Genetic Diversity and Population Structure of 93 Rice Cultivars (Lines) (Oryza Sativa Xian Group) in Qinba in China By 3 Types of Genetic Markers

Background: The Qinba region is the transition region between Indica and Japonica varieties in China. It has a long history of Indica rice planting of more than 7000 years and is also a planting area for fine-quality Indica rice. The aims of this study are to explore different genetic markers applied to the analysis population structure, genetic diversity, selection and optimization of molecular markers of Indica rice, thus providing more information for the protection and utilization on germplasm resources of Indica rice. Methods: 15 phenotypic traits, a core set of 48 SSR markers as well as SNPs data obtained by genotyping-by-sequencing (GBS, NlaIII and MseI digestion, referred to as SNPs-NlaIII and SNPs-MseI, respectively) for this panel of 93 samples using the Illumina HiSeq2000 sequencing platform, were employed to explore the genetic diversity and population structure of 93 samples.Results: The average of coefficient of variation (CV) and diversity index (He) were 29.72% and 1.83 ranging from 3.07% to 137.43%, and from 1.45 to 2.03, respectively. The correlation coefficient between 15 phenotypic traits ranged from 0.984 to -0.604. The first four PCs accounted for 70.693% phenotypic variation based on phenotypic analysis. A total of 379 alleles were obtained using SSR markers, encompassing an average of 8.0 alleles per primer. Polymorphic bands (PPB) and polymorphism information content (PIC) was 88.65% and 0.77, respectively. The Mantel test showed that the correlation between the genetic distance matrix based on SNPs-NlaIII and SNPs-MseI was the largest (R2=0.88), and that based on 15 phenotypic traits and SSR was the smallest (R2=0.09). The 93 samples could be clustered into two subgroups by 3 types of genetic markers. Molecular variance analysis revealed that the genetic variation was 2% among populations and 98% within populations (the Nm was 0.16), Tajima’s D value was 1.66, the FST between the two populations was 0.61 based on 72,824 SNPs. Conclusions: The population genetic variation explained by SNPs was larger than that explained by SSRs. The gene flow of 93 samples used in this study was larger than that of naturally self-pollinated crops, which may be caused by long-term breeding selection of Indica rice in the Qinba region. The genetic structure of the 93 samples was simple and lacked rare alleles.

COEVOLUTION WITH SPATIALLY STRUCTURED RICE LANDRACES MAINTAINS MULTIPLE GENERALIST LINEAGES IN THE RICE BLAST PATHOGEN

Traditional agrosystems, where humans, crops and microbes have coevolved over long periods, can serve as models to understand the eco-evolutionary determinants of disease dynamics and help the engineering of durably resistant agrosystems. Here, we investigated the genetic and phenotypic relationship between rice (Oryza sativa) landraces and their rice blast pathogen (Magnaporthe oryzae) in the traditional Yuanyang terraces of flooded rice paddies in China, where rice landraces have been grown and bred over centuries without significant disease outbreaks. Analyses of genetic subdivision revealed that indica rice plants clustered according to landrace names. Three new diverse lineages of rice blast specific to the Yuanyang terraces coexisted with lineages previously detected at the worldwide scale. Population subdivision in the pathogen population did not mirror pattern of population subdivision in the host. Measuring the pathogenicity of rice blast isolates on landraces revealed generalist life histories. Our results suggest that the implementation of disease control strategies based on the emergence or maintenance of a generalist lifestyle in pathogens may sustainably reduce the burden of disease in crops.

The Physicochemical Properties of Starch Are Affected by Wxlv in Indica Rice

Amylose largely determines rice grain quality profiles. The process of rice amylose biosynthesis is mainly driven by the waxy (Wx) gene, which also affects the diversity of amylose content. The present study assessed the grain quality profiles, starch fine structure, and crystallinity characteristics of the near-isogenic lines Q11(Wxlv), NIL(Wxa), and NIL(Wxb) in the indica rice Q11 background containing different Wx alleles. Q11(Wxlv) rice contained a relatively higher amylose level but very soft gel consistency and low starch viscosity, compared with rice lines carrying Wxa and Wxb. In addition, starch fine structure analysis revealed a remarkable decrease in the relative area ratio of the short amylopectin fraction but an increased amylose fraction in Q11(Wxlv) rice. Chain length distribution analysis showed that Q11(Wxlv) rice contained less amylopectin short chains but more intermediate chains, which decreased the crystallinity and lamellar peak intensity, compared with those of NIL(Wxa) and NIL(Wxb) rice. Additionally, the starches in developing grains showed different accumulation profiles among the three rice lines. Moreover, significant differences in starch gelatinization and retrogradation characteristics were observed between near-isogenic lines, which were caused by variation in starch fine structure. These findings revealed the effects of Wxlv on rice grain quality and the fine structure of starch in indica rice.

Metatranscriptomic Sequencing Suggests the Presence of Novel RNA Viruses in Rice Transmitted by Brown Planthopper

Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.

