A Natural Allele of a Transcription Factor in Rice Confers Broad-Spectrum Blast Resistance.

Grain quality improvement is a key target for rice breeders, along with yield. It is a multigenic trait that is simultaneously influenced by many factors. Over the past few decades, breeding for semi-dwarf cultivars and hybrids has significantly contributed to the attainment of high yield demands but reduced grain quality, which thus needs the attention of researchers. The availability of rice genome sequences has facilitated gene discovery, targeted mutagenesis, and revealed functional aspects of rice grain quality attributes. Some success has been achieved through the application of molecular markers to understand the genetic mechanisms for better rice grain quality; however, researchers have opted for novel strategies. Genomic alteration employing genome editing technologies (GETs) like clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for reverse genetics has opened new avenues of research in the life sciences, including for rice grain quality improvement. Currently, CRISPR/Cas9 technology is widely used by researchers for genome editing to achieve the desired biological objectives, because of its simple targeting. Over the past few years many genes that are related to various aspects of rice grain quality have been successfully edited via CRISPR/Cas9 technology. Interestingly, studies on functional genomics at larger scales have become possible because of the availability of GETs. In this review, we discuss the progress made in rice by employing the CRISPR/Cas9 editing system and its eminent applications. We also elaborate possible future avenues of research with this system, and our understanding regarding the biological mechanism of rice grain quality improvement.

https://www.mdpi.com/1422-0067/20/4/888/pdf

Applications of the CRISPR/Cas9 System for Rice Grain Quality Improvement: Perspectives and Opportunities.

Seed vigor is an important seed quality trait in rice (Oryza sativa L.) that profoundly affects seedling morphogenesis in different environments. Generally, highly vigorous seeds have many advantages in agricultural production, such as resistance to adverse stresses, rapid emergence, and yield improvement. In the present study, one germplasm collection and one recombinant inbred line (RIL) population were used to identify quantitative trait loci (QTLs) for seed vigor. A total of 19 single nucleotide polymorphisms (SNPs) were found to be significantly associated with seed vigor (−log10(P) > 6) in the germplasm collection, which consisted of 200 rice cultivars. Nine loci responsible for seed vigor were identified via QTL mapping by using a high-density bin map. To screen the candidate genes more efficiently, we selected six loci that were co-localized in GWAS and QTL mapping, overlapped previous reports, repeat detected across two seasons, or high contribution rate as reliable loci. A total of 44 differentially expressed genes were obtained from the reliable loci via gene expression profile analysis. Among these 44 genes, Os06g0108600, Os06g0110200, Os06g0253100, Os06g0282000, Os07g0583600, Os07g0592600, and Os09g0432300 were the most promising candidates associated with seed vigor.

Integrating GWAS, QTL, mapping and RNA-seq to identify candidate genes for seed vigor in rice (Oryza sativa L.)

The development of micronutrient dense rice varieties with good agronomic traits is one of the sustainable and cost-effective approaches for reducing malnutrition. Identification of QTLs for high grain Fe and Zn, yield and yield components helps in precise and faster development of high Fe and Zn rice. We carried out a three-season evaluation using IR05F102 x IR69428 derived doubled-haploid population at IRRI. Inclusive composite interval mapping was carried out using SNP markers and Best Linear Unbiased Estimates of the phenotypic traits. A total of 23 QTLs were identified for eight agronomic traits and grain Fe and Zn concentration that explained 7.2 to 22.0% PV. A QTL by environment interaction analysis confirmed the stability of nine QTLs, including two QTLs for Zn on chromosomes 5 and 12. One epistatic interaction for plant height was significant with 28.4% PVE. Moreover, five QTLs were identified for Fe and Zn that harbor several candidate genes, e.g. OsZIP6 on QTL qZn5.1. A number of QTLs were associated with a combination of greater yield and increased grain Zn levels. These results are useful for development of new rice varieties with good agronomic traits and high grain Zn using MAS, and identification of genetic resources with the novel QTLs for grain Zn.

/pdf/genetic-analysis-of-agronomic-traits-and-grain-iron-and-zinc-n94is9l0gv.pdf

Genetic Analysis of Agronomic Traits and Grain Iron and Zinc Concentrations in a Doubled Haploid Population of Rice (Oryza sativa L.).

