Development of activation-tagged gain-of-functional mutants in indica rice line (BPT 5204) for sheath blight resistance.

BACKGROUND: The development of sheath blight (ShB) resistance varieties has been a challenge for scientists for long time in rice. Activation tagging is an efficient gain-of-function mutation approach to create novel phenotypes and to identify their underlying genes. In this study, a mutant population was developed employing activation tagging in the recalcitrant indica rice (Oryza sativa L.) cv. BPT 5204 (Samba Mahsuri) through activation tagging. METHODS AND RESULTS: In this study, we have generated more than 1000 activation tagged lines in indica rice, from these mutant population 38 (GFP- RFP+) stable Ds plants were generated through germinal transposition at T2 generation based on molecular analysis and seeds selected on hygromycin (50 mg/L) containing medium segregation analyses confirmed that the transgene inherited as mendelian segregation ratio of 3:1 (3 resistant: 1 susceptible). Of them, five stable activation tagged Ds lines (M-Ds-1, M-Ds-2, M-Ds-3, M-Ds-4 and M-Ds-5) were selected based on phenotypic observation through screening for sheath blight (ShB) resistance caused by fungal pathogen Rhizoctonia solani (R. solani),. Among them, M-Ds-3 and M-Ds-5 lines showed significant resistance for ShB over other tagged lines and wild type (WT) plants. Furthermore, analysed for launch pad insertion through TAIL-PCR results and mapped on corresponding rice chromosomes. Flanking sequence and gene expression analysis revealed that the upregulation of glycoside hydrolase-OsGH or similar to Class III chitinase homologue (LOC_Os08g40680) in M-Ds-3 and a hypothetical protein gene (LOC_Os01g55000) in M-Ds-5 are potential candidate genes for sheath blight resistance in rice. CONCLUSION: In the present study, we developed Ac-Ds based ShB resistance gain-of-functional mutants through activation tagging in rice. These activation tagged mutant lines can be excellent sources for the development of ShB resistant cultivars in rice.

Uncovering of natural allelic variants of key yield contributing genes by targeted resequencing in rice (Oryza sativa L.).

In rice (Oryza sativa L.), during the course of domestication, numerous beneficial alleles remain untapped in the progenitor wild species and landraces. This study aims at uncovering these promising alleles of six key genes influencing the yield, such as DEP1, Ghd7, Gn1a, GS3, qSW5 and sd1 by targeted resequencing of the 200 rice genotypes. In all, 543 nucleotide variations including single nucleotide polymorphisms and insertion and deletion polymorphisms were identified from the targeted genes. Of them, 225 were novel alleles, which identified in the present study only and 91 were beneficial alleles that showed significant association with the yield traits. Besides, we uncovered 128 population-specific alleles with indica being the highest of 79 alleles. The neutrality tests revealed that pleiotropic gene, Ghd7 and major grain size contributing gene, GS3 showed positive and balanced selection, respectively during the domestication. Further, the haplotype analysis revealed that some of the rice genotypes found to have rare haplotypes, especially the high yielding variety, BPT1768 has showed maximum of three genes such as Gn1a-8, qSW5-12 and GS3-29. The rice varieties with novel and beneficial alleles along with the rare haplotypes identified in the present study could be of immense value for yield improvement in the rice breeding programs.

QTL-Seq-based genetic analysis identifies a major genomic region governing dwarfness in rice (Oryza sativa L.).

KEY MESSAGE: A major dwarfing region for plant height, asd1, was identified employing the next-generation sequencing-based QTL-Seq approach from a dwarf mutant and is demonstrated to be responsible for the dwarf nature with least penalty on yield in rice. The yield plateauing of modern rice is witnessed since many decades due to the narrow genetic base owing to the usage of a single recessive gene, i.e., semi-dwarf-1 (sd-1) for development of short-statured varieties throughout the world. This calls for the searching of alternate sources for short stature in rice. To this end, we made an attempt to uncover yet another, but valuable dwarfing gene employing next-generation sequencing (NGS)-based QTL-Seq approach. Here, we have identified a major QTL governing plant height on chromosome 1, i.e., alternate semi-dwarf 1 (asd1) from an F2 mapping population derived from a cross between a dwarf mutant, LND384, and a tall landrace, INRC10192. Fine mapping of asd1 region employing sequence-based indel markers delimited the QTL region to 67.51 Kb. The sequencing of the QTL region and gene expression analysis predicted a gene that codes for IWS1 (C-terminus family protein). Furthermore, marker-assisted introgression of the asd1 into tall landrace, INRC10192, reduced its plant height substantially while least affecting the yield and its component traits. Hence, this novel dwarfing gene, asd1, has profound implications in rice breeding.

