QTL mapping of shoot and seed traits impacted by Drought in Barley using a recombinant inbred line Population.

BACKGROUND: With ongoing climate change, drought events are severely limiting barley production worldwide and pose a significant risk to the malting, brewing and food industry. The genetic diversity inherent in the barley germplasm offers an important resource to develop stress resiliency. The purpose of this study was to identify novel, stable, and adaptive Quantitative Trait Loci (QTL), and candidate genes associated with drought tolerance. A recombinant inbred line (RIL) population (n = 192) developed from a cross between the drought tolerant 'Otis' barley variety, and susceptible 'Golden Promise'(GP) was subjected to short-term progressive drought during heading in the biotron. This population was also evaluated under irrigated and rainfed conditions in the field for yields and seed protein content. RESULTS: Barley 50k iSelect SNP Array was used to genotype the RIL population to elucidate drought-adaptive QTL. Twenty-three QTL (eleven for seed weight, eight for shoot dry weight and four for protein content) were identified across several barley chromosomes. QTL analysis identified genomic regions on chromosome 2 and 5 H that appear to be stable across both environments and accounted for nearly 60% variation in shoot weight and 17.6% variation in seed protein content. QTL at approximately 29 Mbp on chromosome 2 H and 488 Mbp on chromosome 5 H are in very close proximity to ascorbate peroxidase (APX) and in the coding sequence of the Dirigent (DIR) gene, respectively. Both APX and DIR are well-known key players in abiotic stress tolerance in several plants. In the quest to identify key recombinants with improved tolerance to drought (like Otis) and good malting profiles (like GP), five drought tolerant RILs were selected for malt quality analysis. The selected drought tolerant RILs exhibited one or more traits that were outside the realms of the suggested limits for acceptable commercial malting quality. CONCLUSIONS: The candidate genes can be used for marker assisted selection and/or genetic manipulation to develop barley cultivars with improved tolerance to drought. RILs with genetic network reshuffling necessary to generate drought tolerance of Otis and favorable malting quality attributes of GP may be realized by screening a larger population.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Three ß-Glucuronosyltransferase Genes Involved in Arabinogalactan Biosynthesis Function in Arabidopsis Growth and Development.

Arabinogalactan proteins (AGPs) contain arabinogalactan (AG) polysaccharides that are biologically relevant to plant growth processes. Here, the biochemical and physiological roles of three Golgi localized ß-glucuronosyltransferase genes (GLCAT14A, GLCAT14B and GLCAT14C) in Arabidopsis thaliana, responsible for the addition of glucuronic acid to AG chains, were further investigated using single, double and triple glcat14 mutant plants. These proteins were localized to the Golgi apparatus when transiently expressed in Nicotiana benthamiana. Sugar analysis of AGP extracts from Arabidopsis stem, leaf and siliques showed a consistent reduction in glucuronic acid in glcat14 mutants relative to wild type, with concomitant effects resulting in tissue-specific alterations, especially in arabinose and galactose sugars. Although we observed defects in trichome branching in glca14a/b and glca14a/b/c mutants, scanning electron microscope analysis/energy dispersive microanalysis (SEM/EDX) showed no difference in the calcium content of trichomes in these mutants relative to wild type. Immunoblot analyses of the stem and leaf showed a reduction in AGPs as detected with the LM2 antibody in glcat14a/b and glcat14a/b/c mutants relative to wild type. The current work exemplifies the possibility of conducting structure-function assessment of cell wall biosynthetic genes to identify their physiological roles in plant growth and development.

Two ß-glucuronosyltransferases involved in the biosynthesis of type II arabinogalactans function in mucilage polysaccharide matrix organization in Arabidopsis thaliana.

