new wheat germplasms with improved grain quality in hardness, starch composition and dough colour.

Wheat (Triticum aestivum L.) is one of the most important crops worldwide, and, as a resilient cereal, it grows in various climatic zones. Due to changing climatic conditions and naturally occurring environmental fluctuations, the priority problem in the cultivation of wheat is to improve the quality of the crop. Biotic and abiotic stressors are known factors leading to the deterioration of wheat grain quality and to crop yield reduction. The current state of knowledge on wheat genetics shows significant progress in the analysis of gluten, starch, and lipid genes responsible for the synthesis of the main nutrients in the endosperm of common wheat grain. By identifying these genes through transcriptomics, proteomics, and metabolomics studies, we influence the creation of high-quality wheat. In this review, previous works were assessed to investigate the significance of genes, puroindolines, starches, lipids, and the impact of environmental factors, as well as their effects on the wheat grain quality.

Kernel hardness that is conditioned by puroindoline genes has a profound effect on milling, baking and end-use quality of bread wheat. In this study, 219 landraces and 166 historical cultivars from China and 12 introduced wheats were investigated for their kernel hardness and puroindoline alleles, using molecular and biochemical markers. The results indicated that frequencies of soft, mixed and hard genotypes were 42.7, 24.3, and 33.0%, respectively, in Chinese landraces and 45.2, 13.9, and 40.9% in historical cultivars. The frequencies of PINA null, Pinb-D1b and Pinb-D1p genotypes were 43.8, 12.3, and 39.7%, respectively, in hard wheat of landraces, while 48.5, 36.8, and 14.7%, respectively, in historical hard wheats. A new Pinb-D1 allele, designated Pinb-D1t, was identified in two landraces, Guangtouxianmai and Hongmai from the Guizhou province, with the characterization of a glycine to arginine substitution at position 47 in the coding region of Pinb gene. Surprisingly, a new Pina-D1 allele, designated Pina-D1m, was detected in the landrace Hongheshang, from the Jiangsu province, with the characterization of a proline to serine substitution at position 35 in the coding region of Pina gene; it was the first novel mutation found in bread wheat, resulting in a hard endosperm with PINA expression. Among the PINA null genotypes, an allele designed as Pina-D1l, was detected in five landraces with a cytosine deletion at position 265 in Pina locus; while another novel Pina-D1 allele, designed as Pina-D1n, was identified in six landraces, with the characterization of an amino acid change from tryptophan-43 to a 'stop' codon in the coding region of Pina gene. The study of puroindoline polymorphism in Chinese wheat germplasm could provide useful information for the further understanding of the molecular basis of kernel hardness in bread wheat.

The quantitative level of friabilin 15-kDa protein present on the surface of water-washed starch is highly correlated with wheat grain softness. Friabilin is composed primarily, if not exclusively, of the proteins puroindoline a and b. The transcript levels of these two proteins are similar among hard and soft wheat varieties, and the expression of both is controlled by the short arm of chromosome 5D, also the chromosomal location of the Hardness gene. We report here a glycine to serine sequence change in puroindoline b associated with hard grain texture. This amino acid change results from a single nucleotide mutation and resides in a region thought to be important for the lipid-binding properties of puroindolines. No recombination was observed between the serine puroindoline-b mutation, hard grain texture and low levels of starch surface friabilin among a set of 83 homozygous SD recombinant lines derived from the soft-textured variety 'Chinese Spring' and the substitution line 'Chinese Spring' containing the 5D chromosome of the hard-textured variety 'Cheyenne'. The sequence change reported here may adversely affect the lipid-binding properties of puroindoline-b and so effect hard grain texture. The results suggest that grain hardness results from puroindoline-b functionality such that the Hardness gene is a direct manifestation of puroindoline structure. We are suggesting the tentative molecular marker loci designations of Pinb-D1a and Pinb-D1b for the glycine and serine puroindoline-b types, respectively.

Wheat (Triticum aestivum L.) grain hardness is determined mainly by variations in puroindoline genes (Pina-D1 and Pinb-D1), which are located on the short arm of chromosome 5D. This trait has a direct effect on the technological properties of the flour and the final product quality. The objective of the study was to analyze the mutation frequency in both Pin genes and their influence on grain hardness in 118 modern bread wheat cultivars and breeding lines cultivated in Poland, and 80 landraces from Poland. The PCR products containing the Pin gene coding sequences were sequenced by the Sanger method. Based on detected the SNPs (single-nucleotide polymorphisms) we designed CAPS (cleaved amplified polymorphic sequence) markers for the fast screening of Pinb alleles in a large number of genotypes. All analyzed cultivars, breeding lines, and landraces possess the wild-type Pina-D1a allele. Allelic variation was observed within the Pinb gene. The most frequently occurring allele in modern wheat cultivars and breeding lines (over 50%) was Pinb-D1b. The contribution of the remaining alleles (Pinb-D1a, Pinb-D1c, and Pinb-D1d) was much less (approx. 15% each). In landraces, the most frequent allele was Pinb-D1a (over 70%), followed by Pinb-D1b (21% frequency). Pinb-D1c and Pinb-D1g were found in individual varieties. SKCS (single-kernel characterization system) analysis revealed that grain hardness was strictly connected with Pinb gene allelic variation in most tested cultivars. The mean grain hardness values were significantly greater in cultivars with mutant Pinb variants as compared to those with the wild-type Pinb-D1a allele. Based on grain hardness measured by SKCS, we classified the analyzed cultivars and lines into different classes according to a previously proposed classification system.

