Comparative analysis of infected cassava root transcriptomics reveals candidate genes for root rot disease resistance.

Cassava root-rot incited by soil-borne pathogens is one of the major diseases that reduces root yield. Although the use of resistant cultivars is the most effective method of management, the genetic basis for root-rot resistance remains poorly understood. Therefore, our work analyzed the transcriptome of two contrasting genotypes (BRS Kiriris/resistant and BGM-1345/susceptible) using RNA-Seq to understand the molecular response and identify candidate genes for resistance. Cassava seedlings (resistant and susceptible to root-rot) were both planted in infested and sterilized soil and samples from Initial-time and Final-time periods, pooled. Two controls were used: (i) seedlings collected before planting in infested soil (absolute control) and, (ii) plants grown in sterilized soil (mock treatments). For the differentially expressed genes (DEGs) analysis 23.912 were expressed in the resistant genotype, where 10.307 were differentially expressed in the control treatment, 15 DEGs in the Initial Time-period and 366 DEGs in the Final Time-period. Eighteen candidate genes from the resistant genotype were related to plant defense, such as the MLP-like protein 31 and the peroxidase A2-like gene. This is the first model of resistance at the transcriptional level proposed for the cassava × root-rot pathosystem. Gene validation will contribute to screening for resistance of germplasm, segregating populations and/or use in gene editing in the pursuit to develop most promising cassava clones with resistance to root-rot.

Transcriptome-wide association mapping provides insights into the genetic basis and candidate genes governing flowering, maturity and seed weight in rice bean (Vigna umbellata).

BACKGROUND: Rice bean (Vigna umbellata), an underrated legume, adapts to diverse climatic conditions with the potential to support food and nutritional security worldwide. It is used as a vegetable, minor food crop and a fodder crop, being a rich source of proteins, minerals, and essential fatty acids. However, little effort has been made to decipher the genetic and molecular basis of various useful traits in this crop. Therefore, we considered three economically important traits i.e., flowering, maturity and seed weight of rice bean and identified the associated candidate genes employing an associative transcriptomics approach on 100 diverse genotypes out of 1800 evaluated rice bean accessions from the Indian National Genebank. RESULTS: The transcriptomics-based genotyping of one-hundred diverse rice bean cultivars followed by pre-processing of genotypic data resulted in 49,271 filtered markers. The STRUCTURE, PCA and Neighbor-Joining clustering of 100 genotypes revealed three putative sub-populations. The marker-trait association analysis involving various genome-wide association study (GWAS) models revealed significant association of 82 markers on 48 transcripts for flowering, 26 markers on 22 transcripts for maturity and 22 markers on 21 transcripts for seed weight. The transcript annotation provided information on the putative candidate genes for the considered traits. The candidate genes identified for flowering include HSC80, P-II PsbX, phospholipid-transporting-ATPase-9, pectin-acetylesterase-8 and E3-ubiquitin-protein-ligase-RHG1A. Further, the WRKY1 and DEAD-box-RH27 were found to be associated with seed weight. Furthermore, the associations of PIF3 and pentatricopeptide-repeat-containing-gene with maturity and seed weight, and aldo-keto-reductase with flowering and maturity were revealed. CONCLUSION: This study offers insights into the genetic basis of key agronomic traits in rice bean, including flowering, maturity, and seed weight. The identified markers and associated candidate genes provide valuable resources for future exploration and targeted breeding, aiming to enhance the agronomic performance of rice bean cultivars. Notably, this research represents the first transcriptome-wide association study in pulse crop, uncovering the candidate genes for agronomically useful traits.

Stacked mutations in wheat homologues of rice SEMI-DWARF1 confer a novel semi-dwarf phenotype.

