A prolific and robust whole-genome genotyping method using PCR amplification via primer-template mismatched annealing

Whole-genome genotyping methods are important for breeding.However,it has been challenging to develop a robust method for simultaneous foreground and background genotyping that can easily be adapted to different genes and species.In our study,we accidently discovered that in adapter ligation-mediated PCR,the amplification by primertemplate mismatched annealing(PTMA)along the genome could generate thousands of stable PCR products.Based on this observation,we consequently developed a novel method for simultaneous foreground and background integrated genotyping by sequencing(FBI-seq)using one specific primer,in which foreground genotyping is performed by primer-template perfect annealing(PTPA),while background genotyping employs PTMA.Unlike DNA arrays,multiple PCR,or genome target enrichments,FBI-seq requires little preliminary work for primer design and synthesis,and it is easily adaptable to different foreground genes and species.FBI-seq therefore provides a prolific,robust,and accurate method for simultaneous foreground and background genotyping to facilitate breeding in the postgenomics era.

A 9.5-kb deletion in the 1st intron of OsMADS51 enhances temperature sensitivity in rice

Temperature is an important environmental factor affecting heading date of rice.Despite its importance,genes responsible for temperature-sensitive heading in rice have remained elusive.Our previous study identified a quantitative trait locus qHd1 which advances heading date under high temperatures.A 9.5-kb insertion was found in the first intron of OsMADS51 in indica variety Zhenshan 97(ZS97).However,the function of this natural variant in controlling temperature sensitivity has not been verified.In this study,we used CRISPR/Cas9 to knock out the 9.5-kb insertion in ZS97.Experiments conducted under cotrolled conditions in phytotrons confirmed that deletion increased temperature sensitivity and advanced heading by downregulating the expression level of OsMADS51.One-hybrid assays in yeast,ChIP-quantitative polymerase chain reaction,electrophoretic mobility shift,and luciferase-based transient transactivation assays collectively confirmed that OsMADS51 affects heading date by regulation of heading date gene Ehd1.We further determined that the long non-coding RNA HEATINR is generated from the first intron of OsMADS51,offering an explanation for how the 9.5-kb insertion affects temperature sensitivity.We also found that OsMADS51 was strongly selected in early/late-season rice varieties in South China,possibly accounting for their strong temperature sensitivity.These insights not only advance our understanding of the molecular mechanisms underlying the temperature-responsive regulation of heading date in rice but also provide a valuable genetic target for molecular breeding.

Hairy Leaf 6, an AP2/ERF Transcription Factor, Interacts with OsWOX3B and Regulates Trichome Formation in Rice

Trichome formation has been extensively studied as a mechanistic model for epidermal cell differentiation and cell morphogenesis in plants. However, the genetic and molecular mechanisms underlying trichome formation （i.e., initiation and elongation） in rice remain largely unclear. Here, we report an AP2/ERF transcription factor, Hairy Leaf 6 （HL6）, which controls trichome formation in rice. Functional analyses revealed that HL6 transcriptionally regulates trichome elongation in rice, which is dependent on functional OsWOX3B, a homeodomain-containing protein that acts as a key regulator in trichome initiation. Biochemical and molecular genetic analyses demonstrated that HL6 physically interacts with OsWOX3B, and both of them regulate the expression of some auxin-related genes during trichome formation, in which OsWOX3B likely enhances the binding ability of HL6 with one of its direct target gene, OsYUCCA5. Popu- lation genetic analysis indicated that HL6 was under negative selection during rice domestication. Taken together, our findings provide new insights into the molecular regulatory network of trichome formation in rice.