Identification of Candidate Genes Regulating Drought Tolerance in Pearl Millet

Pearl millet is an important crop of the arid and semi-arid ecologies to sustain food and fodder production. The greater tolerance to drought stress attracts us to examine its cellular and molecular mechanisms via functional genomics approaches to augment the grain yield. Here, we studied the drought response of 48 inbreds representing four different maturity groups at the flowering stage. A set of 74 drought-responsive genes were separated into five major phylogenic groups belonging to eight functional groups, namely ABA signaling, hormone signaling, ion and osmotic homeostasis, TF-mediated regulation, molecular adaptation, signal transduction, physiological adaptation, detoxification, which were comprehensively studied. Among the conserved motifs of the drought-responsive genes, the protein kinases and MYB domain proteins were the most conserved ones. Comparative in-silico analysis of the drought genes across millet crops showed foxtail millet had most orthologs with pearl millet. Of 698 haplotypes identified across millet crops, MyC2 and Myb4 had maximum haplotypes. The protein–protein interaction network identified ABI2, P5CS, CDPK, DREB, MYB, and CYP707A3 as major hub genes. The expression assay showed the presence of common as well as unique drought-responsive genes across maturity groups. Drought tolerant genotypes in respective maturity groups were identified from the expression pattern of genes. Among several gene families, ABA signaling, TFs, and signaling proteins were the prospective contributors to drought tolerance across maturity groups. The functionally validated genes could be used as promising candidates in backcross breeding, genomic selection, and gene-editing schemes in pearl millet and other millet crops to increase the yield in drought-prone arid and semi-arid ecologies.     

Waterlogging-Stress-Responsive LncRNAs,Their Regulatory Relationships with miRNAs and Target Genes in Cucumber (Cucumis sativus L.)

Low oxygen level is a phenomenon often occurring during the cucumber cultivation period. Genes involved in adaptations to stress can be regulated by non-coding RNA. The aim was the identification of long non-coding RNAs (lncRNAs) involved in the response to long-term waterlogging stress in two cucumber haploid lines, i.e., DH2 (waterlogging tolerant—WL-T) and DH4 (waterlogging sensitive—WL-S). Plants, at the juvenile stage, were waterlogged for 7 days (non-primed, 1xH), and after a 14-day recovery period, plants were stressed again for another 7 days (primed, 2xH). Roots were collected for high-throughput RNA sequencing. Implementation of the bioinformatic pipeline made it possible to determine specific lncRNAs for non-primed and primed plants of both accessions, highlighting differential responses to hypoxia stress. In total, 3738 lncRNA molecules were identified. The highest number (1476) of unique lncRNAs was determined for non-primed WL-S plants. Seventy-one lncRNAs were depicted as potentially being involved in acquiring tolerance to hypoxia in cucumber. Understanding the mechanism of gene regulation under long-term waterlogging by lncRNAs and their interactions with miRNAs provides sufficient information in terms of adaptation to the oxygen deprivation in cucumber. To the best of our knowledge, this is the first report concerning the role of lncRNAs in the regulation of long-term waterlogging tolerance by priming application in cucumber.     

Genome-Wide Association Mapping Identifies New Candidate Genes for Cold Stress and Chilling Acclimation at Seedling Stage in Rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a chilling-sensitive staple food crop, and thus, low temperature significantly affects rice growth and yield. Many studies have focused on the cold shock of rice although chilling acclimation is more likely to happen in the field. In this paper, a genome-wide association study (GWAS) was used to identify the genes that participated in cold stress and chilling accumulation. A total of 235 significantly associated single-nucleotide polymorphisms (SNPs) were identified. Among them, we detected 120 and 88 SNPs for the relative shoot fresh weight under cold stress and chilling acclimation, respectively. Furthermore, 11 and 12 quantitative trait loci (QTLs) were identified for cold stress and chilling acclimation, respectively, by integrating the co-localized SNPs. Interestingly, we identified 10 and 15 candidate genes in 11 and 12 QTLs involved in cold stress and chilling acclimation, respectively, and two new candidate genes (LOC_Os01g62410, LOC_Os12g24490) were obviously up-regulated under chilling acclimation. Furthermore, OsMYB3R-2 (LOC_Os01g62410) that encodes a R1R2R3 MYB gene was associated with cold tolerance, while a new C3HC4-type zinc finger protein-encoding gene LOC_Os12g24490 was found to function as a putative E3 ubiquitin-protein ligase in rice. Moreover, haplotype, distribution, and Wright’s fixation index (FST) of both genes showed that haplotype 3 of LOC_Os12g24490 is more stable in chilling acclimation, and the SNP (A > T) showed a difference in latitudinal distribution. FST analysis of SNPs in OsMYB3R-2 (LOC_Os01g62410) and LOC_Os12g24490 indicated that several SNPs were under selection in rice indica and japonica subspecies. This study provided new candidate genes in genetic improvement of chilling acclimation response in rice.     