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F2:F3 populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.

\"Soft\" and \"hard\" are the two main market classes of wheat (Triticum aestivum L.) and are distinguished by expression of the Hardness gene, Friabilin, a marker protein for grain softness (Ha), consists of two proteins, puroindoline a and b (pinA and pinB, respectively) we previously demonstrated that a glycine to serine mutation in pinB is linked inseparably to grain hardness. Here, we report that the pinB serine mutation is present in 9 of 13 additional randomly selected hard wheats and in none of 10 soft wheats. The four exceptional hard wheats not containing the serine mutation in pinB express no pinA, the remaining component of the marker protein friabilin. The absence of pinA protein was linked inseparably to grain hardness among 44 near-isogenic lines created between the soft variety Heron and the hard variety Falcon. Both pinA and pinB apparently are required for the expression of grain softness. The absence of pinA and protein and transcript and a glycine-to-serine mutation in pinB are two highly conserved mutations associated with grain hardness, and these friabilin genes are the suggested tightly linked components of the Hardness gene. A previously described grain hardness related gene termed \"GSP-1\" (grain softness protein) is not controlled by chromosome 5D and is apparently not involved in grain hardness. The association of grain hardness with mutations in both pinA or pinB indicates that these two proteins alone may function together to effect grain softness. Elucidation of the molecular basis for grain hardness opens the way to understanding and eventually manipulating this wheat endosperm property

Wheat grain is sold based upon several physiochemical characteristics, one of the most important being grain texture. Grain texture in wheat directly affects many end use qualities such as milling yield, break flour yield, and starch damage. The hardness (Ha) locus located on the short arm of chromosome 5D is known to control grain hardness in wheat. This locus contains the puroindoline A (pina) and puroindoline B (pinb) genes. All wheats to date that have mutations in pina or pinb are hard textured, while wheats possessing both the 'soft type' pina-D1a and pinb-D1a sequences are soft. Furthermore, it has been shown that complementation of the pinb-D1b mutation in hard spring wheat can restore a soft phenotype. Here, our objective was to identify and characterize the effect the puroindoline genes have on grain texture independently and together. To accomplish this we transformed a hard red spring wheat possessing a pinb-D1b mutation with 'soft type' pina and pinb, creating transgenic isolines that have added pina, pinb, or pina and pinb. Northern blot analysis of developing control and transgenic lines indicated that grain hardness differences were correlated with the timing of the expression of the native and transgenically added puroindoline genes. The addition of PINA decreased grain hardness less than the reduction seen with added PINB. Seeds from lines having more 'soft type' PINB than PINA were the softest. Friabilin abundance was correlated with the presence of both 'soft type' PINA and PINB and did not correlate well with total puroindoline abundance. The data indicates that PINA and PINB interact to form friabilin and together affect wheat grain texture.

The genome organization of the Hardness locus in the tribe Triticeae constitutes an excellent model for studying the mechanisms of evolution that played a role in the preservation and potential functional innovations of duplicate genes. Here we applied the nonsynonymous-synonymous rate ratio (d N /d S or ω) to measure the selective pressures at the paralogous puroindoline-a (Pina), puroindoline-b (Pinb), and grain softness protein-1 (Gsp-1) genes located at this locus. Puroindolines represent the molecular-genetic basis of grain texture. In addition, the puroindoline gene products have antimicrobial properties with potential role in plant defense. We document the complete coding sequences from the Triticum/Aegilops taxa, rye and barley including the A, D, C, H, M, N, R, S, and U genomes of the Triticeae. Maximum likelihood analyses performed on Bayesian phylogenetic trees showed distinct evolutionary patterns among Pina, Pinb, and Gsp-1. Positive diversifying selection appeared to drive the evolution of at least one of the three genes examined, suggesting that adaptive forces have operated at this locus. Results evidenced positive selection (ω > 4) at Pina and detected amino acid residues along the mature PIN-a protein with a high probability (>95%) of having evolved under adaptation. We hypothesized that positive selection at the Pina region is congruent with its role as a plant defense gene.

Grain texture affects many milling characteristics and end-use qualities in wheat (Triticum aestivum L.) such as milling yield, flour particle size, and starch damage. In wheat, grain texture is controlled primarily by the two genes puroindoline a (pina) and puroindoline b (pinb) that reside at the Hardness (Ha) locus. Variation in puroindoline activity and abundance is known to influence a variety of milling and baking traits, and the reconstitution of flour with puroindoline A protein (PINA) has been shown to positively affect loaf volume and crumb grain. To investigate which milling and baking traits are affected by the addition of puroindolines in vivo, we transformed the hard red spring wheat cultivar Hi-Line, with genetic constructs driving the expression of pina-D1a, pinb-D1a, or both pina-D1a and pinb-D1a. Transgenic lines exhibited decreased grain hardness and increased puroindoline content. Selected T3 lines were grown in replicated field trials under dry and irrigated conditions. Harvested grain was then milled and baked. Lines transformed with the puroindolines exhibited decreased total flour yields and increased break flour yields, yielding flour with lower protein and ash content. Decreases in loaf volume, mixograph absorption, and crumb grain scores were also observed in transgenic lines having high puroindoline expression. Decreased loaf volume was also observed in whole wheat bakes of transgenic soft wheats vs. normal hard wheat, indicating a direct effect of puroindolines on loaf volume. Puroindoline content did not affect dough mixing times. These results demonstrate that the puroindolines can be used to profoundly influence a variety of milling and bread baking traits in wheat.