Rice grain yield is a complex and highly variable quantitative trait consisting of several key components, including the grain weight, the effective panicles per unit area, and the grain number per panicle (GNPP). The GNPP is a significant contributor to grain yield controlled by multiple genes (QTL) and is crucial for improvement. Attempts have been made to find genes for this trait, which has always been a challenging and arduous task through conventional methods. We combined a BSA analysis, RNA profiling, and a metabolome analysis in the present study to identify new candidate genes involved in the GNPP. The F₂ population from crossing R4233 (high GNPP) and Ce679 (low GNPP) revealed a frequency distribution fitting two segregated genes. Three pools, including low, middle, and high GNPP, were constructed and a BSA analysis revealed six candidate regions spanning 5.38 Mb, containing 739 annotated genes. Further, a conjunctive analysis of BSA-Seq and RNA-Seq showed 31 differentially expressed genes (DEGs) in the candidate intervals. Subsequently, a metabolome analysis showed 1024 metabolites, with 71 significantly enriched, including 44 up and 27 downregulated in Ce679 vs. R4233. A KEGG enrichment analysis of these 31 DEGs and 71 differentially enriched metabolites (DEMs) showed two genes, Os12g0102100 and Os01g0580500, significantly enriched in the metabolic pathways’ biosynthesis of secondary metabolites, cysteine and methionine metabolism, and fatty acid biosynthesis. Os12g0102100, which encodes for the alcohol dehydrogenase superfamily and a zinc-containing protein, is a novel gene whose contribution to the GNPP is not yet elucidated. This gene coding for mitochondrial trans-2-enoyl-CoA reductase is involved in the biosynthesis of myristic acid, also known as tetradecanoic acid. The Os01g0580500 coding for the enzyme 1-aminoclopropane-1-carboxylate oxidase (OsACO7) is responsible for the final step of the ethylene biosynthesis pathway through the conversion of 1-aminocyclopropane-1-carboxylic acid (ACC) into ethylene. Unlike Os12g0102100, this gene was significantly upregulated in R4233, downregulated in Ce679, and significantly enriched in two of the three metabolite pathways. This result pointed out that these two genes are responsible for the difference in the GNPP in the two cultivars, which has never been identified. Further validation studies may disclose the physiological mechanisms through which they regulate the GNPP in rice.

Fragrance is a valuable trait in rice varieties, with its aroma significantly influencing consumer preference. In this study, we conducted comprehensive metabolome and transcriptome analyses to elucidate the genetic and biochemical basis of fragrance in the Shangsixiangnuo (SSXN) variety, a fragrant indica rice cultivated in Guangxi, China. Through sensory evaluation and genetic analysis, we confirmed SSXN as strongly fragrant, with an 806 bp deletion in the BADH2 gene associated with fragrance production. In the metabolome analysis, a total of 238, 233, 105 and 60 metabolic compounds exhibited significant changes at the seedling (S), reproductive (R), filling (F), and maturation (M) stages, respectively. We identified four compounds that exhibited significant changes in SSXN across all four development stages. Our analyses revealed a significant upregulation of 2-acetyl-1-pyrroline (2AP), the well-studied aromatic compound, in SSXN compared to the non-fragrant variety. Additionally, correlation analysis identified several metabolites strongly associated with 2AP, including ethanone, 1-(1H-pyrrol-2-yl)-, 1H-pyrrole, and pyrrole. Furthermore, Weighted Gene Co-expression Network Analysis (WGCNA) analysis highlighted the magenta and yellow modules as particularly enriched in aroma-related metabolites, providing insights into the complex aromatic compounds underlying the fragrance of rice. In the transcriptome analysis, a total of 5582, 5506, 4965, and 4599 differential expressed genes (DEGs) were identified across the four developmental stages, with a notable enrichment of the common pathway amino sugar and nucleotide sugar metabolism in all stages. In our correlation analysis between metabolome and transcriptome data, the top three connected metabolites, phenol-, 3-amino-, and 2AP, along with ethanone, 1-(1H-pyrrol-2-yl)-, exhibited strong associations with transcripts, highlighting their potential roles in fragrance biosynthesis. Additionally, the downregulated expression of the P4H4 gene, encoding a procollagen-proline dioxygenase that specifically targets proline, in SSXN suggests its involvement in proline metabolism and potentially in aroma formation pathways. Overall, our study provides comprehensive insights into the genetic and biochemical mechanisms underlying fragrance production in rice, laying the foundation for further research aimed at enhancing fragrance quality in rice breeding programs.