Ectopic expression of Arabidopsis Target of Rapamycin (AtTOR) improves water-use efficiency and yield potential in rice.

The target of Rapamycin (TOR) present in all eukaryotes is a multifunctional protein, regulating growth, development, protein translation, ribosome biogenesis, nutrient, and energy signaling. In the present study, ectopic expression of TOR gene of Arabidopsis thaliana in a widely cultivated indica rice resulted in enhanced plant growth under water-limiting conditions conferring agronomically important water-use efficiency (WUE) trait. The AtTOR high expression lines of rice exhibited profuse tillering, increased panicle length, increased plant height, high photosynthetic efficiency, chlorophyll content and low ∆13C. Δ13C, which is inversely related to high WUE, was as low as 17‰ in two AtTOR high expression lines. These lines were also insensitive to the ABA-mediated inhibition of seed germination. The significant upregulation of 15 stress-specific genes in high expression lines indicates their contribution to abiotic stress tolerance. The constitutive expression of AtTOR is also associated with significant transcriptional upregulation of putative TOR complex-1 components, OsRaptor and OsLST8. Glucose-mediated transcriptional activation of AtTOR gene enhanced lateral root formation. Taken together, our findings indicate that TOR, in addition to its multiple cellular functions, also plays an important role in response to abiotic stress and potentially enhances WUE and yield related attributes.

Activation tagging in indica rice identifies ribosomal proteins as potential targets for manipulation of water-use efficiency and abiotic stress tolerance in plants.

We have generated 3900 enhancer-based activation-tagged plants, in addition to 1030 stable Dissociator-enhancer plants in a widely cultivated indica rice variety, BPT-5204. Of them, 3000 were screened for water-use efficiency (WUE) by analysing photosynthetic quantum efficiency and yield-related attributes under water-limiting conditions that identified 200 activation-tagged mutants, which were analysed for flanking sequences at the site of enhancer integration in the genome. We have further selected five plants with low Δ13 C, high quantum efficiency and increased plant yield compared with wild type for a detailed investigation. Expression studies of 18 genes in these mutants revealed that in four plants one of the three to four tagged genes became activated, while two genes were concurrently up-regulated in the fifth plant. Two genes coding for proteins involved in 60S ribosomal assembly, RPL6 and RPL23A, were among those that became activated by enhancers. Quantitative expression analysis of these two genes also corroborated the results on activating-tagging. The high up-regulation of RPL6 and RPL23A in various stress treatments and the presence of significant cis-regulatory elements in their promoter regions along with the high up-regulation of several of RPL genes in various stress treatments indicate that they are potential targets for manipulating WUE/abiotic stress tolerance.

Discovery and mapping of genomic regions governing economically important traits of Basmati rice.

BACKGROUND: Basmati rice, originated in the foothills of Himalayas, commands a premium price in the domestic and international markets on account of its unique quality traits. The complex genetic nature of unique traits of Basmati as well as tedious screening methodologies involved in quality testing have been serious constraints to breeding quality Basmati. In the present study, we made an attempt to identify the genomic regions governing unique traits of Basmati rice. RESULTS: A total of 34 Quantitative Trait Loci (QTLs) for 16 economically important traits of Basmati rice were identified employing F(2), F(3) and Recombinant Inbred Line (RIL) mapping populations derived from a cross between Basmati370 (traditional Basmati) and Jaya (semi-dwarf rice). Out of which, 12 QTLs contributing to more than 15 % phenotypic variance were identified and considered as major effect QTLs. Four major effect QTLs coincide with the already known genes viz., sd1, GS3, alk1 and fgr governing plant height, grain size, alkali spreading value and aroma, respectively. For the remaining major QTLs, candidate genes were predicted as auxin response factor for filled grains, soluble starch synthase 3 for chalkiness and VQ domain containing protein for grain breadth and grain weight QTLs, based on the presence of non-synonymous single nucleotide polymorphism (SNPs) that were identified by comparing Basmati genome sequence with that of Nipponbare. CONCLUSIONS: To the best of our knowledge, the current study is the first attempt ever made to carry out genome-wide mapping for the dissection of the genetic basis of economically important traits of Basmati rice. The promising QTLs controlling important traits in Basmati rice, identified in this study, can be used as candidates for future marker-assisted breeding.