BACKGROUND: Semi-dwarfing alleles are used widely in cereals to confer improved lodging resistance and assimilate partitioning. The most widely deployed semi-dwarfing alleles in rice and barley encode the gibberellin (GA)-biosynthetic enzyme GA 20-OXIDASE2 (GA20OX2). The hexaploid wheat genome carries three homoeologous copies of GA20OX2, and because of functional redundancy, loss-of-function alleles of a single homoeologue would not be selected in wheat breeding programmes. Instead, approximately 70% of wheat cultivars carry gain-of-function mutations in REDUCED HEIGHT 1 (RHT1) genes that encode negative growth regulators and are degraded in response to GA. Semi-dwarf Rht-B1b or Rht-D1b alleles encode proteins that are insensitive to GA-mediated degradation. However, because RHT1 is expressed ubiquitously these alleles have pleiotropic effects that confer undesirable traits in some environments. RESULTS: We have applied reverse genetics to combine loss-of-function alleles in all three homoeologues of wheat GA20OX2 and its paralogue GA20OX1 and evaluated their performance in three years of field trials. ga20ox1 mutants exhibited a mild height reduction (approximately 3%) suggesting GA20OX1 plays a minor role in stem elongation in wheat. ga20ox2 mutants have reduced GA1 content and are 12-32% shorter than their wild-type segregants, comparable to the effect of the Rht-D1b 'Green Revolution' allele. The ga20ox2 mutants showed no significant negative effects on yield components in the spring wheat variety 'Cadenza'. CONCLUSIONS: Our study demonstrates that chemical mutagenesis can expand genetic variation in polyploid crops to uncover novel alleles despite the difficulty in identifying appropriate mutations for some target genes and the negative effects of background mutations. Field experiments demonstrate that mutations in GA20OX2 reduce height in wheat, but it will be necessary to evaluate the effect of these alleles in different genetic backgrounds and environments to determine their value in wheat breeding as alternative semi-dwarfing alleles.

Different control of resistance to two Colletotrichum orbiculare pathogenic races 0 and 1 in cucumber (Cucumis sativus L.).

KEY MESSAGE: Quantitative trait locus analysis identified independent novel loci in cucumbers responsible for resistance to races 0 and 1 of the anthracnose fungal pathogen Colletotrichum orbiculare. Cucumbers have been reported to be vulnerable to Colletotrichum orbiculare, causing anthracnose disease with significant yield loss under favorable conditions. The deployment of a single recessive Cssgr gene in cucumber breeding for anthracnose resistance was effective until a recent report on high-virulent strains infecting cucumbers in Japan conquering the resistance. QTL mapping was conducted to identify the resistance loci in the cucumber accession Ban Kyuri (G100) against C. orbiculare strains 104-T and CcM-1 of pathogenic races 0 and 1, respectively. A single dominant locus An5 was detected in the disease resistance hotspot on chromosome 5 for resistance to 104-T. Resistance to CcM-1 was governed by three loci with additive effects located on chromosomes 2 (An2) and 1 (An1.1 and An1.2). Molecular markers were developed based on variant calling between the corresponding QTL regions in the de novo assembly of the G100 genome and the publicly available cucumber genomes. Multiple backcrossed populations were deployed to fine-map An5 locus and narrow the region to approximately 222 kbp. Accumulation of An2 and An1.1 alleles displayed an adequate resistance to CcM-1 strain. This study provides functional molecular markers for pyramiding resistance loci that confer sufficient resistance against anthracnose in cucumbers.

Natural variant of Rht27, a dwarfing gene, enhances yield potential in wheat.

