Genetic analysis of iron, zinc and grain yield in wheat-Aegilops derivatives using multi-locus GWAS.

BACKGROUND: Wheat is a major staple crop and helps to reduce worldwide micronutrient deficiency. Investigating the genetics that control the concentrations of iron (Fe) and zinc (Zn) in wheat is crucial. Hence, we undertook a comprehensive study aimed at elucidating the genomic regions linked to the contents of Fe and Zn in the grain. METHODS AND RESULTS: We performed the multi-locus genome-wide association (ML-GWAS) using a panel of 161 wheat-Aegilops substitution and addition lines to dissect the genomic regions controlling grain iron (GFeC), and grain zinc (GZnC) contents. The wheat panel was genotyped using 10,825 high-quality SNPs and phenotyped in three different environments (E1-E3) during 2017-2019. A total of 111 marker-trait associations (MTAs) (at p-value < 0.001) were detected that belong to all three sub-genomes of wheat. The highest number of MTAs were identified for GFeC (58), followed by GZnC (44) and yield (9). Further, six stable MTAs were identified for these three traits and also two pleiotropic MTAs were identified for GFeC and GZnC. A total of 1291 putative candidate genes (CGs) were also identified for all three traits. These CGs encode a diverse set of proteins, including heavy metal-associated (HMA), bZIP family protein, AP2/ERF, and protein previously associated with GFeC, GZnC, and grain yield. CONCLUSIONS: The significant MTAs and CGs pinpointed in this current study are poised to play a pivotal role in enhancing both the nutritional quality and yield of wheat, utilizing marker-assisted selection (MAS) techniques.

Application of CRISPR-Mediated Gene Editing for Crop Improvement.

Plant gene editing has become an important molecular tool to revolutionize modern breeding of crops. Over the past years, remarkable advancement has been made in developing robust and efficient editing methods for plants. Despite a variety of available genome editing methods, the discovery of most recent system of clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) has been one of the biggest advancement in this path, with being the most efficient approach for genome manipulation. Until recently, genetic manipulations were confined to methods, like Agrobacterium-mediated transformations, zinc-finger nucleases, and TAL effector nucleases. However this technology supersedes all other methods for genetic modification. This RNA-guided CRISPR-Cas system is being rapidly developed with enhanced functionalities for better use and greater possibilities in biological research. In this review, we discuss and sum up the application of this simple yet powerful tool of CRISPR-Cas system for crop improvement with recent advancement in this technology.

Effect of wheat grain protein composition on end-use quality.

The quality of wheat products has been a new challenge next to wheat production which was achieved substantially during green revolution. The end-use quality of wheat is an essential factor for its commercial demand. The quality of wheat is largely based on the wheat storage proteins which extensively influences the dough properties. High molecular weight glutenin subunits (HMWGS), low molecular weight glutenin subunits (LMWGS) and gliadins significantly influence the end-use quality. Genomics and proteomics study of these gluten proteins of bread and durum wheat have explored new avenues for precise identification of the alleles and their role in end-use quality improvement. Secalin protein of Secale cereale encoded by Sec-1 loci and is associated with 1RS.1BL translocation has been known for deterioration of end-use quality. Chromosomal manipulations using various approaches have led to the development of new recombinant lines of wheat without secalin. Advanced techniques associated with assessment of end-use quality have integrated the knowledge of useful or deteriorating HMWGS/LMWGS alleles and their potential role in end-use quality. This review gives a comprehensive insight of different aspects of the end-use quality perspective for bread making in wheat along with some information on the immunological interference of gluten in celiac disease.

Identification, expression analysis, and molecular modeling of Iron-deficiency-specific clone 3 (Ids3)-like gene in hexaploid wheat.

