T. Mohapatra

The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications

Rice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals. We have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes. Because the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.BackgroundRice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals.ResultsWe have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes.ConclusionBecause the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.

Combining bacterial blight resistance and Basmati quality characteristics by phenotypic and molecular marker-assisted selection in rice

Bacterial Blight (BB) caused by Xanthomonas oryzae pv. oryzae is a major disease of rice in tropical Asia. Since all the Basmati varieties are highly susceptible and the disease is prevalent in the entire Basmati growing region of India, BB is a severe constraint in Basmati rice production. The present study was undertaken with the objective of combining the important Basmati quality traits with resistance to BB by a combination of phenotypic and molecular marker-assisted selection (MAS). Screening of 13 near-isogenic lines of rice against four isolates of the pathogen from Basmati growing regions identified the Xa4, xa8, xa13 and Xa21 effective against all the isolates tested. Two or more of these genes in combination imparted enhanced resistance as expressed by reduced average lesion length in comparison to individual genes. The two-gene pyramid line IRBB55 carrying xa13 and Xa21 was found equally effective as three/four gene pyramid lines. The two BB resistance genes present in IRBB55 were combined with the Basmati quality traits of Pusa Basmati-1 (PB-1), the most popular high yielding Basmati rice variety used as recurrent parent. Phenotypic selection for disease resistance, agronomic and Basmati quality characteristics and marker-assisted selection for the two resistance genes were carried out in BC1F1, BC1F2 and BC1F3 generations. Background analysis using 252 polymorphic amplified fragment length polymorphism (AFLP) markers detected 80.4 to 86.7% recurrent parent alleles in BC1F3 selections. Recombinants having enhanced resistance to BB, Basmati quality and desirable agronomic traits were identified, which can either be directly developed into commercial varieties or used as immediate donors of BB resistance in Basmati breeding programs.

Combining QTL mapping and transcriptome profiling of bulked RILs for identification of functional polymorphism for salt tolerance genes in rice (Oryza sativa L.).

Identification of genes for quantitative traits is difficult using any single approach due to complex inheritance of the traits and limited resolving power of the individual techniques. Here a combination of genetic mapping and bulked transcriptome profiling was used to narrow down the number of differentially expressed salt-responsive genes in rice in order to identify functional polymorphism of genes underlying the quantitative trait loci (QTL). A population of recombinant inbred lines (RILs) derived from cross between salt-tolerant variety CSR 27 and salt-sensitive variety MI 48 was used to map QTL for salt ion concentrations in different tissues and salt stress susceptibility index (SSI) for spikelet fertility, grain weight, and grain yield. Eight significant QTL intervals were mapped on chromosomes 1, 8, and 12 for the salt ion concentrations and a QTL controlling SSI for spikelet fertility was co-located in one of these intervals on chromosome 8. However, there were total 2,681 genes in these QTL intervals, making it difficult to pinpoint the genes responsible for the functional differences for the traits. Similarly, transcriptome profiling of the seedlings of tolerant and sensitive parents grown under control and salt-stress conditions showed 798 and 2,407 differentially expressed gene probes, respectively. By analyzing pools of RNA extracted from ten each of extremely tolerant and extremely sensitive RILs to normalize the background noise, the number of differentially expressed genes under salt stress was drastically reduced to 30 only. Two of these genes, an integral transmembrane protein DUF6 and a cation chloride cotransporter, were not only co-located in the QTL intervals but also showed the expected distortion of allele frequencies in the extreme tolerant and sensitive RILs, and therefore are suitable for future validation studies and development of functional markers for salt tolerance in rice to facilitate marker-assisted breeding.

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa)

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na+/K+ ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5–18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na+/K+ ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.

Identification, characterization and utilization of unigene derived microsatellite markers in tea (Camellia sinensis L.)