KEY MESSAGE: Discovery of Rht27, a dwarf gene in wheat, showed potential in enhancing grain yield by reducing plant height. Plant height plays a crucial role in crop architecture and grain yield, and semi-dwarf Reduced Height (Rht) alleles contribute to lodging resistance and were important in "Green Revolution." However, the use of these alleles is associated with some negative side effects in some environments, such as reduced coleoptile length, low nitrogen use efficiency, and reduced yield. Therefore, novel dwarf gene resources are needed to pave an alternative route to overcome these side effects. In this study, a super-dwarf mutant rht27 was obtained by the mutagenesis of G1812 (Triticum urartu, the progenitor of the A sub-genome of common wheat). Genetic analysis revealed that the dwarf phenotype was regulated by a single recessive genetic factor. The candidate region for Rht27 was narrowed to a 1.55 Mb region on chromosome 3, within which we found two potential candidate genes that showed polymorphisms between the mutant and non-mutagenized G1812. Furthermore, the natural variants and elite haplotypes of the two candidates were investigated in a natural population of common wheat. The results showed that the natural variants affect grain yield components, and the dwarf haplotypes show the potential in improving agronomic traits and grain yield. Although the mutation in Rht27 results in severe dwarf phenotype in T. urartu, the natural variants in common wheat showed desirable phenotype, which suggests that Rht27 has the potential to improve wheat yield by utilizing its weak allelic mutation or fine-tuning its expression level.

Novel PHOTOPERIOD-1 gene variants associate with yield-related and root-angle traits in European bread wheat.

KEY MESSAGE: PHOTOPERIOD-1 homoeologous gene copies play a pivotal role in regulation of flowering time in wheat. Here, we show that their influence also extends to spike and shoot architecture and even impacts root development. The sequence diversity of three homoeologous copies of the PHOTOPERIOD-1 gene in European winter wheat was analyzed by Oxford Nanopore amplicon-based multiplex sequencing and molecular markers in a panel of 194 cultivars representing breeding progress over the past 5 decades. A strong, consistent association with an average 8% increase in grain yield was observed for the PpdA1-Hap1 haplotype across multiple environments. This haplotype was found to be linked in 51% of cultivars to the 2NS/2AS translocation, originally introduced from Aegilops ventricosa, which leads to an overestimation of its effect. However, even in cultivars without the 2NS/2AS translocation, PpdA1-Hap1 was significantly associated with increased grain yield, kernel per spike and kernel per m2 under optimal growth conditions, conferring a 4% yield advantage compared to haplotype PpdA1-Hap4. In contrast to Ppd-B1 and Ppd-D1, the Ppd-A1 gene exhibits novel structural variations and a high number of SNPs, highlighting the evolutionary changes that have occurred in this region over the course of wheat breeding history. Additionally, cultivars carrying the photoperiod-insensitive Ppd-D1a allele not only exhibit earlier heading, but also deeper roots compared to those with photoperiod-sensitive alleles under German conditions. PCR and KASP assays have been developed that can be effectively employed in marker-assisted breeding programs to introduce these favorable haplotypes.

Identification and validation of stable reference genes for RT-qPCR analyses of Kobresia littledalei seedlings.

BACKGROUND: Kobreisa littledalei, belonging to the Cyperaceae family is the first Kobresia species with a reference genome and the most dominant species in Qinghai-Tibet Plateau alpine meadows. It has several resistance genes which could be used to breed improved crop varieties. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) is a popular and accurate gene expression analysis method. Its reliability depends on the expression levels of reference genes, which vary by species, tissues and environments. However, K.littledalei lacks a stable and normalized reference gene for RT-qPCR analysis. RESULTS: The stability of 13 potential reference genes was tested and the stable reference genes were selected for RT-qPCR normalization for the expression analysis in the different tissues of K. littledalei under two abiotic stresses (salt and drought) and two hormonal treatments (abscisic acid (ABA) and gibberellin (GA)). Five algorithms were used to assess the stability of putative reference genes. The results showed a variation amongst the methods, and the same reference genes showed tissue expression differences under the same conditions. The stability of combining two reference genes was better than a single one. The expression levels of ACTIN were stable in leaves and stems under normal conditions, in leaves under drought stress and in roots under ABA treatment. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable in the roots under the control conditions and salt stress and in stems exposed to drought stress. Expression levels of superoxide dismutase (SOD) were stable in stems of ABA-treated plants and in the roots under drought stress. Moreover, RPL6 expression was stable in the leaves and stems under salt stress and in the stems of the GA-treated plants. EF1-alpha expression was stable in leaves under ABA and GA treatments. The expression levels of 28 S were stable in the roots under GA treatment. In general, ACTIN and GAPDH could be employed as housekeeping genes for K. littledalei under different treatments. CONCLUSION: This study identified the best RT-qPCR reference genes for different K. littledalei tissues under five experimental conditions. ACTIN and GAPDH genes can be employed as the ideal housekeeping genes for expression analysis under different conditions. This is the first study to investigate the stable reference genes for normalized gene expression analysis of K. littledalei under different conditions. The results could aid molecular biology and gene function research on Kobresia and other related species.