Review of methods for the detection and quantification of adulteration of rice: Basmati as a case study.

Rice is a staple and widely grown crop endowed with rich genetic diversity. As it is difficult to differentiate seeds of various rice varieties based on visual observation accurately, the harvested seeds and subsequent processed products are highly prone to adulteration with look-alike and low quality seeds by the dishonest traders. To protect the interests of importing countries and consumers, several methods have been employed over the last few decades for unambiguous discrimination of cultivars, accurate quantification of the adulterants, and for determination of cultivated geographical area. With recent advances in biotechnology, DNA based techniques evolved rapidly and proved successful over conventional non-DNA based methods to purge the problem of adulteration at commercial level. In the current review, we made an attempt to summarize the existing methods of adulteration detection and quantification in a comprehensive manner by providing Basmati as a case study to enable the traders to arrive at a quick resolution in choosing the apt method to eliminate the adulteration practice in the global rice industry.

Identification of simple sequence repeat markers for utilizing wide-compatibility genes in inter-subspecific hybrids in rice (Oryza sativa L.).

Although pronounced heterosis in inter-subspecific hybrids was known in rice for a long time, its utilization for hybrid rice breeding has been limited due to their hybrid sterility (HS). For the last two decades, however, a few inter-subspecific hybrids have been developed by incorporating wide-compatibility genes (WCG) that resolve HS, into parental lines of these inter-subspecific hybrids. For effective use of WCG, it is necessary to find convenient markers linked to WCG of practical importance. In this paper, initially a set of simple sequence repeat (SSR) markers in the vicinity of known WCG loci identified based on comparative linkage maps have been surveyed in a population derived from the three-way cross- IR36/Dular//Akihikari, where a known donor of WCG Dular was crossed to a representative indica and japonica cultivar. Of the five parental polymorphic markers, RM253 and RM276 were found to be closely linked to the WCG locus S5 at a distance of 3.0 and 2.8 cM, respectively. Later, loci for HS were examined in three F(2) populations derived from inter-subspecific crosses, with same set of SSR markers. The locus S8 was confirmed to have major influence on HS in the F(2 )population derived from CHMRF-1/Taichung65 since two SSR markers in its vicinity, RM412 and RM141, co-segregated with HS at a map distance of 7.6 and 4.8 cM, respectively. In the F(2) population derived from the cross BPT5204/Taipei309, three SSR markers in the vicinity of S5, RM50, RM276 and RM136 co-segregated with HS at a map distance of 4.2, 3.2 and 7.8 cM, respectively. In the third F(2 )population derived from Swarna/Taipei309, the SSR markers in the vicinity of S5, RM225, RM253, RM50, RM276 and RM136 were identified to co-segregate with HS at a map distance of 3.2, 2.6, 3.4, 2.6 and 6.6 cM, respectively. These results indicated a clear picture of WCG in Dular as well as the predominant role of HS alleles at S5 locus. The identified SSR markers are expected to be used for incorporation of WCG into parental lines in hybrid rice breeding to solve HS in inter-subspecific hybrids.

Identification and mapping of yield and yield related QTLs from an Indian accession of Oryza rufipogon.

