Expression profiling of immature florets of IR58025A, a wild-abortive cytoplasmic male sterile line of rice and its cognate, isonuclear maintainer line, IR58025B.

Interaction between gene products encoded by the cytoplasm and nucleus form the core of wild abortive cytoplasmic male sterile (WA-CMS) system of hybrid breeding in rice. Gaining insights into such interactions can be helpful in the development of better three-line rice hybrids and also identify novel male sterility systems. In the present study, the whole transcriptome profiles of immature florets of IR58025A, a WA-CMS line and its isonuclear maintainer line, IR58025B, collected at pre-anthesis stage were compared to delineate the pathways involved in pollen abortion and male sterility. Among the 774 differentially expressed transcripts (DETs), 496 were down regulated and 278 were up regulated in IR58025A compared to IR58025B. The genes associated with oxidative stress response, defense response, etc. were significantly up-regulated, while those associated with respiration, cell wall modifications, pectinesterase activity, etc. were significantly down-regulated in the WA-CMS line. Gene ontology and pathway enrichment analyses revealed the down-regulation of both nuclear and organellar genes involved in key metabolic processes of cell respiration, photosynthesis and other energy yielding metabolites in IR58025A, relative to IR58025B, indicating a general shift toward conservation of energy and other key resources in the florets of WA-CMS line. The data derived from RNA-Seq analysis were validated through qRT-PCR analysis. Based on the results obtained, it can be hypothesized that pollen abortion principally occurs due to up-regulation of pathways leading to oxidative stress leading to energy starvation conditions in consonance with reduced expression of genes associated with the cell wall formation, respiration, and other key metabolic processes.

Development of Gene-Pyramid Lines of the Elite Restorer Line, RPHR-1005 Possessing Durable Bacterial Blight and Blast Resistance.

RPHR-1005, the stable restorer line of the popular medium slender (MS) grain type rice hybrid, DRRH-3 was improved in this study for resistance against bacterial blight (BB) and blast diseases through marker-assisted backcross breeding (MABB). In this study, four major resistance genes (i.e., Xa21 and Xa33 for BB resistance and Pi2 and Pi54 for blast resistance) have been transferred to RPHR-1005 using RPBio Patho-1 (possessing Xa21 + Pi2), RPBio Patho-2 (possessing Xa21 + Pi54) and FBR1-15EM (possessing Xa33) as the donors. Foreground selection was carried out using PCR-based molecular markers specific for the target resistance genes and the major fertility restorer genes, Rf3 and Rf4, while background selection was carried out using a set of parental polymorphic rice SSR markers and backcrossing was continued uptoBC2 generation. At BC2F2, plants possessing the gene combination- Xa21 + Pi2, Xa21 + Pi54 and Xa33 in homozygous condition and with >92% recovery of the recurrent parent genome (RPG) were identified and intercrossed to combine all the four resistance genes. Twenty-two homozygous, pyramid lines of RPHR-1005 comprising of three single-gene containing lines, six 2-gene containing lines, eight 3-gene containing lines, and five 4-gene containing lines were identified among the double intercross lines at F3 generation (DICF3). They were then evaluated for their resistance against BB and blast, fertility restoration ability and for key agro-morphological traits. While single gene containing lines were resistant to either BB or blast, the 2-gene, 3-gene, and 4-gene pyramid lines showed good level of resistance against both and/or either of the two diseases. Most of the 2-gene, 3-gene, and 4-gene containing pyramid lines showed yield levels and other key agro-morphological and grain quality traits comparable to the original recurrent parent and showed complete fertility restoration ability, with a few showing higher yield as compared to RPHR-1005. Further, the experimental hybrids derived by crossing the gene-pyramid lines of RPHR-1005 with APMS6A (the female parent of DRRH-3), showed heterosis levels equivalent to or higher than DRRH-3. The results of present study exemplify the utility of MABB for targeted improvement of multiple traits in hybrid rice.

Nucleotide diversity of Pita, a major blast resistance gene and identification of its minimal promoter.

Improvement of host plant resistance is one of the best methods to protect the yield from biotic stresses. Incorporation of major resistance genes or their variants into elite rice varieties will enhance the host plant resistance and its durability. Allele mining is a preferred choice to discover the novel allelic variants of major genes from wide range of germplasm. 'True' allele mining includes coding and noncoding regions, which are known to affect the plant phenotype, eventually. In this study, major blast resistance gene, Pita was analyzed by allele and promoter mining strategy and its different allelic variants were discovered from landraces and wild Oryza species. Polymorphisms at allelic sequences as well as transcription factor binding motif (TFBM) level were examined. At motif level, MYB1AT is present in Pita(Tadukan) and other resistance alleles, but was absent in the susceptible allele. Core promoter was demarked with 449 bp, employing serial promoter deletion strategy. Promoter with 1592 bp upstream region could express the gfp two fold higher than the core promoter. The identified Pita resistance allele (Pita(Konibora)) can be directly used in rice blast resistance breeding programs. Moreover, characterization of Pita core promoter led to deeper understanding of resistance gene's regulation and the identified core promoter can be utilized to express similar genes in rice.

Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice.

The cultivation of rice (Oryza sativa L.), a major food crop, requires ample water (30 % of the fresh water available worldwide), and its productivity is greatly affected by drought, the most significant environmental factor. Much research has focussed on identifying quantitative trait loci, stress-regulated genes and transcription factors that will contribute towards the development of climate-resilient/tolerant crop plants in general and rice in particular. The transcription factor DREB1A, identified from the model plant Arabidopsis thaliana, has been reported to enhance stress tolerance against drought stress. We developed transgenic rice plants with AtDREB1A in the background of indica rice cultivar Samba Mahsuri through Agrobacterium-mediated transformation. The AtDREB1A gene was stably inherited and expressed in T1 and T2 plants and in subsequent generations, as indicated by the results of PCR, Southern blot and RT-PCR analyses. Expression of AtDREB1A was induced by drought stress in transgenic rice lines, which were highly tolerant to severe water deficit stress in both the vegetative and reproductive stages without affecting their morphological or agronomic traits. The physiological studies revealed that the expression of AtDREB1A was associated with an increased accumulation of the osmotic substance proline, maintenance of chlorophyll, increased relative water content and decreased ion leakage under drought stress. Most of the homozygous lines were highly tolerant to drought stress and showed significantly a higher grain yield and spikelet fertility relative to the nontransgenic control plants under both stressed and unstressed conditions. The improvement in drought stress tolerance in combination with agronomic traits is very essential in high premium indica rice cultivars, such as Samba Mahsuri, so that farmers can benefit in times of seasonal droughts and water scarcity.

A putative candidate for the recessive gall midge resistance gene gm3 in rice identified and validated.

KEY MESSAGE: We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and its use in marker-assisted selection. The recessive rice gall midge resistance gene, gm3 identified in the rice breeding line RP2068-18-3-5 confers resistance against five of the seven Indian biotypes of the Asian rice gall midge Orseolia oryzae. We report here tagging and fine-mapping of gm3 gene, development of a functional marker for it and demonstrated its use in marker-assisted selection (MAS). A mapping population consisting of 302 F10 recombinant inbred lines derived from the cross TN1 (susceptible)/RP2068-18-3-5, was screened against gall midge biotype 4 (GMB4) and analyzed with a set of 89 polymorphic SSR markers distributed uniformly across the rice genome. Two SSR markers, RM17480 and gm3SSR4, located on chromosome 4L displayed high degree of co-segregation with the trait phenotype and flanked the gene. In silico analysis of the genomic region spanning these two markers contained 62 putatively expressed genes, including a gene encoding an NB-ARC (NBS-LRR) domain containing protein. A fragment of this gene was amplified with the designed marker, NBcloning 0.9 Kb from the two susceptible TN1, Improved Samba Mahsuri (B95-1) and two resistant cultivars, RP 2068-18-3-5 and Phalguna (with Gm2 gene). The amplicons were observed to be polymorphic between the susceptible and resistant genotypes and hence were cloned and sequenced. A new primer, gm3del3, which was designed based on sequence polymorphism, amplified fragments with distinct size polymorphism among RP2068-18-3-5, Phalguna and TN1 and B95-1 and displayed no recombination in the entire mapping population. Expression of the candidate NB-ARC gene in the susceptible TN1 and the resistant RP2068-18-3-5 plants following infestation with GMB4 was analyzed, through real-time reverse transcription PCR. Results showed twofold enhanced expression in RP2068-18-3-5 plants, but not in TN1 plants, 120 h after infestation. Amino acid sequence and structure analysis of the proteins coded by different alleles of gm3 gene showed deletion of eight amino acids due to an early stop codon in RP2068-18-3-5 resulting in a change in the functional domain of the protein. The gm3del3 was used as a functional marker for introgression of gm3 gene into the genetic background of the elite bacterial blight resistant cultivar Improved Samba Mahsuri (B95-1) through MAS.

Mapping and introgression of QTL for yield and related traits in two backcross populations derived from Oryza sativa cv. Swarna and two accessions of O. nivara.