Identification and expression analysis of the Xyloglucan transglycosylase/hydrolase (XTH) gene family under abiotic stress in oilseed (Brassica napus L.).

XTH genes are key genes that regulate the hydrolysis and recombination of XG components and plays role in the structure and composition of plant cell walls. Therefore, clarifying the changes that occur in XTHs during plant defense against abiotic stresses is informative for the study of the plant stress regulatory mechanism mediated by plant cell wall signals. XTH proteins in Arabidopsis thaliana was selected as the seed sequences in combination with its protein structural domains, 80 members of the BnXTH gene family were jointly identified from the whole genome of the Brassica napus ZS11, and analyzed for their encoded protein physicochemical properties, phylogenetic relationships, covariance relationships, and interoperating miRNAs. Based on the transcriptome data, the expression patterns of BnXTHs were analyzed in response to different abiotic stress treatments. The relative expression levels of some BnXTH genes under Al, alkali, salt, and drought treatments after 0, 6, 12 and 24 h were analyzed by using qRT-PCR to explore their roles in abiotic stress tolerance in B. napus. BnXTHs showed different expression patterns in response to different abiotic stress signals, indicating that the response mechanisms of oilseed rape against different abiotic stresses are also different. This paper provides a theoretical basis for clarifying the function and molecular genetic mechanism of the BnXTH gene family in abiotic stress tolerance in rapeseed.

Molecular evolution of Phytocyanin gene and analysis of expression at different coloring periods in apple (Malus domestica).

BACKGROUND: PC (phytocyanin) is a class of copper-containing electron transfer proteins closely related to plant photosynthesis, abiotic stress responses growth and development in plants, and regulation of the expression of some flavonoids and phenylpropanoids, etc., however, compared with other plants, the PC gene family has not been systematically characterized in apple. RESULTS: A total of 59 MdPC gene members unevenly distributed across 12 chromosomes were identified at the genome-wide level. The proteins of the MdPC family were classified into four subfamilies based on differences in copper binding sites and glycosylation sites: Apple Early nodulin-like proteins (MdENODLs), Apple Uclacyanin-like proteins (MdUCLs), Apple Stellacyanin-like proteins (MdSCLs), and Apple Plantacyanin-like proteins (MdPLCLs). Some MdPC members with similar gene structures and conserved motifs belong to the same group or subfamily. The internal collinearity analysis revealed 14 collinearity gene pairs among members of the apple MdPC gene. Interspecific collinearity analysis showed that apple had 31 and 35 homologous gene pairs with strawberry and grape, respectively. Selection pressure analysis indicated that the MdPC gene was under purifying selection. Prediction of protein interactions showed that MdPC family members interacted strongly with the Nad3 protein. GO annotation results indicated that the MdPC gene also regulated the biosynthesis of phenylpropanoids. Chip data analysis showed that (MdSCL3, MdSCL7 and MdENODL27) were highly expressed in mature fruits and peels. Many cis-regulatory elements related to light response, phytohormones, abiotic stresses and flavonoid biosynthetic genes regulation were identified 2000 bp upstream of the promoter of the MdPC gene, and qRT-PCR results showed that gene members in Group IV (MdSCL1/3, MdENODL27) were up-regulated at all five stages of apple coloring, but the highest expression was observed at the DAF13 (day after fruit bag removal) stage. The gene members in Group II (MdUCL9, MdPLCL3) showed down-regulated or lower expression in the first four stages of apple coloring but up-regulated and highest expression in the DAF 21 stage. CONCLUSION: Herein, one objective of these findings is to provide valuable information for understanding the structure, molecular evolution, and expression pattern of the MdPC gene, another major objective in this study was designed to lay the groundwork for further research on the molecular mechanism of PC gene regulation of apple fruit coloration.

