Exploring Genetic Diversity within aus Rice Germplasm: Insights into the Variations in Agro-morphological Traits.

The aus (Oryza sativa L.) varietal group comprises of aus, boro, ashina and rayada seasonal and/or field ecotypes, and exhibits unique stress tolerance traits, making it valuable for rice breeding. Despite its importance, the agro-morphological diversity and genetic control of yield traits in aus rice remain poorly understood. To address this knowledge gap, we investigated the genetic structure of 181 aus accessions using 399,115 SNP markers and evaluated them for 11 morpho-agronomic traits. Through genome-wide association studies (GWAS), we aimed to identify key loci controlling yield and plant architectural traits.Our population genetic analysis unveiled six subpopulations with strong geographical patterns. Subpopulation-specific differences were observed in most phenotypic traits. Principal component analysis (PCA) of agronomic traits showed that principal component 1 (PC1) was primarily associated with panicle traits, plant height, and heading date, while PC2 and PC3 were linked to primary grain yield traits. GWAS using PC1 identified OsSAC1 on Chromosome 7 as a significant gene influencing multiple agronomic traits. PC2-based GWAS highlighted the importance of OsGLT1 and OsPUP4/ Big Grain 3 in determining grain yield. Haplotype analysis of these genes in the 3,000 Rice Genome Panel revealed distinct genetic variations in aus rice.In summary, this study offers valuable insights into the genetic structure and phenotypic diversity of aus rice accessions. We have identified significant loci associated with essential agronomic traits, with GLT1, PUP4, and SAC1 genes emerging as key players in yield determination.

Phenotypic and Genotypic screening of fifty-two rice (Oryza sativa L.) genotypes for desirable cultivars against blast disease.

Magnaporthe oryzae, the rice blast fungus, is one of the most dangerous rice pathogens, causing considerable crop losses around the world. In order to explore the rice blast-resistant sources, initially performed a large-scale screening of 277 rice accessions. In parallel with field evaluations, fifty-two rice accessions were genotyped for 25 major blast resistance genes utilizing functional/gene-based markers based on their reactivity against rice blast disease. According to the phenotypic examination, 29 (58%) and 22 (42%) entries were found to be highly resistant, 18 (36%) and 29 (57%) showed moderate resistance, and 05 (6%) and 01 (1%), respectively, were highly susceptible to leaf and neck blast. The genetic frequency of 25 major blast resistance genes ranged from 32 to 60%, with two genotypes having a maximum of 16 R-genes each. The 52 rice accessions were divided into two groups based on cluster and population structure analysis. The highly resistant and moderately resistant accessions are divided into different groups using the principal coordinate analysis. According to the analysis of molecular variance, the maximum diversity was found within the population, while the minimum diversity was found between the populations. Two markers (RM5647 and K39512), which correspond to the blast-resistant genes Pi36 and Pik, respectively, showed a significant association to the neck blast disease, whereas three markers (Pi2-i, Pita3, and k2167), which correspond to the blast-resistant genes Pi2, Pita/Pita2, and Pikm, respectively, showed a significant association to the leaf blast disease. The associated R-genes might be utilized in rice breeding programmes through marker-assisted breeding, and the identified resistant rice accessions could be used as prospective donors for the production of new resistant varieties in India and around the world.

Multilocus sequence analysis and identification of mating-type idiomorphs distribution in Magnaporthe oryzae population of Karnataka state of India.

