Identification and characterization of RuvBL DNA helicase genes for tolerance against abiotic stresses in bread wheat (Triticum aestivum L.) and related species.

Recombination UVB (sensitivity) like (RuvBL) helicase genes represent a conserved family of genes, which are known to be involved in providing tolerance against abiotic stresses like heat and drought. We identified nine wheat RuvBL genes, one each on nine different chromosomes, belonging to homoeologous groups 2, 3, and 4. The lengths of genes ranged from 1647 to 2197 bp and exhibited synteny with corresponding genes in related species including Ae. tauschii, Z. mays, O. sativa, H. vulgare, and B. distachyon. The gene sequences were associated with regulatory cis-elements and transposable elements. Two genes, namely TaRuvBL1a-4A and TaRuvBL1a-4B, also carried targets for a widely known miRNA, tae-miR164. Gene ontology revealed that these genes were closely associated with ATP-dependent formation of histone acetyltransferase complex. Analysis of the structure and function of RuvBL proteins revealed that the proteins were localized mainly in the cytoplasm. A representative gene, namely TaRuvBL1a-4A, was also shown to be involved in protein-protein interactions with ten other proteins. On the basis of phylogeny, RuvBL proteins were placed in two sub-divisions, namely RuvBL1 and RuvBL2, which were further classified into clusters and sub-clusters. In silico studies suggested that these genes were differentially expressed under heat/drought. The qRT-PCR analysis confirmed that expression of TaRuvBL genes differed among wheat cultivars, which differed in the level of thermotolerance. The present study advances our understanding of the biological role of wheat RuvBL genes and should help in planning future studies on RuvBL genes in wheat including use of RuvBL genes in breeding thermotolerant wheat cultivars.

Comparative analysis of VMT genes/proteins in selected plant species with emphasis on bread wheat (Triticum aestivum L.).

BACKGROUND: In recent years, the study of molecular basis of uptake, transport and utilization of grain Fe/Zn (GFe/GZn) in wheat has been an active area of research. As a result, it has been shown that a number of transporters are involved in uptake and transport of Fe. In a recent study, knockout of a transporter gene OsVMT (VACUOLAR MUGINEIC ACID TRANSPORTER) in rice was shown to be involved in Fe homoeostasis. OBJECTIVE: In this study, we analysed VMT genes among six monocots and three dicots with major emphasis on wheat VMT genes (TaVMTs), taking OsVMT gene as a reference. METHODS AND RESULTS: Using OsVMT gene as a reference, VMT genes were identified and sequence similarities were examined among six monocots and three dicots. Each VMT protein carried one functional domain and 7 to 10 distinct motifs (including 9 novel motifs). The qRT-PCR analysis showed differential expression by all the six TaVMT genes in pairs of contrasting wheat genotypes with high (FAR4 and WB02) and low (K8027 and HD3226) GFe/GZn at two different grain filling stages (14 DAA and 28 DAA). TaVMT1 genes showed up-regulation in high GFe/Zn genotypes relative to low GFe/Zn genotypes, whereas the TaVMT2 genes showed down-regulation or nonsignificant up-regulation in a few cases. CONCLUSIONS: At 14 DAA, each of the six TaVMT genes exhibited higher expression in wheat genotypes with high GFe and GZn relative to those with low GFe and GZn, suggesting major role of VMT genes in improvement of grain Fe/Zn homoeostasis, thus making TaVMT genes useful for improvement in Fe/Zn in wheat grains.

Multi-omics assisted breeding for biotic stress resistance in soybean.