Diversity of the Rysto gene conferring resistance to potato virus Y in wild relatives of potato.

BACKGROUND: Potato virus Y (PVY) is among the economically most damaging viral pathogen in production of potato (Solanum tuberosum) worldwide. The gene Rysto derived from the wild potato relative Solanum stoloniferum confers extreme resistance to PVY. RESULTS: The presence and diversity of Rysto were investigated in wild relatives of potato (298 genotypes representing 29 accessions of 26 tuber-bearing Solanum species) using PacBio amplicon sequencing. A total of 55 unique Rysto-like sequences were identified in 72 genotypes representing 12 accessions of 10 Solanum species and six resistant controls (potato cultivars Alicja, Bzura, Hinga, Nimfy, White Lady and breeding line PW363). The 55 Rysto-like sequences showed 89.87 to 99.98% nucleotide identity to the Rysto reference gene, and these encoded in total 45 unique protein sequences. While Rysto-like26 identified in Alicja, Bzura, White Lady and Rysto-like16 in PW363 encode a protein identical to the Rysto reference, the remaining 44 predicted Rysto-like proteins were 65.93 to 99.92% identical to the reference. Higher levels of diversity of the Rysto-like sequences were found in the wild relatives of potato than in the resistant control cultivars. The TIR and NB-ARC domains were the most conserved within the Rysto-like proteins, while the LRR and C-JID domains were more variable. Several Solanum species, including S. antipoviczii and S. hougasii, showed resistance to PVY. This study demonstrated Hyoscyamus niger, a Solanaceae species distantly related to Solanum, as a host of PVY. CONCLUSIONS: The new Rysto-like variants and the identified PVY resistant potato genotypes are potential resistance sources against PVY in potato breeding. Identification of H. niger as a host for PVY is important for cultivation of this plant, studies on the PVY management, its ecology, and migrations. The amplicon sequencing based on PacBio SMRT and the following data analysis pipeline described in our work may be applied to obtain the nucleotide sequences and analyze any full-length genes from any, even polyploid, organisms.

Cloning and functional validation of DsWRKY6 gene from Desmodium styracifolium.

The purpose of this study was to analyze the role of transcription factor in Desmodium styracifolium, proving that the DsWRKY6 transcription factor was related to the plant phenotypes of Desmodium styracifolium - cv. 'GuangYaoDa1' and it could be used in molecular-assisted breeding. 'GuangYaoDa1' was used as the material and its DNA was the template to clone DsWRKY6, the transgenic Arabidopsis thaliana line was constructed by agrobacterium tumefaciens­mediated transformation. Transgenic Arabidopsis thaliana was cultivated to study phenotype and physiological and biochemical indexes. Phenotypic observation showed that DsWRKY6 transgenic Arabidopsis thaliana had a faster growth rate while compared with the control group, they had longer lengths of main stem, lateral branches of cauline leaves, and root, but a lower number of cauline leaves and lateral branches of cauline leaves. And it also showed that their flowering and fruiting periods were advanced. The results of physiological and biochemical indexes showed that the relative expressions of DsWRKY6 increased and the abscisic acid content significantly increased in DsWRKY6 transgenic Arabidopsis thaliana compared with the control group. According to the above results, DsWRKY6 could regulate the advancing of flowering and fruiting periods caused by the improvement of abscisic acid content, and expression of the DsWRKY6 transcription factor might be the cause of the upright growth of 'GuangYaoDa1'.