Genome-Wide Identification and Characterization of the CC-NBS-LRR Gene Family in Cucumber (Cucumis sativus L.)

The NBS-LRR (NLR) gene family plays a pivotal role in regulating disease defense response in plants. Cucumber is one of the most important vegetable crops in the world, and various plant diseases, including powdery mildew (PM), cause severe losses in both cucumber productivity and quality annually. To characterize and understand the role of the CC-NBS-LRR(CNL) family of genes in disease defense response in cucumber plants, we performed bioinformatical analysis to characterize these genes systematically. We identified 33 members of the CNL gene family in cucumber plants, and they are distributed on each chromosome with chromosome 4 harboring the largest cluster of five different genes. The corresponding CNL family member varies in the number of amino acids and exons, molecular weight, theoretical isoelectric point (pI) and subcellular localization. Cis-acting element analysis of the CNL genes reveals the presence of multiple phytohormone, abiotic and biotic responsive elements in their promoters, suggesting that these genes might be responsive to plant hormones and stress. Phylogenetic and synteny analysis indicated that the CNL proteins are conserved evolutionarily in different plant species, and they can be divided into four subfamilies based on their conserved domains. MEME analysis and multiple sequence alignment showed that conserved motifs exist in the sequence of CNLs. Further DNA sequence analysis suggests that CsCNL genes might be subject to the regulation of different miRNAs upon PM infection. By mining available RNA-seq data followed by real-time quantitative PCR (qRT-PCR) analysis, we characterized expression patterns of the CNL genes, and found that those genes exhibit a temporospatial expression pattern, and their expression is also responsive to PM infection, ethylene, salicylic acid, and methyl jasmonate treatment in cucumber plants. Finally, the CNL genes targeted by miRNAs were predicted in cucumber plants. Our results in this study provided some basic information for further study of the functions of the CNL gene family in cucumber plants.     

CsSWEET2, a Hexose Transporter from Cucumber (Cucumis sativus L.), Affects Sugar Metabolism and Improves Cold Tolerance in Arabidopsis

Sugars, which are critical osmotic compounds and signalling molecules in plants, and Sugars Will Eventually be Exported Transporters (SWEETs), which constitute a novel family of sugar transporters, play central roles in plant responses to multiple abiotic stresses. In the present study, a member of the SWEET gene family from cucumber (Cucumis sativus L.), CsSWEET2, was identified and characterized. Histochemical analysis of β-glucuronidase expression in transgenic Arabidopsis plants showed that CsSWEET2 is highly expressed in the leaves; subcellular localization indicated that CsSWEET2 proteins are localized in the plasma membrane and endoplasmic reticulum. Heterologous expression assays in yeast demonstrated that CsSWEET2 encodes an energy-independent hexose/H⁺ uniporter that can complement both glucose and fructose transport deficiencies. Compared with wild-type Arabidopsis plants, transgenic Arabidopsis plants overexpressing CsSWEET2 had much lower relative electrolyte leakage levels and were much more resistant to cold stress. Sugar content analysis showed that glucose and fructose levels in the transgenic Arabidopsis plants were significantly higher than those in the wild-type plants. Taken together, our results suggest that, by mediating sugar metabolism and compartmentation, CsSWEET2 plays a vital role in improving plant cold tolerance.     

The Formation of Hollow Trait in Cucumber (Cucumis sativus L.) Fruit Is Controlled by CsALMT2