BACKGROUND: Arabinogalactan-proteins (AGPs) are heavily glycosylated with type II arabinogalactan (AG) polysaccharides attached to hydroxyproline residues in their protein backbone. Type II AGs are necessary for plant growth and critically important for the establishment of normal cellular functions. Despite the importance of type II AGs in plant development, our understanding of the underlying role of these glycans/sugar residues in mucilage formation and seed coat epidermal cell development is poorly understood and far from complete. One such sugar residue is the glucuronic acid residues of AGPs that are transferred onto AGP glycans by the action of ß-glucuronosyltransferase genes/enzymes. RESULTS: Here, we have characterized two ß-glucuronosyltransferase genes, GLCAT14A and GLCAT14C, that are involved in the transfer of ß-glucuronic acid (GlcA) to type II AGs. Using a reverse genetics approach, we observed that glcat14a-1 mutants displayed subtle alterations in mucilage pectin homogalacturonan (HG) compared to wild type (WT), while glcat14a-1glcat14c-1 mutants displayed much more severe mucilage phenotypes, including loss of adherent mucilage and significant alterations in cellulose ray formation and seed coat morphology. Monosaccharide composition analysis showed significant alterations in the sugar amounts of glcat14a-1glcat14c-1 mutants relative to WT in the adherent and non-adherent seed mucilage. Also, a reduction in total mucilage content was observed in glcat14a-1glcat14c-1 mutants relative to WT. In addition, glcat14a-1glcat14c-1 mutants showed defects in pectin formation, calcium content and the degree of pectin methyl-esterification (DM) as well as reductions in crystalline cellulose content and seed size. CONCLUSIONS: These results raise important questions regarding cell wall polymer interactions and organization during mucilage formation. We propose that the enzymatic activities of GLCAT14A and GLCAT14C play partially redundant roles and are required for the organization of the mucilage matrix and seed size in Arabidopsis thaliana. This work brings us a step closer towards identifying potential gene targets for engineering plant cell walls for industrial applications.

RNA-seq profiling of skin in temperate and tropical cattle.

Skin is a major thermoregulatory organ in the body controlling homeothermy, a critical function for climate adaptation. We compared genes expressed between tropical- and temperate-adapted cattle to better understand genes involved in climate adaptation and hence thermoregulation. We profiled the skin of representative tropical and temperate cattle using RNA-seq. A total of 214,754,759 reads were generated and assembled into 72,993,478 reads and were mapped to unique regions in the bovine genome. Gene coverage of unique regions of the reference genome showed that of 24,616 genes, only 13,130 genes (53.34%) displayed more than one count per million reads for at least two libraries and were considered suitable for downstream analyses. Our results revealed that of 255 genes expressed differentially, 98 genes were upregulated in tropically-adapted White Fulani (WF; Bos indicus) and 157 genes were down regulated in WF compared to Angus, AG (Bos taurus). Fifteen pathways were identified from the differential gene sets through gene ontology and pathway analyses. These include the significantly enriched melanin metabolic process, proteinaceous extracellular matrix, inflammatory response, defense response, calcium ion binding and response to wounding. Quantitative PCR was used to validate six representative genes which are associated with skin thermoregulation and epithelia dysfunction (mean correlation 0.92; p < 0.001). Our results contribute to identifying genes and understanding molecular mechanisms of skin thermoregulation that may influence strategic genomic selection in cattle to withstand climate adaptation, microbial invasion and mechanical damage.

Computational genome-wide identification of heat shock protein genes in the bovine genome.