Graminaceous plants secrete hydroxylated phytosiderophores encoded by the genes iron-deficiency-specific clone 2 (Ids2) and iron-deficiency-specific clone 3 (Ids3). An effort was made to identify a putative ortholog of Hodeum vulgare Ids3 gene in hexaploid wheat. The protein structure of TaIDS3 was modeled using homology modeling and structural behavior of modeled structure was analyzed at 20 ns. The simulation trajectory using molecular dynamics simulation suggested the model to be stable with no large fluctuations in residues and local domain level RMSF values (< 2.4 Å). In addition, the ProFunc results also predict the functional similarity between the proteins of HvIDS3 and its wheat ortholog (TaIDS3). The TaIds3 gene was assigned to the telomeric region of chromosome arm 7AS which supports the results obtained through bioinformatics analysis. The relative expression analysis of TaIds3 indicated that the detectable expression of TaIds3 is induced after 5th day of Fe starvation and increases gradually up to 15th day, and thereafter, it decreases until 35th day of Fe-starvation. This reflects that Fe deficiency directly regulates the induction of TaIds3 in the roots of hexaploid wheat. The identification of HvIds3-like gene in wheat has opened up new opportunities to enhance the nutrient quality in wheat through biofortification program.

Transfer of HMW glutenin subunits from Aegilops kotschyi to wheat through radiation hybridization.

High molecular weight glutenin subunits (HMWGS) are responsible for dough elasticity and bread making quality of bread wheat. Related wild non-progenitor species, Aegilops kotschyi possesses higher molecular weight x and y glutenin subunits than the bread wheat cultivars. A wheat-Aegilops substitution line with 1U chromosome was used for the transfer of (HMWGS) of 1U to wheat by using pollen radiation hybridization approach. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiling showed different patterns of allelic variations with either the presence or absence of HMWGS, Glu-1A (1, null), Glu-1B (7, 7 + 8, 17 + 18) and Glu-1D (5 + 10, 2 + 12, null). The pollen irradiated wheat-Aegilops derivatives, B-56-1-4-2, B-56-1-4-3, B-14-1 and B-14-2 with Glu1Ux and 1Uy and absence or presence of some Glu-1A and Glu-1B HMWGS showed high micro SDS sedimentation test (MST) values while B-16-1 and B-16-2 had moderate MST values and high protein content. However, B-58-3 with transfer of Glu-1Ux + 1Uy for Glu-1D showed very low MST values indicating that Glu-1Ux + 1Uy enhance MST value only in the presence of Glu1D HMWGS. The transfer/substitution of alien HMW-GS for Glu-1A and or Glu-1B loci only can lead to improved bread making quality of wheat.

Introgression of a leaf rust resistance gene from Aegilops caudata to bread wheat.

Rusts are the most important biotic constraints limiting wheat productivity worldwide. Deployment of cultivars with broad spectrum rust resistance is the only environmentally viable option to combat these diseases. Identification and introgression of novel sources of resistance is a continuous process to combat the ever evolving pathogens. The germplasm of nonprogenitor Aegilops species with substantial amount of variability has been exploited to a limited extent. In the present investigation introgression, inheritance and molecular mapping of a leaf rust resistance gene of Ae. caudata (CC) acc. pau3556 in cultivated wheat were undertaken. An F(2) population derived from the cross of Triticum aestivum cv. WL711 - Ae. caudata introgression line T291-2 with wheat cultivar PBW343 segregated for a single dominant leaf rust resistance gene at the seedling and adult plant stages. Progeny testing in F(3) confirmed the introgression of a single gene for leaf rust resistance. Bulked segregant analysis using polymorphic D-genome-specific SSR markers and the cosegregation of the 5DS anchored markers (Xcfd18, Xcfd78, Xfd81 and Xcfd189) with the rust resistance in the F(2) population mapped the leaf rust resistance gene (LrAC) on the short arm of wheat chromosome 5D. Genetic complementation and the linked molecular markers revealed that LrAC is a novel homoeoallele of an orthologue Lr57 already introgressed from the 5M chromosome of Ae. geniculata on 5DS of wheat.

Development of multiple embryos in polyembryonic insertional mutant OsPE of rice.