Stem rot disease poses a significant challenge in passion fruit production, necessitating the identification of resistant genes for the development of stem rot resistant varieties. In this study, we conducted artificial inoculation of Fusarium solani on leaves of two passion fruit varieties, 'Huangjinguo' and 'Ziguo 7'. Leaf samples were collected at 0 h, 24 h, and 48 h post-inoculation for RNA-sequencing (RNA-seq) analysis, and 3 370, 4 464, and 3 974 differentially expressed genes (DEGs) were identified at these stages. Gene Ontology (GO) analysis revealed associations with functions such as response to reactive oxygen species (ROS), response to hydrogen peroxide, and protein complex oligomerisation. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis highlighted the enrichment of DEGs in the phenylpropanoid biosynthesis pathway, including genes such as ZX.06G0025070, ZX.01G0064640, ZX.04G0011040, ZX.05G0011380, all implicated in lignin biosynthesis. Weighted gene co-expression network analysis (WGCNA) identified three modules significantly associated with passion fruit stem rot resistance. Network analysis highlighted ZX.08G0013660 as the gene with the highest connectivity in these modules, featuring a leucine-rich repeat domain. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis further validated ZX.08G0013660 and other genes as potential candidates for passion fruit stem rot resistance. Overall, genes related to ROS, phenylpropanoid biosynthesis and leucine-rich repeat domain protein likely play critical roles in passion fruit stem rot resistance. This study provides new insights for breeding passion fruit varieties resistant to stem rot disease.

Rice is a salt-sensitive crop, and salt stress can cause significant reduction in rice yield. China's total saline area is large and growing rapidly. Therefore, it is necessary to screen for the salt-tolerant rice germplasm and cultivate these varieties. In this study, we evaluated the relative salt damage rate of 419 core germplasm of Guangxi rice landraces at seed germination stage under 1.5% NaCl salt stress, and identified the salt-tolerant loci by whole-genome association analysis. The results showed that the average germination rate under salt stress was 57.67%, which was significantly 92.55% lower than that of control group, and the Shapiro-Wilk test (0.9301) found that the distribution did not conform to normal distribution. Based on 208,993 SNP markers, 419 core germplasm was divided into 6 subgroups. We used general linear model (GLM) and mixed linear model (MLM) analysis to identify 129 and 1 significantly associated SNPs, respectively, which were distributed on chromosomes 1, 2, 3, 4, 5, 6, 8, 9, and 12. Among the 14 regions that were significantly associated with rice salt tolerance, 13 regions overlapped with previously identified or cloned salt tolerance genes. The significantly associated region Chr. 8:10,564,948-10,733,175 was reported for the first time and named qGR8. There were 53 genes in qGR8 region, 34 of which were compared with transcriptome data to obtain the expression profiles. We compared the expression profiles and speculated that LOC_Os08g17370 was a candidate gene. This gene is a member of transmembrane 9 superfamily and is up-regulated in roots and leaves of salt-tolerant parent. Thus, this gene might be a novel salt-tolerant gene in qGR8 region at germination stage in rice. These results lay a foundation for cloning new genes of salt-tolerant and provide new genetic resources for breeding salt-tolerant rice varieties.

Common gene mapping approaches mainly include QTL-mapping, BSA-seq (QTL-seq) and GWAS. BSA-seq is designed to facilitate the mapping of quantitative trait loci (QTL) in a cost-effective and high-efficiency manner. In order to accommodate the diverse species-specific traits and population genetic architectures, researchers have developed a series of tailored BSA methodologies. In this study, using six wild rices (Oryza rufipogon) as donors and the elite cultivated rice Youzhan 8 (YZ8) as the recipient, we constructed a BC₄F₈ population through hybridization and backcrossing. These six wild rice introgression lines together constitute a nested association mapping (NAM) population. Upon genotyping 1819 lines of the NAM population for the Sd1 gene, we found that among lines harboring the 383 bp deletion, 98.5–99.3% exhibited a low plant height (LP) phenotype, whereas 0.7–1.5% showed a high plant height (HP) phenotype. Based on this phenotypic segregation, we selected a total of 20 HP lines and 20 LP lines from six BC₄F₈ populations to form the H-bulk and L-bulk, respectively. Using BSA-seq, we identified a total of 33 significantly associated candidate intervals. One candidate interval located on chromosome 1 contains D18, a previously reported gene that regulates plant height. Furthermore, we preliminarily identified two major candidate genomic regions on chromosome 8 (4.60–5.76 Mb and 7.29–7.33 Mb). Integrating RNA-seq data, CAFRI-Rice online functional prediction and RT-qPCR validation, two key candidate genes, LOC_Os08g09900 and LOC_Os08g09000, were selected for subsequent functional characterization. The results of this study indicate that the NAM-BSA technology has great potential to detect QTL associated with complex traits, which can provide a novel technical strategy for the genetic dissection of complex traits in rice.

The complex traits are derived from multiple genes and exhibit a large variety of phenotypes. High-throughput sequencing technologies have become the new strategies for mapping the important traits of crops. However, these methods have their own disadvantages and limitations. Here we introduced Pair-wise Comparison Analysis for Multiple Pool-seq (PCAMP) for mapping the candidate genomic regions involved in anthocyanin biosynthesis in rice pericarp. In this protocol, the second filial generation (F ₂ ) populations obtained by crossing two parents with different target traits were divided into n (n>=3) subpopulations according to their phenotypes. Thirty phenotypically identical individuals were selected from each subpopulation and DNA samples were extracted to form a pool for sequencing. Finally, we compared the SNP-index between every two Pool-seqs to map the candidate genomic regions. We applied PCAMP to analyse F ₂ populations and successfully identified five known genes and five new candidate genomic regions for anthocyanin biosynthesis in rice pericarp. These results demonstrate that PCAMP is an efficient new method for dissecting the complex traits of crops.