BACKGROUND: Cultivated rice (Oryza sativa L.) is endowed with a rich genetic variability. In spite of such a great diversity, the modern rice cultivars have narrow genetic base for most of the agronomically important traits. To sustain the demand of an ever increasing population, new avenues have to be explored to increase the yield of rice. Wild progenitor species present potential donor sources for complex traits such as yield and would help to realize the dream of sustained food security. RESULTS: Advanced backcross method was used to introgress and map new quantitative trait loci (QTLs) relating to yield and its components from an Indian accession of Oryza rufipogon. An interspecific BC2 testcross progeny (IR58025A/O. rufipogon//IR580325B///IR58025B////KMR3) was evaluated for 13 agronomic traits pertaining to yield and its components. Transgressive segregants were obtained for all the traits. Thirty nine QTLs were identified using interval mapping and composite interval mapping. In spite of it's inferiority for most of the traits studied, O. rufipogon alleles contributed positively to 74% of the QTLs. Thirty QTLs had corresponding occurrences with the QTLs reported earlier, indicating that these QTLs are stable across genetic backgrounds. Nine QTLs are novel and reported for the first time. CONCLUSION: The study confirms that the progenitor species constitute a prominent source of still unfolded variability for traits of complex inheritance like yield. With the availability of the complete genome sequence of rice and the developments in the field of genomics, it is now possible to identify the genes underlying the QTLs. The identification of the genes constituting QTLs would help us to understand the molecular mechanisms behind the action of QTLs.

Genetic analysis of traditional and evolved Basmati and non-Basmati rice varieties by using fluorescence-based ISSR-PCR and SSR markers.

The objective of the present study was to make use of efficient molecular marker systems to reveal genetic relationships in traditional and evolved Basmati (EB) and semidwarf non-Basmati (NB) rice varieties. A subset of three rice groups was analyzed by using 19 simple sequence repeat (SSR) loci and 12 inter-SSR-PCR primers. A total of 70 SSR alleles and 481 inter-SSR-PCR markers were revealed in 24 varieties from the three groups. The lowest genetic diversity was observed among the traditional Basmati varieties, whereas the EB varieties showed the highest genetic diversity by both the marker assays. The results indicated that the subset of aromatic rice varieties analyzed in the present study is probably derived from a single land race. The traditional Basmati (TB) and semidwarf NB rice varieties used in the present study were clearly delineated by both marker assays. A number of markers, which could unambiguously distinguish the TB varieties used in the present study from the evolved and NB rice varieties, were identified. The potential use of these markers in Basmati rice-breeding programs and authentication of TB varieties used in the present study are envisaged.

Mode of inheritance of dwarf stature and allelic relationships among various spontaneous and induced dwarfs of cultivated rice Oryza sativa L.

Genetic study of spontaneous and induced dwarfs included the mode of inheritance of dwarf stature and the allelic relationships among various dwarfs. Qualitative genetic analysis involving crosses of fourteen dwarfs with a common tall variety 'IARI 11124' showed that the degree of dominance in the F1 hybrids varied with the cross. With the exception of the crosses of IARI 6579 and IARI 10560 with the tall variety, all crosses exhibited incomplete dominance. The segregation pattern in F2 populations of height classes showed dwarfness to be a monogenic recessive trait functioning, however, in association with modifier complexes of varied strength. From F2 behaviour of all possible crosses involving the fourteen different dwarfs, the allelic relationships were deduced. Three major groups of dwarfs could be recognised. Group I, comprised of FF 36, IARI 5842, IARI 5906-2B, IARI 5923, IARI 10061, IARI 10560 and IARI 11445, was allelic to I-geo-tse and Dee-Gee-Woo-Gen with modifiers of predominantly negative effects, while group-2, comprised of dwarfs IARI 5901-2, IARI 5924, IARI 6579 and IARI 7312B, was also allelic to Dee-Gee-Woo-Gen and I-geo-tse but with large and equal number of modifiers of positive and negative effects. The induced mutant, Central Africa Mutant (CAM) which constituted the third group seemed to possess a dwarfing gene that was non-allelic to those of the above mentioned two groups of dwarfs, with equal strength of modifiers of plus and minus effects. Unlike the dwarfs of spontaneous origin, which are invariably allelic to 'Dee-Gee-Woo-Gen', the induced dwarf was nonallelic. Thus, induced mutagenesis appears to give rise to dwarfing genes different from those found in the naturally occurring dwarfs.

Study of interrelationship among A-genome species of the genus Oryza through isoenzyme variation.

