Genetic mapping reveals a dominant awn-inhibiting gene related to differentiation of the variety anathera in the wild diploid wheat <Emphasis Type="Italic">Aegilops tauschii</Emphasis>

Aegilops tauschii, a wild wheat relative, is the D-genome donor of common wheat. Subspecies and varieties of Ae. tauschii are traditionally classified based on differences in their inflorescence architecture. However, the genetic information for their diversification has been quite limited in the wild wheat relatives. The variety anathera has no awn on the lemma, but the genetic basis for this diagnostic character is unknown. Wide variations in awn length traits at the top and middle spikes were found in the Ae. tauschii core collection, and the awn length at the middle spike was significantly smaller in the eastward-dispersed sublineage than in those in other sublineages. To clarify loci controlling the awnless phenotype of var. anathera, we measured awn length of an intervariety F₂ mapping population, and found that the F₂ individuals could be divided into two groups mainly based on the awn length at the middle of spike, namely short and long awn groups, significantly fitting a 3:1 segregation ratio, which indicated that a single locus controls the awnless phenotype. The awnless locus, Anathera (Antr), was assigned to the distal region of the short arm of chromosome 5D. Quantitative trait locus analysis using the awn length data of each F₂ individual showed that only one major locus was at the same chromosomal position as Antr. These results suggest that a single dominant allele determines the awnless diagnostic character in the variety anathera. The Antr dominant allele is a novel gene inhibiting awn elongation in wheat and its relatives.     

The DROOPING LEAF and OsETTIN2 genes promote awn development in rice

The awn is a long needle‐like appendage that, in some grass species, is formed on the lemma that encloses floral organs together with the palea. In rice, most wild species and most strains of Oryza sativa ssp. indica generate an awn, whereas most strains of O. sativa ssp. japonica do not. In japonica, the long‐awn characteristic appears to have been lost during domestication and breeding programs. Here, we found that the genes DROOPING LEAF (DL) and OsETTIN2 (OsETT2) are involved in awn development in the awned indica strain Kasalath. Genetic analyses and RNA‐silencing experiments indicate that DL and OsETT2 act independently in awn formation, and that either gene alone is not sufficient for awn development. Scanning electron microscopy revealed that the top region of the lemma (a putative awn primordium) is larger in an awned floret than in an awnless floret. OsETT2 is expressed in the awn primordium in the awned indica floret, but not in the awnless japonica floret except in the provascular bundle. DL is expressed underneath the primordium at similar levels in both indica and japonica florets, suggesting non‐cell‐autonomous action. We hypothesize that loss of expression of OsETT2 in the awn primordium is probably associated with the failure of awn formation in japonica strains.     

Analysis of the functions of TaGW2 homoeologs in wheat grain weight and protein content traits

GW2 is emerging as a key genetic determinant of grain weight in cereal crops; it has three homoeologs (TaGW2‐A1, ‐B1 and ‐D1) in hexaploid common wheat (Triticum aestivum L.). Here, by analyzing the gene editing mutants that lack one (B1 or D1), two (B1 and D1) or all three (A1, B1 and D1) homoeologs of TaGW2, several insights are gained into the functions of TaGW2‐B1 and ‐D1 in common wheat grain traits. First, both TaGW2‐B1 and ‐D1 affect thousand‐grain weight (TGW) by influencing grain width and length, but the effect conferred by TaGW2‐B1 is stronger than that of TaGW2‐D1. Second, there exists functional interaction between TaGW2 homoeologs because the TGW increase shown by a double mutant (lacking B1 and D1) was substantially larger than that of their single mutants. Third, both TaGW2‐B1 and ‐D1 modulate cell number and length in the outer pericarp of developing grains, with TaGW2‐B1 being more potent. Finally, TaGW2 homoeologs also affect grain protein content as this parameter was generally increased in the mutants, especially in the lines lacking two or three homoeologs. Consistent with this finding, two wheat end‐use quality‐related parameters, flour protein content and gluten strength, were considerably elevated in the mutants. Collectively, our data shed light on functional difference between and additive interaction of TaGW2 homoeologs in the genetic control of grain weight and protein content traits in common wheat, which may accelerate further research on this important gene and its application in wheat improvement.     