AIMS: To investigate the genetic diversity, population structure and mating-type distribution among the eco-distinct isolates of Magnaporthe oryzae from Karnataka, India. METHODS AND RESULTS: A set of 38 isolates of M. oryzae associated with leaf blast disease of rice were collected from different rice ecosystems of Karnataka, India, and analysed for their diversity at actin, ß-tubulin, calmodulin, translation elongation factor 1-α (TEF-1-α), and internal transcribed spacer (ITS) genes/region. The isolates were grouped into two clusters based on the multilocus sequence diversity, the majority being in cluster-IA (n = 37), and only one isolate formed cluster-IB. Population structure was analysed using 123 SNP data to understand the genetic relationship. Based on K = 2 and ancestry threshold of >70%, blast strains were classified into two subgroups (SG1 and SG2) whereas, based on K = 4 and ancestry threshold of >70%, blast strains were classified into four subgroups (SG1, SG2, SG3 and SG4). We have identified 13 haplotype groups where haplotype group 2 was predominant (n = 20) in the population. The Tajima's and Fu's Fs neutrality tests exhibited many rare alleles. Further, the mating-type analysis was also performed using MAT1 gene-specific primers to find the potentiality of sexual reproduction in different ecosystems. The majority of the isolates (54.5%) had MAT1-2 idiomorph, whereas 45.5% of the isolates possessed MAT1-1 idiomorph. CONCLUSIONS: The present study found the genetically homogenous population of M. oryzae by multilocus sequence analysis. Both mating types, MAT1-1 and MAT1-2, were found within the M. oryzae population of Karnataka. SIGNIFICANCE AND IMPACT OF STUDY: The study on the population structure and sexual mating behaviour of M. oryzae is important in developing region-specific blast-resistant rice cultivars. This is the first report of MAT1 idiomorphs distribution in the M. oryzae population in any Southern state of India.

Morpho-molecular diversity and avirulence genes distribution among the diverse isolates of Magnaporthe oryzae from Southern India.

AIMS: To investigate the diversity of eco-distinct isolates of Magnaporthe oryzae for their morphological, virulence and molecular diversity and relative distribution of five Avr genes. METHODS AND RESULTS: Fifty-two M. oryzae isolates were collected from different rice ecosystems of southern India. A majority of them (n = 28) formed a circular colony on culture media. Based on the disease reaction on susceptible cultivar (cv. HR-12), all 52 isolates were classified in to highly virulent (n = 28), moderately virulent (n = 11) and less-virulent (13) types. Among the 52 isolates, 38 were selected for deducing internal transcribed spacer (ITS) sequence diversity. For deducing phylogeny, another set of 36 isolates from other parts of the world was included, which yielded two distinct phylogenetic clusters. We identified eight haplotype groups and 91 variable sites within the ITS sequences, and haplotype-group-2 (Hap_2) was predominant (n = 24). The Tajima's and Fu's Fs neutrality tests exhibited many rare alleles. Furthermore, PCR analysis for detecting the presence of five Avr genes in the different M. oryzae isolates using Avr gene-specific primers in PCR revealed that Avr-Piz-t, Avr-Pik, Avr-Pia and Avr-Pita were present in 73.68%, 73.68%, 63.16% and 47.37% of the isolates studied, respectively; whereas, Avr-Pii was identified only in 13.16% of the isolates. CONCLUSIONS: Morpho-molecular and virulence studies revealed the significant diversity among eco-distinct isolates. PCR detection of Avr genes among the M. oryzae population revealed the presence of five Avr genes. Among them, Avr-Piz-t, Avr-Pik and Avr-Pia were more predominant. SIGNIFICANCE AND IMPACT OF THE STUDY: The study documented the morphological and genetic variability of eco-distinct M. oryzae isolates. This is the first study demonstrating the distribution of the Avr genes among the eco-distinct population of M. oryzae from southern India. The information generated will help plant breeders to select appropriate resistant gene/s combinations to develop blast disease-resistant rice cultivars.

A single nucleotide substitution in the SPDT transporter gene reduced phytic acid and increased mineral bioavailability from Rice grain (Oryza sativa L.).

Phosphorus (P) flow in agricultural land depends on the P taken off from harvested product, its losses through runoff and fertilizer applied to balance the removed P. Phytic acid (PA), the major storage form of phosphorus (P) in cereal grains is a key anti-nutrient for human and non-ruminants leads to eutrophication of waterways. As the natural non-renewable P reserves are limited, enhancing P use efficiency is needed for field crops. SULTR-like phosphorus distribution transporter (SPDT) is a novel rice transporter transfer P to the grain. Any alteration in transporter gene reduce grain P with concomitant rise in the leaves. A low PA (3.0 g/kg) rice Khira was identified where a single nucleotide mutation in LOC_Os06g05160 gene encoding SPDT showed low P transportation to grain. An amino acid change was detected as Valine-330 to Alanine at the 3' end of fifth exon. Highest expression of SPDT was observed in node I of rice as compared to low PA genotype. The mutation in SPDT could significantly affect P and PA accumulation in the grains with increased mineral bioavailability. PRACTICAL APPLICATIONS: Excessive P application in crop leads to higher production cost as well as rapid depletion of limited rock phosphate. Alteration of P transporter function in the rice lower PA and total P accumulation in the grains with increased mineral bioavailability. The re-distributed P in the straw can be applied as manure to the rice field. Thus, less P will be removed from the field, result in the decreased requirement for P fertilizer.