The interrelationships among ten different A-genome species of the genus Oryza were studied based on variations in the electrophoretic pattern of isoenzymes of two non-specific enzymes, esterase and peroxidase. There were 16 isoenzymes of esterase and 14 of peroxidase. The esterase pattern could be classified into 3 different Zymograms 1e, 2e & 3e based on the presence and/or absence of bands at particular Rf values. The pattern le was found exclusively among the species and varietal groups of sativa complex, whereas 2e and 3e were distributed exclusively among the species of the glaberrima complex and related wild forms. The peroxidase pattern also fell into 3 different zymograms viz. 1p, 2p and 3p. Unlike esterase, all three zymograms were present in both the sativa and glaberrima complexes.The similarity indices (S) between the different pairs of entries were computed taking into account the presence as well as the relative intensity of the corresponding isoenzyme bands. The varieties and sub-species of 0.sativa showed very high similarity values with the Asian perennis (O.perennis sub sp. balunga), lending evidence for the probable differentiation of the former from the latter. The African cultivated species O.glaberrima showed very high similarity to the African perennis form O.pevennis sub sp. barthii, O.breviligulata and O.stapfii. The only cubensis form studied had the same esterase and peroxidase pattern as that of the species of the glaberrima complex and also a very high similarity with this group. Thus, the entire A-genome species could be broadly grouped into the sativa and glaberrima complexes, and within the group there was a lot of overlapping in similarity values making it difficult to identify and pin-point species or subspecies based on their isoenzyme patterns and similarity values.

A Study on the nature of genetic divergence in rice from assam and North East Himalayas.

A representative group of 190 rice types collected from North-East India along with four standard varieties, three of which were indicas and one japonica, was studied to understand the nature of genetic divergence. Preliminary grouping was done by canonical analysis and the resultant 42 groups were further classified using the D(2) statistic.The final grouping resulted in nine divergent clusters. The three indica standards were found in three different clusters indicating the wide available variability among them. The japonica standard formed a separate group by itself. A majority of the North-East Indian types formed clusters with indicas, whereas some were intermediate and still others were closer to japonica or indica, thus indicating a series of intergrades bridging indica and japonica.Height followed by leaf area was found to be important for primary and 100-grain weight, followed by amylose content for secondary differentiation. It appears that natural selection as well as human selection might have operated for characters differentiating rice types in Assam and North Eastern Himalayas. Geographical distance was not found to be related to genetic divergence. The study suggests that O. sativa contains innumerable but divergent forms, and its classification into definite varietal groups on an arbitrary basis such as isolation barrier, sexual affinity or geographic distribution would be far from reality.

Study on mutational manipulation of protein characteristics in rice.

A large number of induced mutants isolated from indica and japonica varieties of rice were screened for two different aspects of nutritive quality, namely crude protein content and protein distribution pattern in the endosperm. The study revealed a wide variation for both characteristics. There was no consistent relationship between protein content and test-grain weight or spikelet sterility. It appears from the present study that both these characteristics of protein are easily amenable to mutagen induced changes. The potential use of these characteristics in rice breeding is discussed.

Intra and inter subspecific variation in soluble proteins of Oryza sativa L.

Types representing three subspecies of Oryza sativa, namely, indica, japonica and javanica, and a group of intermediate types collected from North East India, were studied for variation in soluble proteins using acrylamide gel electrophoresis. The study revealed that there was a marked variation within and between varietal groups. Variability for number and intensity of protein bands in indica was wider than in japonica and javanica. Protein pattern in the group comprising N.E. Indian types transgressed that of all three established groups by displaying a wide spectrum. Relative homology, as measured from percentage similarities of N.E. Indian types to the three subspecies, suggested the existence of six different groups. Comparison of varietal groups for the protein mobility pattern showed that japonica and javanica varieties tended to show higher percentages of slow mobility proteins than indica. It appears, from the narrow variability and relatively low percentage of slow mobility proteins, that the japonica and javanica races are of later origin compared to indica. However, the study with a limited number of types suggested the monophyletic origin of varietal groups from an indica-like base predominantly found in the secondary centres of origin of O. sativa.