The absense of a definite gene-specific effect in wheat after seed treatment with exogenous DNA

Summary To reveal gene-specific genetic transformation in wheat experiments have been performed with two inheritable marker characters, namely, the complementing genes of necrosis, ne ₁ and ne ₂, and genes of awness. The awned spikes were found after treatment of seeds of awnless wheat with the DNA isolated from both awned wheat and from awnless wheat. The awned plants conserved their changed character in subsequent generations. In the experiments with necrosis genes no single example of interaction of the ne ₁ and ne ₂ genes was found, although some non specific effects (the decrease in number of germinating plants, the increase in height of plants and the length of spikes, etc) were observed.     

Identification of Novel SNP in Promoter Sequence of TaGW2-6A Associated with Grain Weight and Other Agronomic Traits in Wheat (Triticum aestivum L.)

TaGW2 is an orthologue of rice gene OsGW2, which encodes E3 RING ubiquitin ligase and controls the grain size in rice. In wheat, three copies of TaGW2 have been identified and mapped on wheat homoeologous group 6 viz. TaGW2-6A, TaGW2-6B and TaGW2-6D. In the present study, using as many as 207 Indian wheat genotypes, we identified four SNPs including two novel SNPs (SNP-988 and SNP-494) in the promoter sequence of TaGW2-6A. All the four SNPs were G/A or A/G substitutions (transitions). Out of the four SNPs, SNP-494 was causal, since it was found associated with grain weight. The mean TGW (41.1 g) of genotypes with the allele SNP-494_A was significantly higher than mean TGW (38.6 g) of genotypes with the allele SNP-494_G. SNP-494 also regulates the expression of TaGW2-6A so that the wheat genotypes with SNP-494_G have higher expression and lower TGW and the genotypes with SNP-494_A have lower expression but higher TGW. Besides, SNP-494 was also found associated with grain length-width ratio, awn length, spike length, grain protein content, peduncle length and plant height. This suggested that gene TaGW2-6A not only controls grain size, but also controls other agronomic traits. In the promoter region, SNP-494 was present in ‘CGCG’ motif that plays an important role in Ca²⁺/calmodulin mediated regulation of genes. A user-friendly CAPS marker was also developed to identify the desirable allele of causal SNP (SNP-494) for use in marker-assisted selection for improvement of grain weight in wheat. Using four SNPs, five haplotypes were identified; of these, Hap_5 (G_A_G_A) was found to be a desirable haplotype having significantly higher grain weight (41.13g) relative to other four haplotypes (36.33-39.16 g).     

The temporal and species dynamics of photosynthetic acclimation in flag leaves of rice (Oryza sativa) and wheat (Triticum aestivum) under elevated carbon dioxide

In this study, we tested for the temporal occurrence of photosynthetic acclimation to elevated [CO₂] in the flag leaf of two important cereal crops, rice and wheat. In order to characterize the temporal onset of acclimation and the basis for any observed decline in photosynthetic rate, we characterized net photosynthesis, g_s, g_m, C_i/C_a, C_i/C_c, V_cmax, J_max, cell wall thickness, content of Rubisco, cytochrome (Cyt) f, N, chlorophyll and carbohydrate, mRNA expression for rbcL and petA, activity for Rubisco, sucrose phosphate synthase (SPS) and sucrose synthase (SS) at full flag expansion, mid‐anthesis and the late grain‐filling stage. No acclimation was observed for either crop at full flag leaf expansion. However, at the mid‐anthesis stage, photosynthetic acclimation in rice was associated with RuBP carboxylation and regeneration limitations, while wheat only had the carboxylation limitation. By grain maturation, the decline of Rubisco content and activity had contributed to RuBP carboxylation limitation of photosynthesis in both crops at elevated [CO₂]; however, the sharp decrease of Rubisco enzyme activity played a more important role in wheat. Although an increase in non‐structural carbohydrates did occur during these later stages, it was not consistently associated with changes in SPS and SS or photosynthetic acclimation. Rather, over time elevated [CO₂] appeared to enhance the rate of N degradation and senescence so that by late‐grain fill, photosynthetic acclimation to elevated [CO₂] in the flag leaf of either species was complete. These data suggest that the basis for photosynthetic acclimation with elevated [CO₂] may be more closely associated with enhanced rates of senescence, and, as a consequence, may be temporally dynamic, with significant species variation.     