Differential Expression of Genes at Panicle Initiation and Grain Filling Stages Implied in Heterosis of Rice Hybrids.

RNA-Seq technology was used to analyze the transcriptome of two rice hybrids, Ajay (based on wild-abortive (WA)-cytoplasm) and Rajalaxmi (based on Kalinga-cytoplasm), and their respective parents at the panicle initiation (PI) and grain filling (GF) stages. Around 293 and 302 million high quality paired-end reads of Ajay and Rajalaxmi, respectively, were generated and aligned against the Nipponbare reference genome. Transcriptome profiling of Ajay revealed 2814 and 4819 differentially expressed genes (DEGs) at the PI and GF stages, respectively, as compared to its parents. In the case of Rajalaxmi, 660 and 5264 DEGs were identified at PI and GF stages, respectively. Functionally relevant DEGs were selected for validation through qRT-PCR, which were found to be co-related with the expression patterns to RNA-seq. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated significant DEGs enriched for energy metabolism pathways, such as photosynthesis, oxidative phosphorylation, and carbon fixation, at the PI stage, while carbohydrate metabolism-related pathways, such as glycolysis and starch and sucrose metabolism, were significantly involved at the GF stage. Many genes involved in energy metabolism exhibited upregulation at the PI stage, whereas the genes involved in carbohydrate biosynthesis had higher expression at the GF stage. The majority of the DEGs were successfully mapped to know yield related rice quantitative trait loci (QTLs). A set of important transcription factors (TFs) was found to be encoded by the identified DEGs. Our results indicated that a complex interplay of several genes in different pathways contributes to higher yield and vigor in rice hybrids.

The potentiality of rice microsatellite markers in assessment of cross-species transferability and genetic diversity of rice and its wild relatives.

The main aim of this study is to assess the potentiality of SSR markers for the identification of the cross-species transferability frequency in a large set of the diverse genome types of wild relative rice along with cultivated rice. Here, we used 18 different rice genotypes representing nine different genome types with 70 SSR markers to investigate the potentiality of cross-species transferability rate. The overall cross-species transferability of SSR markers across the 18 rice genotypes ranged from 38.9% (RM280 and RM447) to 100% (RM490, RM318, RM279, RM18877 and RM20033, RM19303) with an average of 76.58%. Also, cross-species transferability across chromosome ranged from 54.4% (chromosome 4) to 86.5% (chromosome 2) with an average of 74.35%. The polymorphism information content of the markers varied from 0.198 (RM263) to 0.868 (RM510) with a mean of 0.549 ± 0.153, showing high discriminatory power. The highest rate of cross-transferability was observed in O. rufipogon (97%), The highest rate of cross-species transferability was in O. rufipogon (97.00%), followed by O. glaberrima (94.20%), O. nivara (92.80%), Swarna (92.80%), O. longistaminata (91.40%), O. eichingeri (90%), O. barthii (88.50%), O. alta (82.80%), O. australiensis (77.10%), O. grandiglumis (74.20%), O. officinalis (74.20%), Zizania latifolia (70.00%), O. latifolia (68.50%), O. brachyantha (62.80%), Leersia perrieri (57.10%) and O. ridleyi (41.40%) with least in O. coarctata (28.50%). A total of 341 alleles from 70 loci were detected with the number of alleles per locus ranged from 2 to 12. Based on dendrogram analysis, the AA genome groups was separated as distinct group from the rest of the genome types. Similarly, principal coordinate analysis and structure analysis clearly separated the AA genome type from the rest of the genome types. Through the analysis of molecular variance, more variance (51%) was observed among the individual, whereas less (14%) was observed among the population. Thus, our findings may offer a valuable resource for studying the genetic diversity and relationship to facilitate the understanding of the complex mechanism of the origin and evolutionary processes of different Oryza species and wild relative rice.