Genomic identification and expression analysis of acid invertase (AINV) gene family in Dendrobium officinale Kimura et Migo.

BACKGROUND: Dendrobium officinale Kimura et Migo, a renowned traditional Chinese orchid herb esteemed for its significant horticultural and medicinal value, thrives in adverse habitats and contends with various abiotic or biotic stresses. Acid invertases (AINV) are widely considered enzymes involved in regulating sucrose metabolism and have been revealed to participate in plant responses to environmental stress. Although members of AINV gene family have been identified and characterized in multiple plant genomes, detailed information regarding this gene family and its expression patterns remains unknown in D. officinale, despite their significance in polysaccharide biosynthesis. RESULTS: This study systematically analyzed the D. officinale genome and identified four DoAINV genes, which were classified into two subfamilies based on subcellular prediction and phylogenetic analysis. Comparison of gene structures and conserved motifs in DoAINV genes indicated a high-level conservation during their evolution history. The conserved amino acids and domains of DoAINV proteins were identified as pivotal for their functional roles. Additionally, cis-elements associated with responses to abiotic and biotic stress were found to be the most prevalent motif in all DoAINV genes, indicating their responsiveness to stress. Furthermore, bioinformatics analysis of transcriptome data, validated by quantitative real-time reverse transcription PCR (qRT-PCR), revealed distinct organ-specific expression patterns of DoAINV genes across various tissues and in response to abiotic stress. Examination of soluble sugar content and interaction networks provided insights into stress release and sucrose metabolism. CONCLUSIONS: DoAINV genes are implicated in various activities including growth and development, stress response, and polysaccharide biosynthesis. These findings provide valuable insights into the AINV gene amily of D. officinale and will aid in further elucidating the functions of DoAINV genes.

High-density genetic map construction and QTL mapping of a zigzag-shaped stem trait in tea plant (Camellia sinensis).

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement.

Genome-wide expression analysis of LACS gene family implies GhLACS25 functional responding to salt stress in cotton.

BACKGROUND: Long-chain acyl-coenzyme A synthetase (LACS) is a type of acylating enzyme with AMP-binding, playing an important role in the growth, development, and stress response processes of plants. RESULTS: The research team identified different numbers of LACS in four cotton species (Gossypium hirsutum, Gossypium barbadense, Gossypium raimondii, and Gossypium arboreum). By analyzing the structure and evolutionary characteristics of the LACS, the GhLACS were divided into six subgroups, and a chromosome distribution map of the family members was drawn, providing a basis for further research classification and positioning. Promoter cis-acting element analysis showed that most GhLACS contain plant hormones (GA, MeJA) or non-biological stress-related cis-elements. The expression patterns of GhLACS under salt stress treatment were analyzed, and the results showed that GhLACS may significantly participate in salt stress response through different mechanisms. The research team selected 12 GhLACSs responsive to salt stress for tissue expression analysis and found that these genes are expressed in different tissues. CONCLUSIONS: There is a certain diversity of LACS among different cotton species. Analysis of promoter cis-acting elements suggests that GhLACS may be involved in regulating plant growth, development and stress response processes. GhLACS25 was selected for in-depth study, which confirmed its significant role in salt stress response through virus-induced gene silencing (VIGS) and induced expression in yeast cells.

Evaluation and validation of suitable reference genes for quantitative real-time PCR analysis in lotus (Nelumbo nucifera Gaertn.).