Background: Heat shock proteins (HSPs) are molecular chaperones known to bind and sequester client proteins under stress. Methods: To identify and better understand some of these proteins, we carried out a computational genome-wide survey of the bovine genome. For this, HSP sequences from each subfamily (sHSP, HSP40, HSP70 and HSP90) were used to search the Pfam (Protein family) database, for identifying exact HSP domain sequences based on the hidden Markov model. ProtParam tool was used to compute potential physico-chemical parameters detectable from a protein sequence. Evolutionary trace (ET) method was used to extract evolutionarily functional residues of a homologous protein family. Results: We computationally identified 67 genes made up of 10, 43, 10 and 4 genes belonging to small HSP, HSP40, HSP70 and HSP90 families respectively. These genes were widely dispersed across the bovine genome, except in chromosomes 24, 26 and 27, which lack bovine HSP genes. We found an uncharacterized outer dense fiber ( ODF1) gene in cattle with an intact alpha crystallin domain, like other small HSPs. Physico-chemical characteristic of aliphatic index was higher in HSP70 and HSP90 gene families, compared to small HSP and HSP40. Grand average hydropathy showed that small HSP (sHSP), HSP40, HSP70 and HSP90 genes had negative values except for DNAJC22, a member of HSP40 gene family. The uniqueness of DNAJA3 and DNAJB13 among HSP40 members, based on multiple sequence alignment, evolutionary trace analysis and sequence identity dendrograms, suggests evolutionary distinct structural and functional features, with unique roles in substrate recognition and chaperone functions. The monophyletic pattern of the sequence identity dendrograms of cattle, human and mouse HSP sequences suggests functional similarities. Conclusions: Our computational results demonstrate the first-pass in-silico identification of heat shock proteins and calls for further investigation to better understand their functional roles and mechanisms in Bovidae.

Genetic variation in N- and C-terminal regions of bovine DNAJA1 heat shock protein gene in African, Asian and American cattle.

DNAJA1 or heat shock protein 40 (Hsp40) is associated with heat adaptation in various organisms. We amplified and sequenced a total of 1,142 bp of bovine Hsp40 gene representing the critical N-terminal (NTR) and C-terminal (CTR) regions in representative samples of African, Asian and American cattle breeds. Eleven and 9 different haplotypes were observed in the NTR in Asian and African breeds respectively while in American Brangus, only two mutations were observed resulting in two haplotypes. The CTR appears to be highly conserved between cattle and yak. In-silico functional analysis with PANTHER predicted putative deleterious functional impact of c.161 T>A; p. V54Q while alignment of bovine and human NTR-J domains revealed that p.Q19H, p.E20Q and p. E21X mutations occurred in helix 2 and p.V54Q missense mutation occurred in helix 3 respectively. The 124 bp insertion found in the yak DNAJA1 ortholog may have significant functional relevance warranting further investigation. Our results suggest that these genetic differences may be concomitant with population genetic history and possible functional consequences for climate adaptation in bovidae.

A novel TaqI polymorphism in the coding region of the ovine TNXB gene in the MHC class III region: morphostructural and physiological influences.

The tenascin-XB (TNXB) gene has antiadhesive effects, functions in matrix maturation in connective tissues, and localizes to the major histocompatibility complex class III region. We hypothesized that it may influence adaptive physiological response through an effect on blood vessel function. We identified a novel g.1324 A→G polymorphism at a TaqI recognition site in a 454 bp fragment of ovine TNXB and genotyped it in 150 Nigerian sheep using PCR-RFLP. The missense mutation changes glutamic acid (GAA) to glycine (GGA). Among SNP genotypes, significant differences (P < 0.05) were observed in body weight and fore cannon bone length. Interaction effects of breed, SNP genotype, and geographic location had a significant effect (P < 0.05) on chest girth. The SNP genotype was significantly (P < 0.05) associated with physiological traits of pulse rate and skin temperature. The observed effect of this novel polymorphism may be mediated through its role in connective tissue biology, requiring further association and functional studies.

Genetic diversity in exon 2 of the major histocompatibility complex class II DQB1 locus in Nigerian goats.

The DQB1 locus is located in the major histocompatibility complex (MHC) class II region and involved in immune response. We identified 20 polymorphic sites in a 228 bp fragment of exon 2, one of the most critical regions of the MHC DQB1 gene, in 60 Nigerian goats. Four sites are located in the peptide binding region, and 10 amino acid substitutions are peculiar to Nigerian goats, compared with published sequences. A significantly higher ratio of nonsynonymous/synonymous substitutions (dN/dS) suggests that allelic sequence evolution is driven by balancing selection (P < 0.01). In silico functional analysis using PANTHER predicted that substitution P56R, with a subPSEC score of -4.00629 (Pdeleterious = 0.73229), is harmful to protein function. The phylogenetic tree from consensus sequences placed the two northern breeds closer to each other than either was to the southern goats. This first report of sequence diversity at the DQB1 locus for any African goat breed may be useful in the search for disease-resistant genotypes.