Correction: Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers.

[This corrects the article DOI: 10.1371/journal.pone.0211061.].

Detection of stable QTLs for grain protein content in rice (Oryza sativa L.) employing high throughput phenotyping and genotyping platforms.

Lack of appropriate donors, non-utilization of high throughput phenotyping and genotyping platforms with high genotype × environment interaction restrained identification of robust QTLs for grain protein content (GPC) in rice. In the present investigation a  BC3F4 mapping population was developed using grain protein donor, ARC10075 and high-yielding cultivar Naveen and 190 lines were genotyped using 40 K Affimetrix custom SNP array with the objective to identify stable QTLs for protein content. Three of the identified QTLs, one for GPC (qGPC1.1) and the other two for single grain protein content (qSGPC2.1, qSGPC7.1) were stable over the environments explaining  13%, 14% and 7.8% of the phenotypic variances, respectively. Stability and repeatability of these additive QTLs were supported by the synergistic additive effects of multi-environmental-QTLs. One epistatic-QTL, independent of  the  main effect QTL was detected over the environment for SGPC. A few functional genes governing seed storage protein were hypothesised inside these identified QTLs. The qGPC1.1 was validated by NIR Spectroscopy-based high throughput phenotyping in BC3F5 population. Higher glutelin content was estimated in high-protein lines with the introgression of qGPC1.1 in telomeric region of short arm of chromosome 1. This was supported by the postulation of probable candidate gene inside this QTL region encoding glutelin family proteins.

Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers.

Understanding of genetic diversity is important to explore existing gene in any crop breeding program. Most of the diversity preserved in the landraces which are well-known reservoirs of important traits for biotic and abiotic stresses. In the present study, the genetic diversity at twenty-four most significant blast resistance gene loci using twenty-eight gene specific markers were investigated in landraces originated from nine diverse rice ecologies of India. Based on phenotypic evaluation, landraces were classified into three distinct groups: highly resistant (21), moderately resistant (70) and susceptible (70). The landraces harbour a range of five to nineteen genes representing blast resistance allele with the frequency varied from 4.96% to 100%. The cluster analysis grouped entire 161 landraces into two major groups. Population structure along with other parameters was also analyzed to understand the evolution of blast resistance gene in rice. The population structure analysis and principal coordinate analysis classified the landraces into two sub-populations. Analysis of molecular variance showed maximum (93%) diversity within the population and least (7%) between populations. Five markers viz; K3957, Pikh, Pi2-i, RM212and RM302 were strongly associated with blast disease with the phenotypic variance of 1.4% to 7.6%. These resistant landraces will serve as a valuable genetic resource for future genomic studies, host-pathogen interaction, identification of novel R genes and rice improvement strategies.

An EMS-induced new sequence variant, TEMS5032, in the coding region of SRS3 gene leads to shorter grain length in rice (Oryza sativa L.).

Grain shape and size influence yield and consumer preferences in rice. In the present study, we characterized and mapped a short and bold grained mutant and named it as TEMS5032, as the mutant is a result of EMS-induced transition from C to T at the 5032nd bp of SRS3 gene, which is known to affect grain size in rice. The substitution led to creation of a stop codon in the motor domain of SRS3, a kinesin 13 family gene, translating into a truncated protein product. However, transcription of this gene remained unaffected in TEMS5032 compared to the wild type, N22. Further, the mutation was found to affect 13 of the 25 cell cycle-related genes as they showed differential expression with respect to N22. Based on rate of grain filling, dry matter accumulation in the endosperm and histological studies, the effect of mutation in TEMS5032 was found to be similar to a known variant, TCM758, but less severe than sar1 mutant. Sequencing of 88 rice germplasm lines in the kinesin motor domain region did not reveal the presence of this mutation, establishing it as a new variant of SRS3 gene.

Effect of multiple allelic combinations of genes on regulating grain size in rice.