The qRT-PCR technique has been regarded as an important tool for assessing gene expression diversity. Selection of appropriate reference genes is essential for validating deviation and obtaining reliable and accurate results. Lotus (Nelumbo nucifera Gaertn) is a common aquatic plant with important aesthetic, commercial, and cultural values. Twelve candidate genes, which are typically used as reference genes for qRT-PCR in other plants, were selected for this study. These candidate reference genes were cloned with, specific primers designed based on published sequences. In particular, the expression level of each gene was examined in different tissues and growth stages of Lotus. Notably, the expression stability of these candidate genes was assessed using the software programs geNorm and NormFinder. As a result, the most efficient reference genes for rootstock expansion were TBP and UBQ. In addition, TBP and EF-1α were the most efficient reference genes in various floral tissues, while ACT and GAPDH were the most stable genes at all developmental stages of the seed. CYP and GAPDH were the best reference genes at different stages of leaf development, but TUA was the least stable. Meanwhile, the gene expression profile of NnEXPA was analyzed to confirm the validity of the findings. It was concluded that, TBP and GAPDH were identified as the best reference genes. The results of this study may help researchers to select appropriate reference genes and thus obtain credible results for further quantitative RT-qPCR gene expression analyses in Lotus.

Analysis of drought and heat stress response genes in rice using co-expression network and differentially expressed gene analyses.

Studies on Oryza sativa (rice) are crucial for improving agricultural productivity and ensuring global sustenance security, especially considering the increasing drought and heat stress caused by extreme climate change. Currently, the genes and mechanisms underlying drought and heat resistance in rice are not fully understood, and the scope for enhancing the development of new strains remains considerable. To accurately identify the key genes related to drought and heat stress responses in rice, multiple datasets from the Gene Expression Omnibus (GEO) database were integrated in this study. A co-expression network was constructed using a Weighted Correlation Network Analysis (WGCNA) algorithm. We further distinguished the core network and intersected it with differentially expressed genes and multiple expression datasets for screening. Differences in gene expression levels were verified using quantitative real-time polymerase chain reaction (PCR). OsDjC53, MBF1C, BAG6, HSP23.2, and HSP21.9 were found to be associated with the heat stress response, and it is also possible that UGT83A1 and OsCPn60a1, although not directly related, are affected by drought stress. This study offers significant insights into the molecular mechanisms underlying stress responses in rice, which could promote the development of stress-tolerant rice breeds.

A method for mining condition-specific co-expressed genes in Camellia sinensis based on k-means clustering.

BACKGROUND: As one of the world's most important beverage crops, tea plants (Camellia sinensis) are renowned for their unique flavors and numerous beneficial secondary metabolites, attracting researchers to investigate the formation of tea quality. With the increasing availability of transcriptome data on tea plants in public databases, conducting large-scale co-expression analyses has become feasible to meet the demand for functional characterization of tea plant genes. However, as the multidimensional noise increases, larger-scale co-expression analyses are not always effective. Analyzing a subset of samples generated by effectively downsampling and reorganizing the global sample set often leads to more accurate results in co-expression analysis. Meanwhile, global-based co-expression analyses are more likely to overlook condition-specific gene interactions, which may be more important and worthy of exploration and research. RESULTS: Here, we employed the k-means clustering method to organize and classify the global samples of tea plants, resulting in clustered samples. Metadata annotations were then performed on these clustered samples to determine the "conditions" represented by each cluster. Subsequently, we conducted gene co-expression network analysis (WGCNA) separately on the global samples and the clustered samples, resulting in global modules and cluster-specific modules. Comparative analyses of global modules and cluster-specific modules have demonstrated that cluster-specific modules exhibit higher accuracy in co-expression analysis. To measure the degree of condition specificity of genes within condition-specific clusters, we introduced the correlation difference value (CDV). By incorporating the CDV into co-expression analyses, we can assess the condition specificity of genes. This approach proved instrumental in identifying a series of high CDV transcription factor encoding genes upregulated during sustained cold treatment in Camellia sinensis leaves and buds, and pinpointing a pair of genes that participate in the antioxidant defense system of tea plants under sustained cold stress. CONCLUSIONS: To summarize, downsampling and reorganizing the sample set improved the accuracy of co-expression analysis. Cluster-specific modules were more accurate in capturing condition-specific gene interactions. The introduction of CDV allowed for the assessment of condition specificity in gene co-expression analyses. Using this approach, we identified a series of high CDV transcription factor encoding genes related to sustained cold stress in Camellia sinensis. This study highlights the importance of considering condition specificity in co-expression analysis and provides insights into the regulation of the cold stress in Camellia sinensis.

