To reveal the allelic differentiations at the two genes for fertility restoration (Rf) on chromosomes 1 (Rf3) and 10 (Rf4), 15 chromosome single segment substitution lines (SSSLs) with the Rf3 locus and 18 SSSLs with the Rf4 locus were crossed with Bobai A (BbA), a cytoplasmic male sterility line with wild abortive type of cytoplasm (WA-CMS), respectively. Based on the pollen and seed fertility of the F 1 hybrids, the Rf3 and Rf4 genes were each classified into four alleles, namely Rf3-1, Rf3-2, Rf3-3, and Rf3-4 for Rf3, and Rf4-1, Rf4-2, Rf4-3, and Rf4-4 for Rf4. Out of the 33 SSSLs, an SSSL W23-19-06-06-11 carrying the genotype Rf3-4Rf3-4/Rf4-4Rf4-4 possessed the strongest restoring ability for BbA. To determine the genetic effects of Rf3 and Rf4 for WA-CMS, one BC 3 F 2 population possessing the genetic background of W23-19-06-06-11 was generated from the cross between W23-19-06-06-11 and BbA by backcrossing and marker-assisted selection. In the BC 3 F 2 population, the plants carrying the Rf3Rf3/Rf4Rf4, Rf3Rf3/rf4rf4, and rf3rf3/Rf4Rf4 genotypes were selected and their phenotyping for pollen and spikelet fertility were evaluated. The result showed that under the genetic background of SSSL W23-19-06-06-11, the effect of Rf4 appeared to be slightly larger than that of Rf3 and their effects were additive for WA-CMS system. These studies will lead to the transfer of Rf genes into adapted cultivars through marker-assisted selection in active hybrid rice breeding programs.

Madhuca pasquieri (Dubard) Lam. is a tree on the International Union for Conservation of Nature Red List and a national key protected wild plant (II) of China, known for its seed oil and timber. However, lacking of genomic and transcriptome data for this species hampers study of its reproduction, utilization, and conservation. Here, single-molecule long-read sequencing (PacBio) and next-generation sequencing (Illumina) were combined to obtain the transcriptome from five developmental stages of M. pasquieri. Overall, 25,339 transcript isoforms were detected by PacBio, including 24,492 coding sequences (CDSs), 9440 simple sequence repeats (SSRs), 149 long non-coding RNAs (lncRNAs), and 182 alternative splicing (AS) events, a majority was retained intron (RI). A further 1058 transcripts were identified as transcriptional factors (TFs) from 51 TF families. PacBio recovered more full-length transcript isoforms with a longer length, and a higher expression level, whereas larger number of transcripts (124,405) was captured in de novo from Illumina. Using Nr, Swissprot, KOG, and KEGG databases, 24,405 transcripts (96.31%) were annotated by PacBio. Functional annotation revealed a role for the auxin, abscisic acid, gibberellin, and cytokinine metabolic pathways in seed germination and post-germination. These findings support further studies on seed germination mechanism and genome of M. pasquieri, and better protection of this endangered species.

The wild population of Madhuca pasquieri (Dubard) H. J. Lam is currently dwindling; its understory seedlings are rare, and there is a lack of molecular studies, which impedes the conservation of this species. This study exploited second-generation sequencing and widely targeted metabolomics analysis to uncover the dynamic changes in differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in five post-germination stages of M. pasquieri whole organism. Notably, the weighted gene co-expression network analysis (WGCNA), transcriptome, and metabolome association analyses all indicated significant enrichment of the flavonoid biosynthesis pathway in stage 4 (two-leaf), and an upregulation of the genes encoding flavonol biosynthesis in this stage. In stage 5 (nine-leaf), the flavonols were significantly accumulated, indicating that the changes in metabolites were driven at the transcript level. According to the significant changes in gene expression encoding auxin transport carriers and their correlation with flavonols during stage 5, the flavonols were speculated to have a direct inhibitory effect on the expression of PIN4 encoding gene, which may inhibit the process of polar auxin transport. The results provided important insights into the molecular network relationships between the transcription and metabolism of this rare and endangered species during the post-germination stages and explained the reasons for the slow growth of its seedlings at the molecular level.