Biotic stress is a critical factor limiting soybean growth and development. Soybean responses to biotic stresses such as insects, nematodes, fungal, bacterial, and viral pathogens are governed by complex regulatory and defense mechanisms. Next-generation sequencing has availed research techniques and strategies in genomics and post-genomics. This review summarizes the available information on marker resources, quantitative trait loci, and marker-trait associations involved in regulating biotic stress responses in soybean. We discuss the differential expression of related genes and proteins reported in different transcriptomics and proteomics studies and the role of signaling pathways and metabolites reported in metabolomic studies. Recent advances in omics technologies offer opportunities to reshape and improve biotic stress resistance in soybean by altering gene regulation and/or other regulatory networks. We suggest using 'integrated omics' to precisely understand how soybean responds to different biotic stresses. We also discuss the potential challenges of integrating multi-omics for the functional analysis of genes and their regulatory networks and the development of biotic stress-resistant cultivars. This review will help direct soybean breeding programs to develop resistance against different biotic stresses.

Meta-QTLs, ortho-MetaQTLs and candidate genes for grain Fe and Zn contents in wheat (Triticum aestivum L.).

Majority of cereals are deficient in essential micronutrients including grain iron (GFe) and grain zinc (GZn), which are therefore the subject of research involving biofortification. In the present study, 11 meta-QTLs (MQTLs) including nine novel MQTLs for GFe and GZn contents were identified in wheat. Eight of these 11 MQTLs controlled both GFe and GZn. The confidence intervals of the MQTLs were narrower (0.51-15.75 cM) relative to those of the corresponding QTLs (0.6 to 55.1 cM). Two ortho-MQTLs involving three cereals (wheat, rice and maize) were also identified. Results of MQTLs were also compared with the results of earlier genome wide association studies (GWAS). As many as 101 candidate genes (CGs) underlying MQTLs were also identified. Twelve of these CGs were prioritized; these CGs encoded proteins with important domains (zinc finger, RING/FYVE/PHD type, flavin adenine dinucleotide linked oxidase, etc.) that are involved in metal ion binding, heme binding, iron binding, etc. qRT-PCR analysis was conducted for four of these 12 prioritized CGs using genotypes which have differed for GFe and GZn. Significant differential expression in these genotypes was observed at 14 and 28 days after anthesis. The MQTLs/CGs identified in the present study may be utilized in marker-assisted selection (MAS) for improvement of GFe/GZn contents and also for understanding the molecular basis of GFe/GZn homeostasis in wheat. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01149-9.

WheatQTLdb V2.0: a supplement to the database for wheat QTL.

We recently developed a database for hexaploid wheat QTL (WheatQTLdb; www.wheatqtldb.net), which included 11,552 QTL affecting various traits of economic importance. However, that database did not include valuable QTL from other wheat species and/or progenitors of hexaploid wheat. Therefore, an updated and improved version of wheat QTL database (WheatQTLdb V2.0) was developed, which now includes information on hexaploid wheat (Triticum aestivum) and the following seven other related species: T. durum, T. turgidum, T. dicoccoides, T. dicoccum, T. monococcum, T. boeoticum, and Aegilops tauschii. WheatQTLdb V2.0 includes a much-improved list of QTL, including 27,518 main effect QTL, 202 epistatic QTL, and 1321 metaQTL. This newly released WheatQTLdb V2.0 also has additional valuable options to search and choose the QTL, category-wise, and trait-wise data for their use in research or breeding programs.

Meta-QTLs and candidate genes for stripe rust resistance in wheat.

In bread wheat, meta-QTL analysis was conducted using 353 QTLs that were available from earlier studies. When projected onto a dense consensus map comprising 76,753 markers, only 184 QTLs with the required information, could be utilized leading to identification of 61 MQTLs spread over 18 of the 21 chromosomes (barring 5D, 6D and 7D). The range for mean R2 (PVE %) was 1.9% to 48.1%, and that of CI was 0.02 to 11.47 cM; these CIs also carried 37 Yr genes. Using these MQTLs, 385 candidate genes (CGs) were also identified. Out of these CGs, 241 encoded known R proteins and 120 showed differential expression due to stripe rust infection at the seedling stage; the remaining 24 CGs were common in the sense that they encoded R proteins as well as showed differential expression. The proteins encoded by CGs carried the following widely known domains: NBS-LRR domain, WRKY domains, ankyrin repeat domains, sugar transport domains, etc. Thirteen breeders' MQTLs (PVE > 20%) including four pairs of closely linked MQTLs are recommended for use in wheat molecular breeding, for future studies to understand the molecular mechanism of stripe rust resistance and for gene cloning.