Photosynthesis and conductance of spring wheat ears: field response to free-air CO2 enrichment and limitations in water and nitrogen supply

The mid-day responses of wheat ear CO₂ and water vapour exchange to full-season CO₂ enrichment were investigated using a Free-Air CO₂ Enrichment (FACE) apparatus. Spring wheat [Triticum aestivum (L). cv. Yecora Rojo] was grown in two experiments under ambient and elevated atmospheric CO₂ (C_a) concentrations (approximately 370 μ mol mol ^{− 1} and 550 μ mol mol ^{− 1}, respectively) combined first with two irrigation (Irr) schemes (Wet: 100% and Dry: 50% replacement of evapotranspiration) and then with two levels of nitrogen (N) fertilization (High: 350, Low: 70 kg ha ^{− 1} N). Blowers were used for C_a enrichment. Ambient C_a plots were exposed to blower induced winds as well the C_a × N but not in the C_a × Irr experiment. The net photosynthesis for the ears was increased by 58% and stomatal conductance (g_s) was decreased by 26% due to elevated C_a under ample water and N supply when blowers were applied to both C_a treatments. The use of blowers in the C_a-enriched plots only during the C_a × Irr experiment (blower effect) and Low N supply restricted the enhancement of net photosynthesis of the ear due to higher C_a. In the latter case, the increase of net photosynthesis of the ear amounted to 26%. The decrease in g_s caused by higher C_a was not affected by the blower effect and N treatment. The mid-day enhancement of net photosynthesis due to elevated C_a was higher for ears than for flag leaves and this effect was most pronounced under ample water and N supply. The contribution of ears to grain filling is therefore likely to increase in higher C_a environments in the future. In the comparison between Wet and Dry, the higher C_a did not alter the response of net photosynthesis of the ear and g_s to Irr. However, C_a enrichment increased the drought tolerance of net photosynthesis of the glume and delayed the increase of the awn portion of net photosynthesis of the ear during drought. Therefore, the role of awns for maintaining high net photosynthesis of the ear under drought may decrease when C_a increases.     

An-1 Encodes a Basic Helix-Loop-Helix Protein That Regulates Awn Development,Grain Size,and Grain Number in Rice

Long awns are important for seed dispersal in wild rice (Oryza rufipogon), but are absent in cultivated rice (Oryza sativa). The genetic mechanism involved in loss-of-awn in cultivated rice remains unknown. We report here the molecular cloning of a major quantitative trait locus, An-1, which regulates long awn formation in O. rufipogon. An-1 encodes a basic helix-loop-helix protein, which regulates cell division. The nearly-isogenic line (NIL-An-1) carrying a wild allele An-1 in the genetic background of the awnless indica Guangluai4 produces long awns and longer grains, but significantly fewer grains per panicle compared with Guangluai4. Transgenic studies confirmed that An-1 positively regulates awn elongation, but negatively regulates grain number per panicle. Genetic variations in the An-1 locus were found to be associated with awn loss in cultivated rice. Population genetic analysis of wild and cultivated rice showed a significant reduction in nucleotide diversity of the An-1 locus in rice cultivars, suggesting that the An-1 locus was a major target for artificial selection. Thus, we propose that awn loss was favored and strongly selected by humans, as genetic variations at the An-1 locus that cause awn loss would increase grain numbers and subsequently improve grain yield in cultivated rice.     