Advanced backcross QTL (AB-QTL) analysis was carried out in two Oryza nivara-derived BC2F2 populations. For nine traits, we identified 28 QTL in population 1 and 26 QTL in population 2. The two most significant yield-enhancing QTL, yldp9.1 and yldp2.1 showed an additive effect of 16 and 7 g per plant in population 1, while yld2.1 and yld11.1 showed an additive effect of 11 and 10 g per plant in population 2. At least one O. nivara-derived QTL with a phenotypic variance of >15% was detected for seven traits in population 1 and three traits in population 2. The O. nivara-derived QTL ph1.1, nt12.1, nsp1.1, nfg1.1, bm11.1, yld2.1 and yld11.1 were conserved at the same chromosomal locations in both populations. Two major QTL clusters were detected at the marker intervals RM488-RM431 and RM6-RM535 on chromosomes 1 and 2, respectively. The colocation of O. nivara-derived yield QTL with yield meta-QTL on chromosomes 1, 2 and 9 indicates their accuracy and consistency. The major-effect QTL reported in this study are useful for marker-assisted breeding and are also suitable for further fine mapping and candidate gene identification.

Identification of abiotic stress miRNA transcription factor binding motifs (TFBMs) in rice.

Plant growth and yield are affected by many abiotic stresses like salinity, drought, cold and heavy metal; these stresses trigger up and down-regulate several genes through various transcription factors (TFs). Transcription factor binding motifs (TFBMs), located in the upstream region of the genes, associate with TFs to regulate the gene expression. Many factors, including the activation of miRNAs, which are encoded by genes having independent transcription units, regulate the gene expression. TFBMs in the regulatory region of miRNA sequences influence the miRNA expression, which in turn influences the expression of other genes in the cell. However, the current level of information available on TFBMs of miRNA involved in abiotic stress related defense pathway(s) is limited and in-depth studies in this direction may lead to a better understanding of their role in expression and regulation of defense responses in plants. In this study, various aspects related to genomic positions of pre-miRNA, prediction of TSS and TATA box positions and identification of known, unique motifs at regulatory regions of all the reported miRNAs of rice associated with different abiotic stresses are discussed. Sixteen motifs were identified in this study, of which nine are known cis-regulatory elements associated with various stresses, two strong motifs, (CGCCGCCG, CGGCGGCG) and five unique motifs which might play a vital role in the regulation of abiotic stresses related miRNA genes. Common motifs shared by miRNAs that are involved in more than one abiotic stresses were also identified. The motifs identified in this study will be a resource for further functional validation.

The complete genome sequence of a south Indian isolate of Rice tungro spherical virus reveals evidence of genetic recombination between distinct isolates.

In this study, complete genome of a south Indian isolate of Rice tungro spherical virus (RTSV) from Andhra Pradesh (AP) was sequenced, and the predicted amino acid sequence was analysed. The RTSV RNA genome consists of 12,171 nt without the poly(A) tail, encoding a putative typical polyprotein of 3,470 amino acids. Furthermore, cleavage sites and sequence motifs of the polyprotein were predicted. Multiple alignment with other RTSV isolates showed a nucleotide sequence identity of 95% to east Indian isolates and 90% to Philippines isolates. A phylogenetic tree based on complete genome sequence showed that Indian isolates clustered together, while Vt6 and PhilA isolates of Philippines formed two separate clusters. Twelve recombination events were detected in RNA genome of RTSV using the Recombination Detection Program version 3. Recombination analysis suggested significant role of 5' end and central region of genome in virus evolution. Further, AP and Odisha isolates appeared as important RTSV isolates involved in diversification of this virus in India through recombination phenomenon. The new addition of complete genome of first south Indian isolate provided an opportunity to establish the molecular evolution of RTSV through recombination analysis and phylogenetic relationship.

Mapping QTLs and candidate genes for iron and zinc concentrations in unpolished rice of Madhukar×Swarna RILs.

Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. 168 F(7) RILs derived from Madhukar×Swarna were used to map QTLs for iron and zinc concentrations in unpolished rice grains. Iron ranged from 0.2 to 224 ppm and zinc ranged from 0.4 to 104ppm. Genome wide mapping using 101 SSRs and 9 gene specific markers showed 5 QTLs on chromosomes 1, 3, 5, 7 and 12 significantly linked to iron, zinc or both. In all, 14 QTLs were identified for these two traits. QTLs for iron were co-located with QTLs for zinc on chromosomes 7 and 12. In all, ten candidate genes known for iron and zinc homeostasis underlie 12 of the 14 QTLs. Another 6 candidate genes were close to QTLs on chromosomes 3, 5 and 7. Thus the high priority candidate genes for high Fe and Zn in seeds are OsYSL1 and OsMTP1 for iron, OsARD2, OsIRT1, OsNAS1, OsNAS2 for zinc and OsNAS3, OsNRAMP1, Heavy metal ion transport and APRT for both iron and zinc together based on our genetic mapping studies as these genes strictly underlie QTLs. Several elite lines with high Fe, high Zn and both were identified.

QTL analysis for grain quality traits in 2 BC2F2 populations derived from crosses between Oryza sativa cv Swarna and 2 accessions of O. nivara.