A T-DNA insertional mutant OsPE of rice gives twin and triplet seedlings in up to 20 % of the seeds. Detailed cytological and histological analysis of OsPE indicated normal male and female gametogenesis in the OsPE mutant. Confocal laser scanning microscopic (CLSM) analysis of the developing seeds of OsPE showed multiple embryo development in up to 60 % of the ovules. The multiple embryos, mostly twins and triplets, and rarely quadruplets, developed through sequential cleavage from a single zygotic embryo in each ovule. The reduced number of multiple seedlings compared with multiple embryos observed in CLSM study may be attributed to their inability to develop further due to competition in a single embryo sac. Key message Multiple seedlings in the OsPE mutant are due to sequential proliferation and cleavage of the zygotic embryos. The nucellar tissue was not involved in multiple embryo development.

Reduced tillering in Basmati rice T-DNA insertional mutant OsTEF1 associates with differential expression of stress related genes and transcription factors.

A T-DNA insertional mutant OsTEF1 of rice gives 60-80% reduced tillering, retarded growth of seminal roots, and sensitivity to salt stress compared to wild type Basmati 370. The insertion occurred in a gene encoding a transcription elongation factor homologous to yeast elf1, on chromosome 2 of rice. Detailed transcriptomic profiling of OsTEF1 revealed that mutation in the transcription elongation factor differentially regulates the expression of more than 100 genes with known function and finely regulates tillering process in rice by inducing the expression of cytochrome P450. Along with different transcription factors, several stress associated genes were also affected due to a single insertion. In silico analysis of the TEF1 protein showed high conservation among different organisms. This transcription elongation factor predicted to interact with other proteins that directly or indirectly positively regulate tillering in rice.

Abnormal endosperm development causes female sterility in rice insertional mutant OsAPC6.

A T-DNA insertional mutant OsAPC6 of rice, with gibberellic acid insensitivity and reduced height, had up to 45% reduced seed set. The insertion occurred on chromosome 3 of rice in the gene encoding one of the subunits of anaphase promoting complex/Cyclosome APC6. The primary mother cells of the mutant plants had normal meiosis, male gametophyte development and pollen viability. Confocal laser scanning microscopic (CLSM) studies of megagametophyte development showed abnormal mitotic divisions with reduced number or total absence of polar nuclei in about 30-35% megagametophytes of OsAPC6 mutant leading to failure of endosperm and hence embryo and seed development. Abnormal female gametophyte development, high sterility and segregation of tall and gibberellic acid sensitive plants without selectable marker Hpt in the selfed progeny of OsAPC6 mutant plants indicate that the mutant could be maintained in heterozygous condition. The abnormal mitotic divisions during megagametogenesis could be attributed to the inactivation of the APC6/CDC16 of anaphase promoting complex of rice responsible for cell cycle progression during megagametogenesis. Functional validation of the candidate gene through transcriptome profiling and RNAi is in progress.

QTL mapping of 1000-kernel weight, kernel length, and kernel width in bread wheat (Triticum aestivum L.).

Kernel size and morphology influence the market value and milling yield of bread wheat (Triticum aestivum L.). The objective of this study was to identify quantitative trait loci (QTLs) controlling kernel traits in hexaploid wheat. We recorded 1000-kernel weight, kernel length, and kernel width for 185 recombinant inbred lines from the cross Rye Selection 111 × Chinese Spring grown in 2 agro-climatic regions in India for many years. Composite interval mapping (CIM) was employed for QTL detection using a linkage map with 169 simple sequence repeat (SSR) markers. For 1000-kernel weight, 10 QTLs were identified on wheat chromosomes 1A, 1D, 2B, 2D, 4B, 5B, and 6B, whereas 6 QTLs for kernel length were detected on 1A, 2B, 2D, 5A, 5B and 5D. Chromosomes 1D, 2B, 2D, 4B, 5B and 5D had 9 QTLs for kernel width. Chromosomal regions with QTLs detected consistently for multiple year-location combinations were identified for each trait. Pleiotropic QTLs were found on chromosomes 2B, 2D, 4B, and 5B. The identified genomic regions controlling wheat kernel size and shape can be targeted during further studies for their genetic dissection.