Differential CO2 effect on primary carbon metabolism of flag leaves in durum wheat (Triticum durum Desf.)

C sink/source balance and N assimilation have been identified as target processes conditioning crop responsiveness to elevated CO₂. However, little is known about phenology‐driven modifications of C and N primary metabolism at elevated CO₂ in cereals such as wheat. Here, we examined the differential effect of elevated CO₂ at two development stages (onset of flowering, onset of grain filling) in durum wheat (Triticum durum, var. Sula) using physiological measurements (photosynthesis, isotopes), metabolomics, proteomics and ¹⁵N labelling. Our results show that growth at elevated CO₂ was accompanied by photosynthetic acclimation through a lower internal (mesophyll) conductance but no significant effect on Rubisco content, maximal carboxylation or electron transfer. Growth at elevated CO₂ altered photosynthate export and tended to accelerate leaf N remobilization, which was visible for several proteins and amino acids, as well as lysine degradation metabolism. However, grain biomass produced at elevated CO₂ was larger and less N rich, suggesting that nitrogen use efficiency rather than photosynthesis is an important target for improvement, even in good CO₂‐responsive cultivars.     

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

Grain size and weight are important components of a suite of yield‐related traits in crops. Here, we showed that the CRISPR‐Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1‐recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double‐copy mutant showing larger effect than the respective single copy mutants. The TaGW7‐centered gene co‐expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co‐localization of TaGW7 with α‐ and β‐tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7‐associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR‐Cas9 system with cross‐species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.     

Genome‐wide association study of six quality traits reveals the association of the TaRPP13L1 gene with flour colour in Chinese bread wheat

Flour colour, kernel hardness, grain protein content and wet gluten content are important quality properties that determine end use in bread wheat. Here, a wheat 90K genotyping assay was used for a genome‐wide association study (GWAS) of the six quality‐related traits in Chinese wheat cultivars in eight environments over four years. A total of 846 significant single nucleotide polymorphisms (SNPs) were identified, explaining approximately 30% of the phenotypic variation on average, and 103 multienvironment‐significant SNPs were detected in more than four environments. Quantitative trait loci (QTL) mapping in the biparent population confirmed some important SNP loci. Moreover, it was determined that some important genes were associated with the six quality traits, including some known functional genes and annotated unknown functional genes. Of the annotated unknown functional genes, it was verified that TaRPP13L1 was associated with flour colour. Wheat cultivars or lines with TaRPP13L1‐B1a showed extremely significantly higher flour redness and lower yellowness than those with TaRPP13L1‐B1b in the Chinese wheat natural population and the doubled haploid (DH) population. Two tetraploid wheat lines with premature stop codons of the TaRPP13L1 gene mutagenized by ethyl methanesulfonate (EMS) showed extremely significantly higher flour redness and lower yellowness than wild type. Our data suggest that the TaRPP13L1 gene plays an important role in modulating wheat flour colour. This study provides useful information for further dissection of the genetic basis of flour colour and also provides valuable genes or genetic loci for marker‐assisted selection to improve the process of breeding quality wheat in China.     

Mutation of RGG2, which encodes a type B heterotrimeric G protein γ subunit,increases grain size and yield production in rice