The hollow trait is crucial for commercial quality of cucumber (Cucumis sativus L.) fruit, and its molecular regulatory mechanism is poorly understood due to its environmental sensitivity. In the previous research, we obtained the hollow and the non-hollow materials of ecotype cucumbers of South China, which were not easily affected by the external environment through a systematic breeding method. In this study, first, we proposed to use the percentage of the hollow area as the criterion to compare the hollow characteristics between two materials, and to analyze the formation mechanism of early hollow trait from the perspective of cytology. The results showed that the hollow trait occurred in the early stage of fruit development, and formed with the opening of carpel ventral zipped bi-cell layer, which formed rapidly from 2 to 4 days, and then slowed to a constant rate from 14 to 16 days. Meanwhile, the different genetic populations were constructed using these materials, and fine mapping was performed by bulked segregant analysis (BSA) and kompetitive allele specific PCR (KASP) method. The Csa1G630860 (CsALMT2), encoding protein ALMT2, was determined as a candidate gene for regulating the hollow trait in fruit. Furthermore, the expression profile of CsALMT2 was analyzed by qRT-PCR and fluorescence in situ hybridization. The expression of CsALMT2 had obvious tissue specificity, and it was abundantly expressed in the ovule development zone inside the fruit. In the hollow material of cucumber fruit, the expression of CsALMT2 was significantly downregulated. The subcellular localization in tobacco leaves indicated that CsALMT2 was distributed on the plasma membrane. In conclusion, in this study, for the first time, we found the regulatory gene of hollow trait in cucumber fruit, which laid the foundation for subsequent research on the molecular mechanism of hollow trait formation in cucumber fruit, and made it possible to apply this gene in cucumber breeding.     

Genome-Wide Association Study on Root System Architecture and Identification of Candidate Genes in Wheat (Triticum aestivum L.)

The root tissues play important roles in water and nutrient acquisition, environmental adaptation, and plant development. In this study, a diversity panel of 388 wheat accessions was collected to investigate nine root system architecture (RSA) traits at the three-leaf stage under two growing environments: outdoor pot culture (OPC) and indoor pot culture (IPC). Phenotypic analysis revealed that root development was faster under OPC than that under IPC and a significant correlation was observed between the nine RSA traits. The 660K single-nucleotide polymorphism (SNP) chip was used for a genome-wide association study (GWAS). Significant SNPs with a threshold of −log₁₀ (p-value) ≥ 4 were considered. Thus, 36 quantitative trait loci (QTLs), including 13 QTL clusters that were associated with more than one trait, were detected, and 31 QTLs were first identified. The QTL clusters on chromosomes 3D and 5B were associated with four and five RSA traits, respectively. Two candidate genes, TraesCS2A01G516200 and TraesCS7B01G036900, were found to be associated with more than one RSA trait using haplotype analysis, and preferentially expressed in the root tissues. These favourable alleles for RSA traits identified in this study may be useful to optimise the root system in wheat.     

Identification of Candidate Genes for Root Traits Using Genotype–Phenotype Association Analysis of Near-Isogenic Lines in Hexaploid Wheat (Triticum aestivum L.)

Global wheat (Triticum aestivum L.) production is constrained by different biotic and abiotic stresses, which are increasing with climate change. An improved root system is essential for adaptability and sustainable wheat production. In this study, 10 pairs of near-isogenic lines (NILs)—targeting four genomic regions (GRs) on chromosome arms 4BS, 4BL, 4AS, and 7AL of hexaploid wheat—were used to phenotype root traits in a semi-hydroponic system. Seven of the 10 NIL pairs significantly differed between their isolines for 11 root traits. The NIL pairs targeting qDSI.4B.1 GR varied the most, followed by the NIL pair targeting qDT.4A.1 and QHtscc.ksu-7A GRs. For pairs 5–7 targeting qDT.4A.1 GR, pair 6 significantly differed in the most root traits. Of the 4 NIL pairs targeting qDSI.4B.1 GR, pairs 2 and 4 significantly differed in 3 and 4 root traits, respectively. Pairs 9 and 10 targeting QHtscc.ksu-7A GR significantly differed in 1 and 4 root traits, respectively. Using the wheat 90K Illumina iSelect array, we identified 15 putative candidate genes associated with different root traits in the contrasting isolines, in which two UDP-glycosyltransferase (UGT)-encoding genes, TraesCS4A02G185300 and TraesCS4A02G442700, and a leucine-rich repeat receptor-like protein kinase (LRR-RLK)-encoding gene, TraesCS4A02G330900, also showed important functions for root trait control in other crops. This study characterized, for the first time, that these GRs control root traits in wheat, and identified candidate genes, although the candidate genes will need further confirmation and validation for marker-assisted wheat breeding.     