The grain size is one of the complex trait of rice yield controlled by a plethora of interaction of several genes in different pathways. The present study was undertaken to investigate the influence of seven known grain size regulating genes: DEP1, GS7, GS3, GW8, GL7, GS5 and GW2. A wide phenotypic variation for grain length, grain width and grain length-width ratio were observed in 89 germplasm. The correlation analysis showed a strong association among these three grain traits viz. GL, GW, GLWR and TGW which play important roles in determining the final rice grain size. Except for GW2, all six genes showed strong association with grain size traits. A total of 21 alleles were identified with an average of 2.1 allele/locus in 89 germplasm of which seven alleles were found to be favourable alleles for improving the grain size with the frequency range of 24 (26.97%) to 82 (92.13%); the largest was found in GS5 followed by GW8, GL7, DEP1, GS3 and GS7 genes. Through ANOVA, four markers (GS3-PstI, S9, GID76 and GID711) of three genes (GS3, DEP1 and GL7) were found significantly associated with all the three traits (GL, GLWR and TGW). Concurrent results of significant associations of grain size traits with other markers were observed in both analysis of variance and genetic association through the general linear model. Besides, the population structure analysis, cluster analysis and PCoA divided the entire germplasm into three sub-groups with the clear-cut demarcation of long and medium grain types. The present results would help in formulating strategies by selecting suitable candidate markers/genes for obtaining preferred grain shape/size and improving grain yield through marker-assisted breeding.

Correction: Use of molecular markers in identification and characterization of resistance to rice blast in India.

[This corrects the article DOI: 10.1371/journal.pone.0176236.].

Use of molecular markers in identification and characterization of resistance to rice blast in India.

Rice blast disease caused by Magnaporthe oryzae is one of the most destructive disease causing huge losses to rice yield in different parts of the world. Therefore, an attempt has been made to find out the resistance by screening and studying the genetic diversity of eighty released rice varieties by National Rice Research Institute, Cuttack (NRVs) using molecular markers linked to twelve major blast resistance (R) genes viz Pib, Piz, Piz-t, Pik, Pik-p, Pikm Pik-h, Pita/Pita-2, Pi2, Pi9, Pi1 and Pi5. Out of which, nineteen varieties (23.75%) showed resistance, twenty one were moderately resistant (26.25%) while remaining forty varieties (50%) showed susceptible in uniform blast nursery. Rice varieties possessing blast resistance genes varied from four to twelve and the frequencies of the resistance genes ranged from 0 to 100%. The cluster analysis grouped the eighty NRVs into two major clusters at 63% level of genetic similarity coefficient. The PIC value for seventeen markers varied from 0 to 0.37 at an average of 0.20. Out of seventeen markers, only five markers, 195R-1, Pi9-i, Pita3, YL155/YL87 and 40N23r corresponded to three broad spectrum R genes viz. Pi9, Pita/Pita2 and Pi5 were found to be significantly associated with the blast disease with explaining phenotypic variance from 3.5% to 7.7%. The population structure analysis and PCoA divided the entire 80 NRVs into two sub-groups. The outcome of this study would help to formulate strategies for improving rice blast resistance through genetic studies, plant-pathogen interaction, identification of novel R genes, development of new resistant varieties through marker-assisted breeding for improving rice blast resistance in India and worldwide.

Development and validation of cross-transferable and polymorphic DNA markers for detecting alien genome introgression in Oryza sativa from Oryza brachyantha.

African wild rice Oryza brachyantha (FF), a distant relative of cultivated rice Oryza sativa (AA), carries genes for pests and disease resistance. Molecular marker assisted alien gene introgression from this wild species to its domesticated counterpart is largely impeded due to the scarce availability of cross-transferable and polymorphic molecular markers that can clearly distinguish these two species. Availability of the whole genome sequence (WGS) of both the species provides a unique opportunity to develop markers, which are cross-transferable. We observed poor cross-transferability (~0.75 %) of O. sativa specific sequence tagged microsatellite (STMS) markers to O. brachyantha. By utilizing the genome sequence information, we developed a set of 45 low cost PCR based co-dominant polymorphic markers (STS and CAPS). These markers were found cross-transferrable (84.78 %) between the two species and could distinguish them from each other and thus allowed tracing alien genome introgression. Finally, we validated a Monosomic Alien Addition Line (MAAL) carrying chromosome 1 of O. brachyantha in O. sativa background using these markers, as a proof of concept. Hence, in this study, we have identified a set molecular marker (comprising of STMS, STS and CAPS) that are capable of detecting alien genome introgression from O. brachyantha to O. sativa.