Heterotrimeric G proteins, which consist of G_α, G_β and G_γ subunits, function as molecular switches that regulate a wide range of developmental processes in plants. In this study, we characterised the function of rice RGG2, which encodes a type B G_γ subunit, in regulating grain size and yield production. The expression levels of RGG2 were significantly higher than those of other rice G_γ‐encoding genes in all tissues tested, suggesting that RGG2 plays essential roles in rice growth and development. By regulating cell expansion, overexpression of RGG2 in Nipponbare (NIP) led to reduced plant height and decreased grain size. By contrast, two mutants generated by the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) system in the Zhenshan 97 (ZS97) background, zrgg2‐1 and zrgg2‐2, exhibited enhanced growth, including elongated internodes, increased 1000‐grain weight and plant biomass and enhanced grain yield per plant (+11.8% and 16.0%, respectively). These results demonstrate that RGG2 acts as a negative regulator of plant growth and organ size in rice. By measuring the length of the second leaf sheath after gibberellin (GA₃) treatment and the GA‐induced α‐amylase activity of seeds, we found that RGG2 is also involved in GA signalling. In summary, we propose that RGG2 may regulate grain and organ size via the GA pathway and that manipulation of RGG2 may provide a novel strategy for rice grain yield enhancement.     

AtZFP1, encoding Arabidopsis thaliana C2H2 zinc-finger protein 1, is expressed downstream of photomorphogenic activation

C₂H₂ zinc-finger proteins play important roles in plant development including floral organogenesis, leaf initiation, lateral shoot initiation, gametogenesis and seed development. The gene for one such protein from Arabidopsis, AtZFP1 (Arabidopsis thalianazinc-finger protein 1), is expressed at high levels in the shoot apex, including the apical meristem, developing leaves and the developing vascular system. In light-grown seedlings, AtZFP1 expression is induced about three days after germination, before the expansion of the true leaves. Dark-grown plants, in which photomorphogenesis is repressed, have no detectable AtZFP1 expression in the shoot apex. Under conditions which induce or mimic photomorphogenic development including growth in the light, shifting dark-grown plants to continuous light or growth on cytokinin in the dark, high levels of AtZFP1 expression are detected. Furthermore, AtZFP1 expression does not depend on active photosynthesis as shown by analysis of plants grown on the carotenoid biosynthetic inhibitor norflurazon. These results are discussed in relation to a possible role for AtZFP1 in shoot development, downstream of photomorphogenic activation.     

Awn contribution to gas exchanges of barley ears

The effects of awn removal on ear gas exchange in four barley lines (Morex, Harrington, Steptoe, and TR306) were studied under a controlled environment using a Before-After Control-Impact Paired (BACIP) experimental design. From ear emergence to grain maturity, plants were grown in pots at either 60 or 90 % of soil water holding capacity. Gas-exchange measurements of ears were made 9 and 10 d after anthesis (DAA). On 11 DAA, awn removal was performed on half of the ears in each pot, followed by measurements on both intact and de-awned ears on 12 and 13 DAA. Net photosynthetic (P _N) and transpiration (E) rates decreased significantly with awn removal, but dark respiration (R _D) rate was not affected. We estimated for each ear a temperature-adjusted respiration rate (R _a) from R _D. When we corrected P _N with R _a, we found that rates of spikelet photosynthesis were largely underestimated. Moderate water stress had minimal effect on gas exchange of bracts and awns of the barley ear. Barley lines did not differ for any individual gas-exchange parameter.     

Genetic analysis of tetraploid and hexaploid wheat by utilization of monopentaploid hybrids