WheatQTLdb: a QTL database for wheat.

During the last three decades, QTL analysis in wheat has been conducted for a variety of individual traits, so that thousands of QTL along with the linked markers, their genetic positions and contribution to phenotypic variation (PV) for concerned traits are now known. However, no exhaustive database for wheat QTL is currently available at a single platform. Therefore, the present database was prepared which is an exhaustive information resource for wheat QTL data from the published literature till May, 2020. QTL data from both interval mapping and genome-wide association studies (GWAS) have been included for the following classes of traits: (i) morphological traits, (ii) N and P use efficiency, (iii) traits for biofortification (Fe, K, Se, and Zn contents), (iv) tolerance to abiotic stresses including drought, water logging, heat stress, pre-harvest sprouting and salinity, (v) resistance to biotic stresses including those due to bacterial, fungal, nematode and insects, (vi) quality traits, and (vii) a variety of physiological traits, (viii) developmental traits, and (ix) yield and its related traits. For the preparation of the database, literature was searched for data on QTL/marker-trait associations (MTAs), curated and then assembled in the form of WheatQTLdb. The available information on metaQTL, epistatic QTL and candidate genes, wherever available, is also included in the database. Information on QTL in this WheatQTLdb includes QTL names, traits, associated markers, parental genotypes, crosses/mapping populations, association mapping panels and other useful information. To our knowledge, WheatQTLdb prepared by us is the largest collection of QTL (11,552), epistatic QTL (107) and metaQTL (330) data for hexaploid wheat to be used by geneticists and plant breeders for further studies involving fine mapping, cloning, and marker-assisted selection (MAS) during wheat breeding.

Population structure and genome-wide association studies in bread wheat for phosphorus efficiency traits using 35 K Wheat Breeder's Affymetrix array.

Soil bioavailability of phosphorus (P) is a major concern for crop productivity worldwide. As phosphatic fertilizers are a non-renewable resource associated with economic and environmental issues so, the sustainable option is to develop P use efficient crop varieties. We phenotyped 82 diverse wheat (Triticum aestivum L.) accessions in soil and hydroponics at low and sufficient P. To identify the genic regions for P efficiency traits, the accessions were genotyped using the 35 K-SNP array and genome-wide association study (GWAS) was performed. The high-quality SNPs across the genomes were evenly distributed with polymorphic information content values varying between 0.090 and 0.375. Structure analysis revealed three subpopulations (C1, C2, C3) and the phenotypic responses of these subpopulations were assessed for P efficiency traits. The C2 subpopulation showed the highest genetic variance and heritability values for numerous agronomically important traits as well as strong correlation under both P levels in soil and hydroponics. GWAS revealed 78 marker-trait associations (MTAs) but only 35 MTAs passed Bonferroni Correction. A total of 297 candidate genes were identified for these MTAs and their annotation suggested their involvement in several biological process. Out of 35, nine (9) MTAs were controlling polygenic trait (two controlling four traits, one controlling three traits and six controlling two traits). These multi-trait MTAs (each controlling two or more than two correlated traits) could be utilized for improving bread wheat to tolerate low P stress through marker-assisted selection (MAS).

Identification and characterization of SET domain family genes in bread wheat (Triticum aestivum L.).

SET domain genes (SDGs) that are involved in histone methylation have been examined in many plant species, but have never been examined in bread wheat; the histone methylation caused due to SDGs is associated with regulation of gene expression at the transcription level. We identified a total of 166 bread wheat TaSDGs, which carry some interesting features including the occurrence of tandem/interspersed duplications, SSRs (simple sequence repeats), transposable elements, lncRNAs and targets for miRNAs along their lengths and transcription factor binding sites (TFBS) in the promoter regions. Only 130 TaSDGs encoded proteins with complete SET domain, the remaining 36 proteins had truncated SET domain. The TaSDG encoded proteins were classified into six classes (I-V and VII). In silico expression analysis indicated relatively higher expression (FPKM > 20) of eight of the 130 TaSDGs in different tissues, and downregulation of 30 TaSDGs under heat and drought at the seedling stage. qRT-PCR was also conducted to validate the expression of seven genes at the seedling stage in pairs of contrasting genotypes in response to abiotic stresses (water and heat) and biotic stress (leaf rust). These genes were generally downregulated in response to the three stresses examined.