Purple carrots can accumulate large quantities of anthocyanins in their roots and -in some genetic backgrounds- petioles, and therefore they represent an excellent dietary source of antioxidant phytonutrients. In a previous study, using linkage analysis in a carrot F2 mapping population segregating for root and petiole anthocyanin pigmentation, we identified a region in chromosome 3 with co-localized QTL for all anthocyanin pigments of the carrot root, whereas petiole pigmentation segregated as a single dominant gene and mapped to one of these "root pigmentation" regions conditioning anthocyanin biosynthesis. In the present study, we performed fine mapping combined with gene expression analyses (RNA-Seq and RT-qPCR) to identify candidate genes controlling anthocyanin pigmentation in the carrot root and petiole. Fine mapping was performed in four carrot populations with different genetic backgrounds and patterns of pigmentation. The regions controlling root and petiole pigmentation in chromosome 3 were delimited to 541 and 535 kb, respectively. Genome wide prediction of transcription factor families known to regulate the anthocyanin biosynthetic pathway coupled with orthologous and phylogenetic analyses enabled the identification of a cluster of six MYB transcription factors, denominated DcMYB6 to DcMYB11, associated with the regulation of anthocyanin biosynthesis. No anthocyanin biosynthetic genes were present in this region. Comparative transcriptome analysis indicated that upregulation of DcMYB7 was always associated with anthocyanin pigmentation in both root and petiole tissues, whereas DcMYB11 was only upregulated with pigmentation in petioles. In the petiole, the level of expression of DcMYB11 was higher than DcMYB7. DcMYB6, a gene previously suggested as a key regulator of carrot anthocyanin biosynthesis, was not consistently associated with pigmentation in either tissue. These results strongly suggest that DcMYB7 is a candidate gene for root anthocyanin pigmentation in all the genetic backgrounds included in this study. DcMYB11 is a candidate gene for petiole pigmentation in all the purple carrot sources in this study. Since DcMYB7 is co-expressed with DcMYB11 in purple petioles, the latter gene may act also as a co-regulator of anthocyanin pigmentation in the petioles. This study provides linkage-mapping and functional evidence for the candidacy of these genes for the regulation of carrot anthocyanin biosynthesis.

/pdf/a-cluster-of-myb-transcription-factors-regulates-anthocyanin-34ocg7vydh.pdf

A Cluster of MYB Transcription Factors Regulates Anthocyanin Biosynthesis in Carrot (Daucus carota L.) Root and Petiole.

Grain appearance quality is a main determinant of market value in rice and one of the highly important traits requiring improvement in breeding programs. The genetic basis of grain shape and endosperm chalkiness have been given significant attention because of their importance in affecting grain quality. Meanwhile, the introduction of NGS (Next Generation Sequencing) has a significant part to play in the area of genomics, and offers the possibility for high-resolution genetic map construction, population genetics analysis and systematic expression profile study. A RIL population derived from an inter-subspecific cross between indica rice PYZX and japonica rice P02428 was generated, based on the significant variations for the grain morphology and cytological structure between these two parents. Using the Genotyping-By-Sequencing (GBS) approach, 2711 recombination bin markers with an average physical length of 137.68 kb were obtained, and a high-density genetic map was constructed. Global genetic mapping of QTLs affecting grain shape and chalkiness traits was performed across four environments and the newly identified stable loci were obtained. Twelve important QTL clusters were detected, four of which were coincident with the genomic regions of cloned genes or fine mapped QTL reported. Eight novel QTL clusters (including six for grain shape, one for chalkiness, and one for both grain shape and chalkiness) were firstly obtained and highlighted the value and reliability of the QTL analysis. The important QTL cluster on chromosome 5 affects multiple traits including circularity (CS), grain width (GW), area size of grain (AS), percentage of grains with chalkiness (PGWC) and degree of endosperm chalkiness (DEC), indicating some potentially pleiotropic effects. The transcriptome analysis demonstrated an available gene expression profile responsible for the development of chalkiness, and several DEGs (differentially expressed genes) were co-located nearby the three chalkiness-related QTL regions on chromosomes 5, 7, and 8. Candidate genes were extrapolated, which were suitable for functional validation and breeding utilization. QTLs affecting grain shape (grain width, grain length, length-width ratio, circularity, area size of grain, and perimeter length of grain) and chalkiness traits (percentage of grains with chalkiness and degree of endosperm chalkiness) were mapped with the high-density GBS-SNP based markers. The important differentially expressed genes (DEGs) were co-located in the QTL cluster regions on chromosomes 5, 7 and 8 affecting PGWC and DEC parameters. Our research provides a crucial insight into the genetic architecture of rice grain shape and chalkiness, and acquired potential candidate loci for molecular cloning and grain quality improvement.

/pdf/high-resolution-qtl-mapping-for-grain-appearance-traits-and-pnlkk2gom4.pdf

Jiyong Zou

Papers

High-resolution QTL mapping for grain appearance traits and co-localization of chalkiness-associated differentially expressed candidate genes in rice