Summary This report deals with a method of analysis which uses existing hexaploid wheat monosomics to establish gene-chromosome associations in a tetraploid variety. Monosomics of Triticum aestivum cv. Chinese Spring belonging to the 14 lines of A and B genomes were crossed as female parents with Triticum durum cv. Capeiti, a spring type at present widely grown in Italy. For each line, two F ₁ populations were obtained, normal pentaploids (2 n = 35) and monopentaploid (2 n = 34), in which, in turn, the monosomic A or B chromosome present was supplied by the tetraploid wheat. The morphological and physiological differences observed in the monopentaploid lines are attributed to differential expression of the genetic information concerning the character investigated, carried by the chromosome present in hemizygous condition. Then, only recessive or partially dominant alleles of the variety to be tested can be identified and attributed to a specific chromosome in the F ₁ generation.Eight parameters were analyzed: culm and spike length, length and width of 1st (flag) and 2nd uppermost leaves, days from germination to heading and awn development.As far as culm length is concerned, although heterotic effect is present, seven chromosomes seem to be responsible for the modification of this character (1A, 2A, 2B, 3B, 4B, 5B, and 6 A); chromosomes 2A and 2B in particular, carry major factor (s) for plant height. A similar picture is presented by spike length which seems to be controlled by factors located in several chromosomes belonging to homoeologous groups 1, 2, 3 and 5, as well as the chromosome 4B.Leaf length, also, shows a complex pattern of inheritance. Monosomic conditions for chromosomes 1A and 1B increased, while monosomy for 5A and 5B significantly decreased, leaf length. A highly significant correlation was found between the mean lengths of the 1st and 2 nd leaves (= 0.74). Some monosomic lines (4A, 4B, 5A; 5B; 6A; 7A and 7B) had leaves significantly narrower than in the control and only monosomic 2A had broader leaves. The period from germination to heading seems to be influenced by at least 6 chromosomes. Three monosomic lines are significantly earlier (mono 1A, 7A and 5B) and three (mono 5A, 2B and 7B) are significantly later than the hybrid control.Finally, 8 monosomic lines were found to interfere significantly with awn development. Three lines (mono 2A, 2B and 7A) show a decrease and 5 (mono 1B; 3A, 3B; 4B and 6B) show an increase in awn development. On the basis of evidence in the literature and our own results, it appears that this analysis fits previous results perfectly and actually adds to the picture two further awn-promoting factors, A9 and A10, located on the 7A and 1B chromosomes respectively.Contribution n. 220 from the Laboratorio per le Applicazioni in Agricultura del C. N. E. N., Centro Studi Nucleari della Casaccia, S. Maria di Galeria, Roma, Italy.With the technical assistance for cytological and statistical analyses of P. Mannino.     

Genome‐wide association study for reproductive traits in a Large White pig population

Using the PorcineSNP80 BeadChip, we performed a genome‐wide association study for seven reproductive traits, including total number born, number born alive, litter birth weight, average birth weight, gestation length, age at first service and age at first farrowing, in a population of 1207 Large White pigs. In total, we detected 12 genome‐wide significant and 41 suggestive significant SNPs associated with six reproductive traits. The proportion of phenotypic variance explained by all significant SNPs for each trait ranged from 4.46% (number born alive) to 11.49% (gestation length). Among them, 29 significant SNPs were located within known QTL regions for swine reproductive traits, such as corpus luteum number, stillborn number and litter size, of which one QTL region associated with litter size contained the ALGA0098819 SNP for total number born. Subsequently, we found that 376 functional genes contained or were near these significant SNPs. Of these, 14 genes—BHLHA15, OCM2, IL1B2, GCK, SMAD2, HABP2, PAQR5, GRB10, PRELID2, DMKN, GPI, GPIHBP1, ADCY2 and ACVR2B—were considered important candidates for swine reproductive traits based on their critical roles in embryonic development, energy metabolism and growth development. Our findings contribute to the understanding of the genetic mechanisms for reproductive traits and could have a positive effect on pig breeding programs.     

Photosynthetic Characteristics of Non-Leaf Organs of Winter Wheat Cultivars Differing in Ear Type and their Relationship with Grain Mass Per Ear

Chlorophyll content, photosystem 2 functioning (F_v/F_m, F_v/F₀), activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, and net photosynthetic rates (P _N) of flag leaf blade, sheath, peduncle, and ear organs were assessed in large-ear type (Pin 7) and small-ear type (ND93) wheat cultivars. Some differences were found in photosynthetic properties between different green plant parts, the values of all studied parameters in ear parts being higher in Pin7 than in ND93. Furthermore, ear surface areas and ear P _N in 26 wheat genotypes measured at anthesis showed highly significant positive correlation with grain mass per ear. Hence a greater capability of ear photosynthesis may result in a greater grain yield in large-ear type cultivars.