Studies on optimization of ripening techniques for banana.

Fruits of banana (Musa spp) cultivar 'Grand Naine' were harvested at physiological green mature stage. The first lot of fruit was exposed to ethylene gas (100 ppm) for 24 h in ripening chamber. The second lot was treated with different concentrations of aqueous solution of ethephon (250, 500, 750, 1000 ppm) each for 5 min. The fruits were packed in plastic crates and stored in ripening chamber maintained at 16-18°C and 90-95% RH. Treatment with ethylene gas (100 ppm) or ethephon (500 ppm) resulted in adequate ripening of fruits after 4 days with uniform colour, pleasant flavour, desirable firmness and acceptable quality and better shelf-life. The untreated control fruits were hard textured and poor in colour and quality. The ripening with ethylene gas or ethephon treatment seems to hold promise in reducing postharvest losses and boosting the economy of banana growers and traders.

Effect of pre-harvest calcium sprays on post-harvest life of winter guava (Psidium guajava L.).

Influence of pre-harvest foliar application of calcium nitrate on quality attributes of winter guava cv. 'Sardar' during different interval of cold storage and post cold storage shelf-life under ambient conditions was investigated. Plants were sprayed with calcium nitrate solutions (0.5, 1.0 and 1.5%) at colour break stage of fruit and a fruit with no treatment was control. The fruits were harvested at optimum maturity. Bruise and disease free fruits were packed in corrugated fibre board boxes with newspaper lining and stored at 6-8 °C and 90-95% RH. The fruits were evaluated after 10, 20, 30 and 40 days of storage and during shelf-life at 2 and 4 days interval. Physiological loss in weight and spoilage of fruits increased and firmness, ascorbic acid and acidity decreased continuously during storage. Fruits treated with 1% Ca(NO3)2 effectively reduced spoilage, maintained higher firmness, total soluble solid and ascorbic acid up to 2 days under ambient conditions after 30 days of cold storage and remained moderately acceptable up to the 40 days of storage.

Mapping of adult plant stripe rust resistance genes in diploid A genome wheat species and their transfer to bread wheat.

Stripe rust, caused by Puccinia striiformis West. f.sp. tritici, is one of the most damaging diseases of wheat worldwide. Forty genes for stripe rust resistance have been catalogued so far, but the majority of them are not effective against emerging pathotypes. Triticum monococcum and T. boeoticum have excellent levels of resistance to rusts, but so far, no stripe rust resistance gene has been identified or transferred from these species. A set of 121 RILs generated from a cross involving T. monococcum (acc. pau14087) and T. boeoticum (acc. pau5088) was screened for 3 years against a mixture of pathotypes under field conditions. The parental accessions were susceptible to all the prevalent pathotypes at the seedling stage, but resistant at the adult plant stage. Genetic analysis of the RIL population revealed the presence of two genes for stripe rust resistance, with one gene each being contributed by each of the parental lines. A linkage map with 169 SSR and RFLP loci generated from a set of 93 RILs was used for mapping these resistance genes. Based on phenotypic data for 3 years and the pooled data, two QTLs, one each in T. monococcum acc. pau14087 and T. boeoticum acc. pau5088, were detected for resistance in the RIL population. The QTL in T. monococcum mapped on chromosome 2A in a 3.6 cM interval between Xwmc407 and Xwmc170, whereas the QTL from T. boeoticum mapped on 5A in 8.9 cM interval between Xbarc151 and Xcfd12 and these were designated as QYrtm.pau-2A and QYrtb.pau-5A, respectively. Based on field data for 3 years, their R2 values were 14 and 24%, respectively. T. monococcum acc. pau14087 and three resistant RILs were crossed to hexaploid wheat cvs WL711 and PBW343, using T. durum as a bridging species with the objective of transferring these genes into hexaploid wheat. The B genome of T. durum suppressed resistance in the F1 plants, but with subsequent backcrossing one resistance gene could be transferred from one of the RILs to the hexaploid wheat background. This gene was derived from T. boeoticum acc. pau5088 as indicated by co-introgression of T. boeoticum sequences linked to stripe rust resistance QTL, QYrtb.pau-5A. Homozygous resistant progenies with 40-42 chromosomes have been identified.