BackgroundDespite great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in tea (Camellia sinensis L.). The development of microsatellite markers will have a major impact on genetic analysis, gene mapping and marker assisted breeding. Unigene derived microsatellite (UGMS) markers identified from publicly available sequence database have the advantage of assaying variation in the expressed component of the genome with unique identity and position. Therefore, they can serve as efficient and cost effective alternative markers in such species.ResultsConsidering the multiple advantages of UGMS markers, 1,223 unigenes were predicted from 2,181 expressed sequence tags (ESTs) of tea (Camellia sinensis L.). A total of 109 (8.9%) unigenes containing 120 SSRs were identified. SSR abundance was one in every 3.55 kb of EST sequences. The microsatellites mainly comprised of di (50.8%), tri (30.8%), tetra (6.6%), penta (7.5%) and few hexa (4.1%) nucleotide repeats. Among the dinucleotide repeats, (GA)n.(TC)n were most abundant (83.6%). Ninety six primer pairs could be designed form 83.5% of SSR containing unigenes. Of these, 61 (63.5%) primer pairs were experimentally validated and used to investigate the genetic diversity among the 34 accessions of different Camellia spp. Fifty one primer pairs (83.6%) were successfully cross transferred to the related species at various levels. Functional annotation of the unigenes containing SSRs was done through gene ontology (GO) characterization. Thirty six (60%) of them revealed significant sequence similarity with the known/putative proteins of Arabidopsis thaliana. Polymorphism information content (PIC) ranged from 0.018 to 0.972 with a mean value of 0.497. The average heterozygosity expected (HE) and observed (Ho) obtained was 0.654 and 0.413 respectively, thereby suggesting highly heterogeneous nature of tea. Further, test for IAM and SMM models for the UGMS loci showed excess heterozygosity and did not show any bottleneck operating in the tea population.ConclusionUGMS markers identified and characterized in this study provided insight about the abundance and distribution of SSR in the expressed genome of C. sinensis. The identification and validation of 61 new UGMS markers will not only help in intra and inter specific genetic diversity assessment but also be enriching limited microsatellite markers resource in tea. Further, the use of these markers would reduce the cost and facilitate the gene mapping and marker-aided selection in tea. Since, 36 of these UGMS markers correspond to the Arabidopsis protein sequence data with known functions will offer the opportunity to investigate the consequences of SSR polymorphism on gene functions.

Molecular fingerprinting of hybrids and assessment of genetic purity of hybrid seeds in rice using microsatellite markers

Microsatellite markers were used for fingerprinting of hybrids, assessing variation within parental lines and testing the genetic purity of hybrid seed lot in rice. Ten sequence tagged microsatellite sites (STMS) markers were employed for fingerprinting 11 rice hybrids and their parental lines. Nine STMS markers were found polymorphic across the hybrids and produced unique fingerprint for the 11 hybrids. A set of four markers (RM 206, RM 216, RM 258 and RM 263) differentiated all the hybrids from each other, which can be used as referral markers for unambiguous identification and protection of these hybrids. Cluster analysis based on Jaccards similarity coefficient using UPGMA grouped the hybrids into three clusters. Within the cluster all the hybrids shared a common cytoplasmic male sterile line as female parent. The genetic similarity between the hybrids ranged from 0.33 to 0.92 with an average similarity index of 0.63. The analysis of plant-to-plant variation within the parental lines of the hybrid Pusa RH 10, using informative markers indicated residual heterozygosity at two marker loci. This highlights the importance of STMS markers in maintaining the genetic purity of the parental lines. The unique value of the restorer gene linked marker for testing the genetic purity of hybrid seeds is demonstrated for the first time.

Suitability of mapped sequence tagged microsatellite site markers for establishing distinctness, uniformity and stability in aromatic rice