Morphological and microsatellite DNA diversity of Nigerian indigenous sheep.

BACKGROUND: Sheep is important in the socio-economic lives of people around the world. It is estimated that more than half of our once common livestock breeds are now endangered. Since genetic characterization of Nigerian sheep is still lacking, we analyzed ten morphological traits on 402 animals and 15 microsatellite DNA markers in 384 animals of the 4 Nigerian sheep breeds to better understand genetic diversity for breeding management and germplasm conservation. RESULTS: Morphological traits of Uda and Balami were significantly (P < 0.05) higher than Yankasa, which were both higher than West African Dwarf (WAD) sheep. Stepwise discriminant analysis showed tail length, rump height, chest girth, ear length and chest depth as the most discriminating variables for classification. Mahalanobis distances show the least differentiation between Uda and Balami and the largest between WAD and Balami sheep. While 93.3% of WAD sheep were correctly assigned to their source genetic group, 63.9% of Yankasa, 61.2% of Balami and 45.2% of Uda were classified correctly by nearest neighbour discriminant analysis. The overall high Polymorphism Information Content (PIC) of all microsatellite markers ranged from 0.751 to 0.927 supporting their use in genetic characterization. Expected heterozygosity was high for all loci (0.783 to 0.93). Mean heterozygote deficiency across all populations (0.171 to 0.534) possibly indicate significant inbreeding (P < 0.05). Mean values for FST, FIT and FIS statistics across all loci were 0.088, 0.394 and 0.336 respectively. Yankasa and Balami are the most closely related breeds (DA = 0.184) while WAD and Balami are the farthest apart breeds (DA = 0.665), which is coincident with distance based on morphological analysis and population structure assessed by STRUCTURE. CONCLUSIONS: These results suggest that within-breed genetic variation in Nigerian sheep is higher than between-breeds and may be a valuable tool for genetic improvement and conservation. The higher genetic variability in Yankasa suggests the presence of unique ancestral alleles reflecting the presence of certain functional genes which may result in better adaptability in more agro-ecological zones of Nigeria. These genetic characteristics are potentially useful in planning improvement and conservation strategies in Nigerian indigenous sheep.

Multivariate analysis of sexual size dimorphism in local turkeys (Meleagris gallopavo) in Nigeria.

Sexual size dimorphism is a key evolutionary feature that can lead to important biological insights. To improve methods of sexing live birds in the field, we assessed sexual size dimorphism in Nigerian local turkeys (Meleagris gallopavo) using multivariate techniques. Measurements were taken on 125 twenty-week-old birds reared under the intensive management system. The body parameters measured were body weight, body length, breast girth, thigh length, shank length, keel length, wing length and wing span. Univariate analysis revealed that toms (males) had significantly (P < 0.05) higher mean values than hens (females) in all the measured traits. Positive phenotypic correlations between body weight and body measurements ranged from 0.445 to 0.821 in toms and 0.053-0.660 in hens, respectively. Three principal components (PC1, PC2 and PC3) were extracted in toms, each accounting for 63.70%, 19.42% and 5.72% of the total variance, respectively. However, four principal components (PC1, PC2, PC3 and PC4) were extracted in hens, which explained 54.03%, 15.29%, 11.68% and 6.95%, respectively of the generalised variance. A stepwise discriminant function analysis of the eight morphological traits indicated that body weight, body length, tail length and wing span were the most discriminating variables in separating the sexes. The single discriminant function obtained was able to correctly classify 100% of the birds into their source population. The results obtained from the present study could aid future management decisions, ecological studies and conservation of local turkeys in a developing economy.