Genetic Dissection of the Breakdown of Durable Resistance in Indica Rice Variety PTB33 to Brown Planthoppers Nilaparvata Lugens (Stål)

BackgroundTo avoid and delay the resistance breakdown of varieties against pathogens and insect pests, broad-spectrum and durable resistance by multiple genes pyramiding are expected to be one of the practical approaches. The indica rice variety PTB33 (Oryza sativa L.) shows high and durable resistance to the brown planthopper (BPH, Nilaparvata lugens Stål). However, this variety gradually lost its resistance against the recent virulence development of BPH. However, breakdown processes are not fully elucidated by individual genetic loci. ResultsEffective QTLs were explored across the whole genomic region against four BPH populations collected in Japan in 1988, 1989, 1999, and 2013 using high-density single-nucleotide polymorphism (SNP) markers obtained by genotyping-by-sequencing. Among seven genomic regions of PTB33 likely conferring BPH resistance, four QTLs, qFSA4a, qFSA6, qFSA11, and qFSA12 on chromosomes 4, 6, 11, and 12, respectively, were validated as BPH resistance QTLs. The PTB33 alleles at the four QTLs positively contributed to BPH resistance. Infestation of monogenic segregating lines showed that the PTB33 alleles at qFSA11, qFSA12, and qFSA4a lost resistance effects at least in 1989, 1999, and 2013, respectively. ConclusionThis study showed breakdown of durable resistance in PTB33 was explained by step-by-step losses of genetic effects at each resistance locus and probably multiple acquisitions of virulence genes in BPH in a gene-by-gene specific manner.

Devising an Innovative, Genotype-free Transformation Protocol for Recalcitrant Indica Rice Genotypes: Samba Mahsuri and Sundarbans’ Salt-tolerant Indica Rice Cultivars White Getu and Hamilton to Establish an Ingenious Platform for CRISPR/Cas9-mediated Genome Editing

BackgroundPlant genetic transformation involves in vitro callus induction & regeneration strategies that are quintessential for introduction of novel agronomical traits employing CRISPR/Cas9-based genome editing. However, lack of effective regeneration and transformation techniques for indica rice cultivars pose as the foremost hurdle towards genetic improvement in rice crop. We devised an astounding road-map to genotype-independent and efficacious in vitro callus induction, transformation and shoot regeneration protocol that emerges as an optimal therapy, universally adaptable to invariably any rice cultivar, in order to establish an ingenious CRISPR/Cas9-based genome editing platform in this crop.ResultsWe developed a genotype-independent regeneration and transformation protocol employing mature seed-derived calli for indica rice (one mega variety- Samba Mahsuri and two salt tolerant wild genotypes- White Getu & Hamilton) genotypes to introduce important agronomical traits via CRISPR/Cas9-based genome editing system. MS- and N6-salt based media reinforced with 2,4-D (2.5 mg/L); dicamba (1.5 mg/L); TDZ (0.1 mg/L), proline (1000 mg/L), and glutamine (2.5 mg/L) exhibited highest percentage (95-98%) of embryogenic calli initiation and development. Employing this novel protocol, we achieved unparalleled regeneration efficiencies within untransformed calli (90-94%) and transformed calli (81-86%) in these recalcitrant indica genotypes and significantly enhanced number of shoots (18-20) on MS medium containing BAP (1.5 mg/L), NAA (0.5 mg/L), TDZ (1.0 mg/L), zeatin (0.2 mg/L) and proline (500 mg/L). We successfully transformed rice calli with pCAMBIA1300-based marker- free NICTK-1_pCRISPR-Cas9 vector harbouring the cassette of plant codon optimized Cas9 via biolistic approach that exhibited notably enhanced transformation efficiencies (67-69%). The integration of Cas9 gene into rice genome was validated by PCR, Southern blotting and Sanger sequencing analyses. The transgenic lines were phenotypically indistinguishable from the wild type as no significant differences in phenotypic performances were revealed between transgenic and wild type lines. ConclusionWe devised a promising, time-efficient, universally adaptable, optimal hormonal-media therapy for triggering enhanced embryogenic callus formation, regeneration and transformation efficiencies, across recalcitrant indica rice genotypes.

Optimization of selected plant growth regulators on callus induction of Oryza sativa L. var MR 219

MR 219, an indica rice from a cross between MR 137 and MR 151 is a long-grain rice variety of high-yielding, good quality of shape and taste, short maturation as well as resistance to blast and bacterial leaf blight. However, it is regarded as a recalcitrant variety due to its low ratio of callogenesis and regeneration in in vitro culturing. This would be a hindrance to crop improvements such as genetic transformation and other crop improvement methods. The objective of this study was to optimize the concentrations and combinations of plant growth regulators (PGRs) which were 2,4-Dichlorophenoxyacetic acid (2,4-D), Kinetin (KIN) and Naphthalene acetic acid (NAA) on its callus induction that ranging from 0.5 to 15.0 mg/L. The callus was induced from the mature seeds of MR 219 on the Murashige and Skoog (MS) media supplemented with 4.4 g/L of MS powder with vitamins, 30.0 g/L of sucrose and 3.5 g/L of gelrite with the addition of 2,4-D, KIN and NAA in different concentrations and combinations. All media supplemented with 2,4- D had successfully induced the callus and 2.0 mg/L of 2,4-D was the best concentration for the callus induction with a 100% success rate. The addition of 15.0 mg/L of NAA, provided less time taken for callus induction with better callus morphology which resulted in fewer browning problems.