Integrative Analysis of Metabolomic,Proteomic and Genomic Data to Reveal Functional Pathways and Candidate Genes for Drip Loss in Pigs

The aim of this study was to integrate multi omics data to characterize underlying functional pathways and candidate genes for drip loss in pigs. The consideration of different omics levels allows elucidating the black box of phenotype expression. Metabolite and protein profiling was applied in Musculus longissimus dorsi samples of 97 Duroc × Pietrain pigs. In total, 126 and 35 annotated metabolites and proteins were quantified, respectively. In addition, all animals were genotyped with the porcine 60 k Illumina beadchip. An enrichment analysis resulted in 10 pathways, amongst others, sphingolipid metabolism and glycolysis/gluconeogenesis, with significant influence on drip loss. Drip loss and 22 metabolic components were analyzed as intermediate phenotypes within a genome-wide association study (GWAS). We detected significantly associated genetic markers and candidate genes for drip loss and for most of the metabolic components. On chromosome 18, a region with promising candidate genes was identified based on SNPs associated with drip loss, the protein “phosphoglycerate mutase 2” and the metabolite glycine. We hypothesize that association studies based on intermediate phenotypes are able to provide comprehensive insights in the genetic variation of genes directly involved in the metabolism of performance traits. In this way, the analyses contribute to identify reliable candidate genes.     

miRNA Profiling and Its Role in Multi-Omics Regulatory Networks Connected with Somaclonal Variation in Cucumber (Cucumis sativus L.)

The role of miRNAs in connection with the phenomenon of somaclonal variation, which occurs during plant in vitro culture, remains uncertain. This study aims to investigate the possible role of miRNAs in multi-omics regulatory pathways in cucumber somaclonal lines. For this purpose, we performed sRNA sequencing (sRNA-seq) from cucumber fruit samples identified 8, 10 and 44 miRNAs that are differentially expressed between somaclones (S1, S2, S3 lines) and the reference B10 line of Cucumis sativus. For miRNA identification, we use ShortStack software designed to filter miRNAs from sRNAs according to specific program criteria. The identification of predicted in-silico targets revealed 2,886 mRNAs encoded by 644 genes. The functional annotation of miRNA’s target genes and gene ontology classification revealed their association with metabolic processes, response to stress, multicellular organism development, biosynthetic process and catalytic activity. We checked with bioinformatic analyses for possible interactions at the level of target proteins, differentially expressed genes (DEGs) and genes affected by genomic polymorphisms. We assume that miRNAs can indirectly influence molecular networks and play a role in many different regulatory pathways, leading to somaclonal variation. This regulation is supposed to occur through the process of the target gene cleavage or translation inhibition, which in turn affects the proteome, as we have shown in the example of molecular networks. This is a new approach combining levels from DNA-seq through mRNA-seq, sRNA-seq and in silico PPI in the area of plants’ somaclonal variation.     

Insights into the Genomic Regions and Candidate Genes of Senescence-Related Traits in Upland Cotton via GWAS

Senescence is the last stage of plant development and is controlled by both internal and external factors. Premature senescence significantly affects the yield and quality of cotton. However, the genetic architecture underlying cotton senescence remains unclear. In this study, genome-wide association studies (GWAS) were performed based on 3,015,002 high-quality SNP markers from the resequencing data of 355 upland cotton accessions to detect genomic regions for cotton senescence. A total of 977 candidate genes within 55 senescence-related genomic regions (SGRs), SGR1–SGR55, were predicted. Gene ontology (GO) analysis of candidate genes revealed that a set of biological processes was enriched, such as salt stress, ethylene processes, and leaf senescence. Furthermore, in the leaf senescence GO term, one candidate gene was focused on: Gohir.A12G270900 (GhMKK9), located in SGR36, which encodes a protein of the MAP kinase kinase family. Quantitative real-time PCR (qRT-PCR) analysis showed that GhMKK9 was up-regulated in old cotton leaves. Overexpression of GhMKK9 in Arabidopsis accelerated natural leaf senescence. Virus-induced gene silencing (VIGS) of GhMKK9 in cotton increased drought tolerance. These results suggest that GhMKK9 is a positive regulator and might be involved in drought-induced senescence in cotton. The results provide new insights into the genetic basis of cotton senescence and will be useful for improving cotton breeding in the future.     