An integrated molecular linkage map of diploid wheat based on a Triticum boeoticum x T. monococcum RIL population.

Diploid A genome species of wheat harbour immense variability for biotic stresses and productivity traits, and these could be transferred efficiently to hexaploid wheat through marker assisted selection, provided the target genes are tagged at diploid level first. Here we report an integrated molecular linkage map of A genome diploid wheat based on 93 recombinant inbred lines (RILs) derived from Triticum boeoticum x Triticum monococcum inter sub-specific cross. The parental lines were analysed with 306 simple sequence repeat (SSR) and 194 RFLP markers, including 66 bin mapped ESTs. Out of 306 SSRs tested for polymorphism, 74 (24.2%) did not show amplification (null) in both the parents. Overall, 171 (73.7%) of the 232 remaining SSR and 98 (50.5%) of the 194 RFLP markers were polymorphic. Both A and D genome specific SSR markers showed similar transferability to A genome of diploid wheat species. The 176 polymorphic markers, that were assayed on a set of 93 RILs, yielded 188 polymorphic loci and 177 of these as well as two additional morphological traits mapped on seven linkage groups with a total map length of 1,262 cM, which is longer than most of the available A genome linkage maps in diploid and hexaploid wheat. About 58 loci showed distorted segregation with majority of these mapping on chromosome 2A(m). With a few exceptions, the position and order of the markers was similar to the ones in other maps of the wheat A genome. Chromosome 1A(m) of T. monococcum and T. boeoticum showed a small paracentric inversion relative to the A genome of hexaploid wheat. The described linkage map could be useful for gene tagging, marker assisted gene introgression from diploid into hexaploid wheat as well as for map based cloning of genes from diploid A genome species and orthologous genes from hexaploid wheat.

Identification and mapping of a tiller inhibition gene (tin3) in wheat.

Tillering is one of the most important agronomic traits in cereal crops because tiller number per plant determines the number of spikes or panicles per plant, a key component of grain yield and/or biomass. In order to characterize the underlying genetic variation for tillering, we have isolated mutants that are compromised in tillering ability using ethyl methanesulphonate (EMS)-based mutagenesis in diploid wheat (Triticum monococcum subsp. monococcum). The tillering mutant, tiller inhibition (tin3) produces only one main culm compared to the wild type with many tillers. The monoculm phenotype of tin3 is due to a single recessive mutation. Genetic and molecular mapping in an F(2) population of diploid wheat located the tin3 gene on the long arm of chromosome 3A(m). One codominant RFLP marker Xpsr1205 cosegregated with tin3 in the F(2) population. Physical mapping of PSR1205 in a set of Chinese Spring deletion lines of group-3 chromosomes placed the tin3 gene in the distal 10% of the long arm of chromosome 3A, which is a recombination-rich region in wheat. The implications of the mapping of tin3 on chromosome arm 3A(m)L are discussed with respect to putative orthologs of tin3 in the 3L colinear regions across various cereal genomes and other tillering traits in grasses.

Patterns of antifungal prescribing in general dental practice.

OBJECTIVE: To examine the current practice of antifungal prescribing by GDPs in the United Kingdom. Design A postal questionnaire circulated to a random selection of 400 dentists. OUTCOME MEASURES: The questionnaires were analysed and the responses expressed as absolute and relative frequencies. RESULTS: Responses to the questionnaire were received from 297 (74.3%) GDPs. Nystatin was the most popular choice of antifungal agent that GDPs would use, followed by miconazole, amphotericin B and fluconazole. The likelihood of use of miconazole was positively linked to recent date of graduation. Lack of knowledge regarding contraindications and problems with azole antibiotics was found in a significant minority of practitioners (36%). CONCLUSIONS: The present study indicates that azole antifungal agents (especially miconazole) are becoming more widely used by GDPs, but that knowledge regarding potential problems with their use is sub-optimal. Nystatin remains the most popular choice of antifungal agent.

