Development and verification of SSR markers from drought stress-responsive miRNAs in Dongxiang wild rice (Oryza rufipogon Griff.).

Rice production worldwide has continued to decline due to various environmental stresses, with drought stress being a prominent factor, as rice is a semi-aquatic plant. Thus, development of drought stress-resistant rice varieties is of great importance for rice production. In our previous study, we found that microRNAs (miRNAs) play a crucial role in the response to drought stress in Dongxiang wild rice (DXWR) (Oryza rufipogon Griff.). Developing drought stress-responsive miRNA-based single sequence repeat (SSR) markers for DXWR will be of great value for the efficient identification and utilization of miRNA genes to breed drought stress-resistant rice varieties. In this study, ninety-nine novel SSR markers were developed based on the drought stress-responsive miRNAs of DXWR. These markers were distributed in all 12 rice chromosomes, and most were in chromosomes 2 and 6, with di- and tri-nucleotides being the most abundant repeat motifs. Twelve out of fourteen synthesized markers displayed high levels of genetic diversity in the genomes of three populations of DXWR and 40 modern rice varieties worldwide. The number of alleles per locus ranged from 2 to 7, with an average of 4.67; the genetic diversity index ranged from 0.21 to 0.76, with an average of 0.58; and the polymorphism information content value ranged from 0.18 to 0.72, with an average of 0.53. These novel molecular markers developed from the drought stress-responsive miRNAs of DXWR could be additional tools for mapping elite miRNA genes and breeding drought stress-resistant rice varieties.

Development and Characterization of Chromosome Segment Substitution Lines Derived from Oryza rufipogon in the Background of the Oryza sativa indica Restorer Line R974

Dongxiang wild rice (DXWR) (O. rufipogon Griff.), which has the northernmost worldwide distribution of a wild rice species, is a valuable genetic resource with respect to improving stress tolerance in cultivated rice (Oryza sativa L.). In the three-line hybrid rice breeding system, restorer lines play important roles in enhancing the tolerance of hybrid rice. However, restorer lines have yet to be used as a genomic background for development of substitution lines carrying DXWR chromosome segments. We developed a set of 84 chromosome segment substitution lines (CSSLs) from a donor parent DXWR × recurrent parent restorer line R974 (Oryza sativa indica) cross. On average, each CSSL carried 6.27 introgressed homozygous segments, with 93.37% total genome coverage. Using these CSSLs, we identified a single QTL, qDYST-1, associated with salt stress tolerance on chromosome 3. Furthermore, five CSSLs showing strong salt stress tolerance were subjected to whole-genome single-nucleotide polymorphism chip analyses, during which we detected a common substitution segment containing qDYST-1 in all five CSSLs, thereby implying the validity and efficacy of qDYST-1. These novel CSSLs could make a significant contribution to detecting valuable DXWR QTLs, and provide important germplasm resources for breeding novel restorer lines for use in hybrid rice breeding systems.

Identification, Analysis, and Confirmation of Seed Storability-Related Loci in Dongxiang Wild Rice (Oryza rufipogon Griff.).

Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) has strong seed storability and identifying its elite gene resources may facilitate genetic improvements in rice seed storability. In this study, we developed two backcross inbred lines (BILs) populations, with DXWR as a common donor parent and two rice varieties (F6 and R974) as recipient parents. Bulked segregant analysis via whole genome sequencing (BSA-seq) was used to identify seed storability-related loci in the DXWR and F6 population. Two main genomic regions containing 18,550,000-20,870,000 bp on chromosome 4 and 7,860,000-9,780,000 bp on chromosome 9 were identified as candidate loci of DXWR seed storability; these overlapped partially with seed storability-related quantitative trait loci (QTLs) discovered in previous studies, suggesting that these loci may provide important regions for isolating the responsible genes. In total, 448 annotated genes were predicted within the identified regions, of which 274 and 82 had nonsynonymous and frameshift mutations, respectively. We detected extensive metabolic activities and cellular processes during seed storability and confirmed the effects of the seed storability-related candidate loci using four BILs from DXWR and R974. These results may facilitate the cloning of DXWR seed storability-related genes, thereby elucidating rice seed storability and its improvement potential.