CsPAO2 Improves Salt Tolerance of Cucumber through the Interaction with CsPSA3 by Affecting Photosynthesis and Polyamine Conversion

Polyamine oxidases (PAOs) are key enzymes in polyamine metabolism and are related to the tolerance of plants to abiotic stresses. In this study, overexpression of cucumber (Cucumis sativus L.) PAO2 (CsPAO2) in Arabidopsis resulted in increased activity of the antioxidant enzyme and accelerated conversion from Put to Spd and Spm, while malondialdehyde content (MDA) and electrolyte leakage (EL) was decreased when compared with wild type, leading to enhanced plant growth under salt stress. Photosystem Ⅰ assembly 3 in cucumber (CsPSA3) was revealed as an interacting protein of CsPAO2 by screening yeast two-hybrid library combined with in vitro and in vivo methods. Then, CsPAO2 and CsPSA3 were silenced in cucumber via virus-mediated gene silencing (VIGS) with pV190 as the empty vector. Under salt stress, net photosynthetic rate (Pn) and transpiration rate (Tr) of CsPAO2-silencing plants were lower than pV190-silencing plants, and EL in root was higher than pV190-silencing plants, indicating that CsPAO2-silencing plants suffered more serious salt stress damage. However, photosynthetic parameters of CsPSA3-silencing plants were all higher than those of CsPAO2 and pV190-silencing plants, thereby enhancing the photosynthesis process. Moreover, CsPSA3 silencing reduced the EL in both leaves and roots when compared with CsPAO2-silencing plants, but the EL only in leaves was significantly lower than the other two gene-silencing plants, and conversion from Put to Spd and Spm in leaf was also promoted, suggesting that CsPSA3 interacts with CsPAO2 in leaves to participate in the regulation of salt tolerance through photosynthesis and polyamine conversion.     

Relationship between Key Environmental Factors and the Architecture of Fruit Shape and Size in Near-Isogenic Lines of Cucumber (Cucumis sativus L.)

Fruit shape and size are complex traits influenced by numerous factors, especially genetics and environment factors. To explore the mechanism of fruit shape and size development in cucumber, a pair of near-isogenic lines (NIL) Ln35 and Ln37 were used. The fruit length and diameter, cell length and diameter, and related gene expression were measured. Both the fruit length, diameter, and cell length and diameter showed sigmate curves in the two lines. The cell length and diameter were significantly positively correlated with fruit length and diameter both in two lines. The expression of CsACS2 and CsLNG showed significant positive correlations with fruit length and diameter increment in Ln35, and there was no correlation in Ln37. Furthermore, there were significant positive correlations between fruit size and thermal effectiveness (TE), as well as between fruit size and photosynthetic active radiation (PAR), both in two lines. Two models using logistic regression were formulated to assess the relationships among fruit length and diameter in Ln35 and Ln37, respectively, based on thermal effectiveness and photosynthetic active radiation (TEP). The coefficient R² values of the models were 0.977 and 0.976 in Ln35, and 0.987 and 0.981 in Ln37, respectively. The root mean square error (RMSE) was 12.012 mm and 4.338 mm in Ln35, and 5.17 mm and 7.082 mm in Ln37, respectively, which illustrated the accurate and efficient of these models. These biologically interpreted parameters will provide precision management for monitoring fruit growth and forecasting the time of harvesting under different temperatures and light conditions.     

Genome-Wide Association Studies of Seven Root Traits in Soybean (Glycine max L.) Landraces

Soybean [Glycine max (L.) Merr.], an important oilseed crop, is a low-cost source of protein and oil. In Southeast Asia and Africa, soybeans are widely cultivated for use as traditional food and feed and industrial purposes. Given the ongoing changes in global climate, developing crops that are resistant to climatic extremes and produce viable yields under predicted climatic conditions will be essential in the coming decades. To develop such crops, it will be necessary to gain a thorough understanding of the genetic basis of agronomic and plant root traits. As plant roots generally lie beneath the soil surface, detailed observations and phenotyping throughout plant development present several challenges, and thus the associated traits have tended to be ignored in genomics studies. In this study, we phenotyped 357 soybean landraces at the early vegetative (V2) growth stages and used a 180 K single-nucleotide polymorphism (SNP) soybean array in a genome-wide association study (GWAS) conducted to determine the phenotypic relationships among root traits, elucidate the genetic bases, and identify significant SNPs associated with root trait-controlling genomic regions/loci. A total of 112 significant SNP loci/regions were detected for seven root traits, and we identified 55 putative candidate genes considered to be the most promising. Our findings in this study indicate that a combined approach based on SNP array and GWAS analyses can be applied to unravel the genetic basis of complex root traits in soybean, and may provide an alternative high-resolution marker strategy to traditional bi-parental mapping. In addition, the identified SNPs, candidate genes, and diverse variations in the root traits of soybean landraces will serve as a valuable basis for further application in genetic studies and the breeding of climate-resilient soybeans characterized by improved root traits.