An interpretable model of pre-mRNA splicing for animal and plant genes.

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing.

Clpf encodes pentatricopeptide repeat protein (PPR5) and regulates pink flesh color in watermelon (Citrullus lanatus L.).

KEY MESSAGE: The gene controlling pink flesh in watermelon was finely mapped to a 55.26-kb region on chromosome 6. The prime candidate gene, Cla97C06G122120 (ClPPR5), was identified through forward genetics. Carotenoids offer numerous health benefits; while, they cannot be synthesized by the human body. Watermelon stands out as one of the richest sources of carotenoids. In this study, genetic generations derived from parental lines W15-059 (red flesh) and JQ13-3 (pink flesh) revealed the presence of the recessive gene Clpf responsible for the pink flesh (pf) trait in watermelon. Comparative analysis of pigment components and microstructure indicated that the disparity in flesh color between the parental lines primarily stemmed from variations in lycopene content, as well as differences in chromoplast number and size. Subsequent bulk segregant analysis (BSA-seq) and genetic mapping successfully narrowed down the Clpf locus to a 55.26-kb region on chromosome 6, harboring two candidate genes. Through sequence comparison and gene expression analysis, Cla97C06G122120 (annotated as a pentatricopeptide repeat, PPR) was predicted as the prime candidate gene related to pink flesh trait. To further investigate the role of the PPR gene, its homologous gene in tomato was silenced using a virus-induced system. The resulting silenced fruit lines displayed diminished carotenoid accumulation compared with the wild-type, indicating the potential regulatory function of the PPR gene in pigment accumulation. This study significantly contributes to our understanding of the forward genetics underlying watermelon flesh traits, particularly in relation to carotenoid accumulation. The findings lay essential groundwork for elucidating mechanisms governing pigment synthesis and deposition in watermelon flesh, thereby providing valuable insights for future breeding strategies aimed at enhancing fruit quality and nutritional value.

Investigating the genetic control of plant development in spring barley under speed breeding conditions.

KEY MESSAGE: This study found that the genes, PPD-H1 and ELF3, control the acceleration of plant development under speed breeding, with important implications for optimizing the delivery of climate-resilient crops. Speed breeding is a tool to accelerate breeding and research programmes. Despite its success and growing popularity with breeders, the genetic basis of plant development under speed breeding remains unknown. This study explored the developmental advancements of barley genotypes under different photoperiod regimes. A subset of the HEB-25 Nested Association Mapping population was evaluated for days to heading and maturity under two contrasting photoperiod conditions: (1) Speed breeding (SB) consisting of 22 h of light and 2 h of darkness, and (2) normal breeding (NB) consisting of 16 h of light and 8 h of darkness. GWAS revealed that developmental responses under both conditions were largely controlled by two loci: PPDH-1 and ELF3. Allelic variants at these genes determine whether plants display early flowering and maturity under both conditions. At key QTL regions, domesticated alleles were associated with late flowering and maturity in NB and early flowering and maturity in SB, whereas wild alleles were associated with early flowering under both conditions. We hypothesize that this is related to the dark-dependent repression of PPD-H1 by ELF3 which might be more prominent in NB conditions. Furthermore, by comparing development under two photoperiod regimes, we derived an estimate of plasticity for the two traits. Interestingly, plasticity in development was largely attributed to allelic variation at ELF3. Our results have important implications for our understanding and optimization of speed breeding protocols particularly for introgression breeding and the design of breeding programmes to support the delivery of climate-resilient crops.