Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio

Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.

Diversity of Purple Rice (Oryza sativa L.) Landraces in Northern Thailand

Purple rice is a functional food with health benefits and industrial potentials. In northern Thailand, purple rice landraces are grown either as wetland or upland rice, in the lowlands and highlands, in small amounts along with the staple rice. This study examined diversity of 37 accessions of purple rice collected from farmers with InDel cytoplasm markers for subspecies differentiation, 16 SSRs markers for within and between accessions variation, and selected grain quality features, namely, anthocyanin, iron, zinc, and gamma oryzanol content, when grown together as wetland rice. Most of the purple rice, originally grown as upland rice in both the lowlands and highlands, were identified as tropical japonica, except the wetland accessions from the lowlands that almost all belonged to the indica group. A high degree of genetic differentiation was found between the upland and wetland ecotypes, but none between those from the lowlands and highlands. A highland origin of the purple upland rice populations in the lowlands, possibly with adaptation to the upland rice cultivation of the tropical japonica, is suggested by the close genetic affinity between the highland and lowland populations of the upland ecotype. Grown in a much smaller area than the staple unpigmented rice, purple rice landraces are also less diverse genetically. Identification of purple rice landrace populations with exceptional anthocyanin and gamma oryzanol contents demonstrates that purple rice landraces can be a source of agronomically useful traits, while being an important cultural heritage, and contributing to the genetic diversity of the local rice germplasm.

Haplotype Variations and Evolutionary Analysis of the Granule-Bound Starch Synthase I Gene in the Korean World Rice Collection

Granule-bound starch synthase I (GBSSI) is responsible for Waxy gene encoding the, which is involved in the amylose synthesis step of starch biosynthesis. We investigated the genotypic and haplotypic variations of GBSSI (Os06g0133000) gene, including its evolutionary relatedness in the nucleotide sequence level using single-nucleotide polymorphisms (SNPs), indels, and structural variations (SVs) from 475 Korean World Rice Collection (KRICE_CORE), which comprised 54 wild rice and 421 cultivated represented by 6 ecotypes (temperate japonica, indica, tropical japonica, aus, aromatic, and admixture) or in another way by 3 varietal types (landrace, weedy, and bred). The results revealed that 27 of 59 haplotypes indicated a total of 12 functional SNPs (fSNPs), identifying 9 novel fSNPs. According to the identified novel fSNPs, we classified the entire rice collection into three groups: cultivated, wild, and mixed (cultivated and wild) rice. Five novel fSNPs were localized in wild rice: four G/A fSNPs in exons 2, 9, and 12 and one T/C fSNP in exon 13. We also identified the three previously reported fSNPs, namely, a G/A fSNP (exon 4), an A/C fSNP (exon 6), and a C/T fSNP (exon 10), which were observed only in cultivated rice, whereas an A/G fSNP (exon 4) was observed exclusively in wild rice. All-against-all comparison of four varietal types or six ecotypes of cultivated rice with wild rice showed that the GBSSI diversity was higher only in wild rice (π = 0.0056). The diversity reduction in cultivated rice can be useful to encompass the origin of this gene GBSSI during its evolution. Significant deviations of positive (wild and indica under balancing selection) and negative (temperate and tropical japonica under purifying selection) Tajima's D values from a neutral model can be informative about the selective sweeps of GBSSI genome insights. Despite the estimation of the differences in population structure and principal component analysis (PCA) between wild and subdivided cultivated subgroups, an inbreeding effect was quantified by FST statistic, signifying the genetic relatedness of GBSSI. Our findings of a novel wild fSNPS can be applicable for future breeding of waxy rice varieties. Furthermore, the signatures of selective sweep can also be of informative into further deeper insights during domestication.

Genomic divergence during feralization reveals both conserved and distinct mechanisms of parallel weediness evolution

Agricultural weeds are the most important biotic constraints to global crop production, and chief among these is weedy rice. Despite increasing yield losses from weedy rice in recent years worldwide, the genetic basis of weediness evolution remains unclear. Using whole-genome sequence analyses, we examined the origins and adaptation of Japanese weedy rice. We find evidence for a weed origin from tropical japonica crop ancestry, which has not previously been documented in surveys of weedy rice worldwide. We further show that adaptation occurs largely through different genetic mechanisms between independently-evolved temperate japonica- and tropical japonica-derived strains; most genomic signatures of positive selection are unique within weed types. In addition, some weedy rice strains have evolved through hybridization between weedy and cultivated rice with adaptive introgression from the crop. Surprisingly, introgression from cultivated rice confers not only crop-like adaptive traits (such as shorter plant height, facilitating crop mimicry) but also weedy-like traits (such as seed dormancy). These findings reveal how hybridization with cultivated rice can promote persistence and proliferation of weedy rice.

GW10, a Member of P450 Subfamily Regulates Grain Size and Grain Number in Rice

Grain size and grain number play extremely important roles in rice grain yield. Here, we identify GW10 , which encodes a P450 subfamily protein and controlls grain size and grain number by using Lemont ( tropical japonica ) as donor parent and HJX74 ( indica ) as recipient parent. The GW10 locus was mapped into a 20.1 kb region on the long arm of Chromosome 10. Lower expression of the gw10 in panicle is contributed to the shorter and narrower rice grain, and the increased number of grains per panicle. Furthermore, the higher expression levels of some of the brassinosteroid (BR) biosynthesis and response genes are associated with the NIL- GW10 , which strongly suggests that the GW10 is a key node in the brassinosteroid-mediated regulation of rice grain shape and grain number.

Epistasis and Quantitative Resistance to Pyricularia oryzae Revealed by GWAS in Advanced Rice Breeding Populations

Rice blast caused by Pyricularia oryzae is a major rice disease worldwide. Despite the detailed knowledge on major resistance genes available to date, little is known about how these genes interact with quantitative blast resistance loci and with the genetic background. Knowledge on these interactions is crucial for assessing the usefulness of introgressed resistance loci in breeding germplasm. Our goal was to identify quantitative trait loci (QTL) for blast resistance in rice breeding populations and to describe how they interact among each other and with the genetic background. To that end, resistance to blast was mapped by genome-wide association study (GWAS) in two advanced rice breeding subpopulations, one made of 305 indica type inbred lines, and the other of 245 tropical japonica inbred lines. The interactions and main effects of blast resistance loci were assessed in a multilocus model. Well known, major effect blast resistance gene clusters were detected in both tropical japonica (Pii/Pi3/Pi5) and indica (Piz/Pi2/Pi9) subpopulations with the GWAS scan 1. When these major effect loci were included as fixed cofactors in subsequent GWAS scans 2 and 3, additional QTL and more complex genetic architectures were revealed. The multilocus model for the tropical japonica subpopulation showed that Pii/Pi3/Pi5 had significant interaction with two QTL in chromosome 1 and one QTL in chromosome 8, together explaining 64% of the phenotypic variance. In the indica subpopulation a significant interaction among the QTL in chromosomes 6 and 4 and the genetic background, together with Piz/Pi2/Pi9 and QTL in chromosomes 1, 4 and 7, explained 35% of the phenotypic variance. Our results suggest that epistatic interactions can play a major role modulating the response mediated by major effect blast resistance loci such as Pii/Pi3/Pi5. Furthermore, the additive and epistatic effects of multiple QTL bring additional layers of quantitative resistance with a magnitude comparable to that of major effect loci. These findings highlight the need of genetic background-specific validation of markers for molecular assisted blast resistance breeding and provide insights for developing quantitative resistance to blast disease in rice.