At present, testing for distinctness, uniformity and stability (DUS) of crop varieties relies on a set of morphological characters. These characters suffer fromthe limitations of number, interaction with the environment in which the variety grows and subjectivity in decision-making. The potential of DNA-based markers such as sequence tagged microsatellite site (STMS), for establishing DUS merits investigation. In the present study, a set of 55 mapped STMS markers, selected from 12 linkage groups of rice genome, was used to examine distinctness of 23 aromatic rice genotypes including the commercially important Basmati varieties. Forty-one of these markers (74.5%) showed polymorphism between the varieties. The number of alleles per locus ranged from 2–4 with an average of 2.3. The polymorphism information content (PIC) of the markers varied from 0.083 to 0.665 with an average of 0.338. All the varieties could be differentiated from each other at a low probability (0.07×10-13) of identical match by chance. The marker-based clustering of the varieties corresponded with the known phenotypic classification, thereby providing confidence in the distinctness established by the mapped STMS markers. The utility of these markers to study uniformity and stability was analysed using a commercially important crossbred Basmati rice variety Pusa Basmati 1(IET-10364) that contributes about 40–50% of Basmati rice export from India. Genotyping of twenty individual plants, grown from the nucleus, breeder, foundation, certified and farmers saved seed samples using all the 55 markers revealed no variation among the plants. These observations suggested that the set of mapped markers employed in this study could be further used for establishing distinctness of aromatic rice varieties and for studying DUS of the important commercial variety Pusa Basmati 1.

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes.

AFLP Analysis of the Phenetic Organization and Genetic Diversity in the Sugarcane Complex, Saccharum and Erianthus

Amplified fragment length polymorphism (AFLP) markers were evaluated for determining the phylogenetic relationships, and the diversity in the Saccharum complex using 30 clones belonging to S. officinarum, S. robustum, S. spontaneum, S. barberi, S. sinense and the related genus Erianthus. The phenetic tree of the species clones based on AFLP data was consistent with the known taxonomical relationships. AFLP gave higher resolution of closely related species into discrete groups than that by RAPD and RFLP markers, reported earlier. The levels of diversity within the various Saccharum species were also found to be higher than those obtained previously with the same set of clones using RAPD markers. The intraspecies similarity in S. barberi and S. sinense was much higher than interspecies similarity suggesting a clear separation of the two, which are considered ‘horticultural species’. The genetic similarity matrix derived from a single primer combination highly correlated (r = 0.980) with that obtained from all the 12 primer combination used in the study, thus highlighting the efficiency of a single primer combination in delineating species relationships. All the primer combinations could identify markers that are specific to each of the species and the genus Erianthus. Among the species, specific markers were highest in S. spontaneum followed by S. robustum, S. barberi, S. officinarum and S. sinense. Erianthus had a distinct profile with 30% of the total amplified fragments being specific to it. This offers great scope for identifying intergeneric hybrids, which has been very difficult using morphological traits and RAPD markers. High degree of correspondence between the results from the cluster analysis based on Jaccards similarity index, Neighbour Joining tree based on Sokal and Michener distance matrix and AFTD (Analyses Factorielle on Table of Distances) analysis clearly demonstrated that AFLP markers would be an appropriate tool in providing better information about the relationships among the species, estimation of diversity, and in revealing species and genus specific markers that could be directly applied in sugarcane breeding programmes.

Identification and classification of aromatic rices based on DNA fingerprinting

Aromatic rices are preferred by the consumers all over the world due to its flavour and palatability. Although a large number of these collections are available, little systematic analysis of genetic diversity has been carried out. With the objective of identification and classification of aromatic rice genotypes, RAPD profiling was employed using 58 random decamer primers. Most of these primers (96.5%) detected polymorphism among the genotypes. Of the 465 amplified bands, 314 were polymorphic. Cluster analysis based on Jaccards similarity coefficient using UPGMA grouped all the traditional tall, photosensitive, low yielding, long grained ‘basmati’ aromatics together. The short grained aromatic cultivars, formed a different cluster with high level of average similarity among themselves. The dendrogram based on 58 primers was highly similar to that based on 10 and 15 primers with matrix correlation (r) of 0.88 and 0.91, respectively. This suggested that a set of 10 primers can be employed for an initial assessment of genetic diversity in a large number of collections. All the rice genotypes included in the study could be distinguished from each other at the level of 19 to 186 polymorphic bands between individuals in pair wise comparison over all the 58 primers. Probability of identical profiles by chance suggested that about 1041 genotypes can be unambiguously differentiated by RAPD fingerprints obtained by 58 primers. A diagrammatic mode of presentation of DNA fingerprints of the aromatic rices based on 10 of the informative primers was developed.