A study of CCD8 genes/proteins in seven monocots and eight dicots.

In plants, the enzyme CCD8 (carotenoid cleavage dioxygenase 8) is involved in the synthesis of an important hormone, strigolactone, and therefore, plays an important role in controlling growth and development. Using cDNA and protein sequence derived from the gene ZmCCD8 from maize, we identified putative orthologs of the gene encoding CCD8 in six other monocots and eight dicots; the sequence similarity ranged from 52-75.9% at the gene level and 60.9-93.7% at the protein level. The average length of the gene was ~3.3 kb (range: 2.08 to 3.98 kb), although the number of introns within the genes differed (4 or 5 in dicots and 3 or 4 in monocots, except in T. urartu with 6 introns). Several cis-acting regulatory elements were identified in the promoters of CCD8 genes, which are known to respond to biotic and abiotic stresses. The N-terminal end (up to ~70 amino acids) of CCD8 proteins was highly variable due to insertions, deletions and mismatches. The variation in genes and proteins were particularly conspicuous in T. urartu and Ae. tauschii among the monocots and A. thaliana and P. persica among the dicots. In CCD8 proteins, 12 motifs were also identified, of which 6 were novel; 4 of these novel motifs occurred in all the 15 species. The 3D structures of proteins had the characteristic features of the related enzyme apocarotenoid oxygenase (ACO) of Synechocystis (a representative of cyanobacteria). The results of qRT-PCR in wheat revealed that under phosphorous (P)-starved condition (relative to expression under optimum P used as control), the expression of TaCCD8 genes increased ~37 fold in root tissue of the cultivar C306 and ~33 fold in shoot tissue of the cultivar HUW468 (the two cultivars differed in their P-use efficiency). This suggested that expression of TaCCD8 genes is genotype-dependent and tissue-specific and is regulated under different levels of P supply.

Cloning, Sequencing and In Silico Analysis of phbC Gene from Pseudomonas spp.

We report here isolation and analysis of PCR amplified phbC gene from Pseudomonas spp. strain phbmbb15-B3. This strain was previously developed from mutations of landfill isolates and found to be an efficient Poly Hydroxy butyrate (PHB) producer. The fragment was cloned into pTZ57R/T cloning vector and then the gene has been sequenced and submitted to GenBank (Accession Number KT933807). The sequence results confirmed the clone to be phbC homologue and the ORF was 910 base pairs long and coded for 303 amino acids, which shared 92-99% amino acid sequence identity with the available bacterial sequences in Gene Bank. We could also predict the primary and secondary structural features of the expected phbC protein. Phylogenetic analysis also revealed its similarity with several pseudomonads. The results of the present study shall provide a stable foundation for further research on modeling studies of PHB synthase and developing PHB a commercial technology.

Genome-wide identification and characterization of gene family for RWP-RK transcription factors in wheat (Triticum aestivum L.).