QTL analysis for grain protein content using SSR markers and validation studies using NILs in bread wheat.

QTL interval mapping for grain protein content (GPC) in bread wheat was conducted for the first time, using a framework map based on a mapping population, which was available in the form of 100 recombinant inbred lines (RILs). The data on GPC for QTL mapping was recorded by growing the RILs in five different environments representing three wheat growing locations from Northern India; one of these locations was repeated for 3 years. Distribution of GPC values followed normal distributions in all the environments, which could be explained by significant g x e interactions observed through analyses of variances, which also gave significant effects due to genotypes and environments. Thirteen (13) QTLs were identified in individual environments following three methods (single-marker analysis or SMA, simple interval mapping or SIM and composite interval mapping or CIM) and using LOD scores that ranged from 2.5 to 6.5. Threshold LOD scores (ranging from 3.05 to 3.57), worked out and used in each case, however, detected only seven of the above 13 QTLs. Only four (QGpc.ccsu-2B.1; QGpc.ccsu-2D.1; QGpc.ccsu-3D.1 and QGpc.ccsu-7A.1) of these QTLs were identified either in more than one location or following one more method other than CIM; another QTL (QGpc.ccsu-3D.2), which was identified using means for all the environments, was also considered to be important. These five QTLs have been recommended for marker-assisted selection (MAS). The QTLs identified as above were also validated using ten NILs derived from three crosses. Five of the ten NILs possessed 38 introgressed segments from 16 chromosomes and carried 42 of the 173 markers that were mapped. All the seven QTLs were associated with one or more of the markers carried by the above introgressed segments, thus validating the corresponding markers. More markers associated with many more QTLs to be identified should become available in the future by effective MAS for GPC improvement.

Mapping of a resistance gene effective against Karnal bunt pathogen of wheat.

A set of 130 wheat recombinant inbred lines (RILs) developed from a cross between parents susceptible (WL711) and resistant (HD29) to Karnal bunt (caused by Tilletia indica), were screened for 3 years with the pathogen populations prevalent in northern India. When 90 simple sequence repeats (SSRs) and 81 amplified fragment length polymorphism (AFLP) loci were mapped on the RILs, markers on chromosomes 2A, 4B and 7B accounted collectively for about one-third of the variation in the disease reaction. The genomic region of largest effect, identified on the long arm of chromosome 4B, reduced Karnal bunt disease by half in three different experiments and accounted for up to 25% of the phenotypic variation for KB reaction. A closely linked SSR marker, GWM538, may be useful in marker-assisted selection for Karnal bunt resistance in wheat.

Microsatellite markers reveal chimeric origin of redesignated chromosome 4A of wheat from Triticum urartu and other species.

Although a new nomenclature has been adopted for wheat in which chromosome 4A (4AO) has been renamed 4B (4BN) and chromosome 4B (4BO) has been renamed 4A (4AN), their specific origin remains uncertain. The use of wheat microsatellite (WMS) markers mapped to chromosomes 4AN and 4BN in a set of polyploid wheats and diploid genome donors has unequivocally indicated that the entire short arm of 4AN, some part of 4ANL. and a segment of 4BNL were derived from Triticum urartu. The presence of a T. urartu-specific allele at locus gwm368 on 4BNL and of an Aegilops speltoides allele at locus gwm397 on 4ANL suggests the possibility of a reciprocal translocation between 4ANL and 4BNL. The subcentromeric and telomeric regions of 4ANL corresponding to heterochromatic C-bands were derived neither from diploid wheats nor from Ae. speltoides or Aegilops longissima.