RWP-RKs represent a small family of transcription factors (TFs) that are unique to plants and function particularly under conditions of nitrogen starvation. These RWP-RKs have been classified in two sub-families, NLPs (NIN-like proteins) and RKDs (RWP-RK domain proteins). NLPs regulate tissue-specific expression of genes involved in nitrogen use efficiency (NUE) and RKDs regulate expression of genes involved in gametogenesis/embryogenesis. During the present study, using in silico approach, 37 wheat RWP-RK genes were identified, which included 18 TaNLPs (2865 to 7340 bp with 4/5 exons), distributed on 15 chromosomes from 5 homoeologous groups (with two genes each on 4B,4D and 5A) and 19 TaRKDs (1064 to 5768 bp with 1 to 6 exons) distributed on 12 chromosomes from 4 homoeologous groups (except groups 1, 4 and 5); 2-3 splice variants were also available in 9 of the 37 genes. Sixteen (16) of these genes also carried 24 SSRs (simple sequence repeats), while 11 genes had targets for 13 different miRNAs. At the protein level, MD simulation analysis suggested their interaction with nitrate-ions. Significant differences were observed in the expression of only two (TaNLP1 and TaNLP2) of the nine representative genes that were used for in silico expression analysis under varying levels of N at post-anthesis stage (data for other genes was not available for in silico expression analysis). Differences in expression were also observed during qRT-PCR, when expression of four representative genes (TaNLP2, TaNLP7, TaRKD6 and TaRKD9) was examined in roots and shoots of seedlings (under different conditions of N supply) in two contrasting genotypes which differed in NUE (C306 with low NUE and HUW468 with high NUE). These four genes for qRT-PCR were selected on the basis of previous literature, level of homology and the level of expression (in silico study). In particular, the TaNLP7 gene showed significant up-regulation in the roots and shoots of HUW468 (with higher NUE) during N-starvation; this gene has already been characterized in Arabidopsis and tobacco, and is known to be involved in nitrate-signal transduction pathway.

Comparative Analysis of AGPase Genes and Encoded Proteins in Eight Monocots and Three Dicots with Emphasis on Wheat.

ADP-glucose pyrophosphorylase (AGPase) is a heterotetrameric enzyme with two large subunits (LS) and two small subunits (SS). It plays a critical role in starch biosynthesis. We are reporting here detailed structure, function and evolution of the genes encoding the LS and the SS among monocots and dicots. "True" orthologs of maize Sh2 (AGPase LS) and Bt2 (AGPase SS) were identified in seven other monocots and three dicots; structure of the enzyme at protein level was also studied. Novel findings of the current study include the following: (i) at the DNA level, the genes controlling the SS are more conserved than those controlling the LS; the variation in both is mainly due to intron number, intron length and intron phase distribution; (ii) at protein level, the SS genes are more conserved relative to those for LS; (iii) "QTCL" motif present in SS showed evolutionary differences in AGPase belonging to wheat 7BS, T. urartu, rice and sorghum, while "LGGG" motif in LS was present in all species except T. urartu and chickpea; SS provides thermostability to AGPase, while LS is involved in regulation of AGPase activity; (iv) heterotetrameric structure of AGPase was predicted and analyzed in real time environment through molecular dynamics simulation for all the species; (v) several cis-acting regulatory elements were identified in the AGPase promoters with their possible role in regulating spatial and temporal expression (endosperm and leaf tissue) and also the expression, in response to abiotic stresses; and (vi) expression analysis revealed downregulation of both subunits under conditions of heat and drought stress. The results of the present study have allowed better understanding of structure and evolution of the genes and the encoded proteins and provided clues for exploitation of variability in these genes for engineering thermostable AGPase.

Striae and pelvic relaxation: two disorders of connective tissue with a strong association.

Pelvic relaxation, a weakening of pelvic support structures, is an under-reported condition that affects a multitude of women. In the United States alone, more than 338,000 procedures for prolapse are performed annually. Decreased collagen content has been noted in the tissues of women affected by this condition. Interestingly, biopsy specimens of women with striae also show a diminution of collagen. Using self-reported anonymous data, we compared the prevalence of striae in women with and without pelvic relaxation to see if an association between these two disorders of connective tissue existed. More than half the women with prolapse (54.7%) (n = 41) reported striae, whereas only 25.0% of women in the non-prolapse group (n = 8) reported striae (P < 0.01). Multivariate logistic regression analysis confirmed striae as a significant risk factor for the development of clinical prolapse (odds ratio 3.12, P < 0.05). There appears to be a strong association between the presence of striae and the development of pelvic relaxation, which is unrelated to conventionally cited risk factors, such as age, weight, number of pregnancies, or postmenopausal status.