The appearance and cooking quality of rice determine its acceptability and price to a large extent. Quantitative trait loci (QTLs) for 12 grain quality traits were mapped in 2 mapping populations derived from Oryza sativa cv Swarna × O. nivara. The BC(2)F(2) population of the cross Swarna × O. nivara IRGC81848 (population 1) was evaluated during 2005 and that from Swarna × O. nivara IRGC81832 (population 2) was evaluated during 2006. Linkage maps were constructed using 100 simple sequence repeat (SSR) markers in population 1 and 75 SSR markers in population 2. In all, 21 QTLs were identified in population 1 (43% from O. nivara) and 37 in population 2 (38% QTLs from O. nivara). The location of O. nivara-derived QTLs mp1.2 for milling percent, kw6.1 for kernel width, and klac12.1 for kernel length after cooking coincided in the 2 populations and appear to be useful for Marker Assisted Selection (MAS). Four QTLs for milling percent, 1 QTL each for amylose content, water uptake, elongation ratio, 2 QTLs for kernel width, and 3 QTLs for gel consistency, each explained more than 20% phenotypic variance. Three QTL clusters for grain quality traits were close to the genes/QTLs for shattering and seed dormancy. QTLs for 4 quality traits were associated with 5 of the 7 major yield QTLs reported in the same 2 mapping populations. Useful introgression lines have been developed for several agronomic traits. It emerges that 40% O. nivara alleles were trait enhancing in both populations, and QTLs for grain quality overlapped with yield meta-QTLs and QTLs for dormancy and seed shattering.

Genetic variation of coat protein gene among the isolates of Rice tungro spherical virus from tungro-endemic states of the India.

Rice tungro disease, one of the major constraints to rice production in South and Southeast Asia, is caused by a combination of two viruses: Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus (RTBV). The present study was undertaken to determine the genetic variation of RTSV population present in tungro endemic states of Indian subcontinent. Phylogenetic analysis based on coat protein sequences showed distinct divergence of Indian RTSV isolates into two groups; one consisted isolates from Hyderabad (Andhra Pradesh), Cuttack (Orissa), and Puducherry and another from West Bengal, Coimbatore (Tamil Nadu), and Kanyakumari (Tamil Nadu). The results obtained from phylogenetic study were further supported with the SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. In addition, sequence difference count matrix revealed 2-68 nucleotides differences among all the Indian RTSV isolates taken in this study. However, at the protein level these differences were not significant as revealed by Ka/Ks ratio calculation. Sequence identity at nucleotide and amino acid level was 92-100% and 97-100%, respectively, among Indian isolates of RTSV. Understanding of the population structure of RTSV from tungro endemic regions of India would potentially provide insights into the molecular diversification of this virus.

Identification and fine-mapping of Xa33, a novel gene for resistance to Xanthomonas oryzae pv. oryzae.

Broadening of the genetic base for identification and transfer of genes for resistance to insect pests and diseases from wild relatives of rice is an important strategy in resistance breeding programs across the world. An accession of Oryza nivara, International Rice Germplasm Collection (IRGC) accession number 105710, was identified to exhibit high level and broad-spectrum resistance to Xanthomonas oryzae pv. oryzae. In order to study the genetics of resistance and to tag and map the resistance gene or genes present in IRGC 105710, it was crossed with the bacterial blight (BB)-susceptible varieties 'TN1' and 'Samba Mahsuri' (SM) and then backcrossed to generate backcross mapping populations. Analysis of these populations and their progeny testing revealed that a single dominant gene controls resistance in IRGC 105710. The BC(1)F(2) population derived from the cross IRGC 105710/TN1//TN1 was screened with a set of 72 polymorphic simple-sequence repeat (SSR) markers distributed across the rice genome and the resistance gene was coarse mapped on chromosome 7 between the SSR markers RM5711 and RM6728 at a genetic distance of 17.0 and 19.3 centimorgans (cM), respectively. After analysis involving 49 SSR markers located between the genomic interval spanned by RM5711 and RM6728, and BC(2)F(2) population consisting of 2,011 individuals derived from the cross IRGC 105710/TN1//TN1, the gene was fine mapped between two SSR markers (RMWR7.1 and RMWR7.6) located at a genetic distance of 0.9 and 1.2 cM, respectively, from the gene and flanking it. The linkage distances were validated in a BC(1)F(2) mapping population derived from the cross IRGC 105710/SM//2 × SM. The BB resistance gene present in the O. nivara accession was identified to be novel based on its unique map location on chromosome 7 and wider spectrum of BB resistance; this gene has been named Xa33. The genomic region between the two closely flanking SSR markers was in silico analyzed for putatively expressed candidate genes. In total, eight genes were identified in the region and a putative gene encoding serinethreonine kinase appears to be a candidate for the Xa33 gene.

Inheritance of bacterial blight resistance in the rice cultivar Ajaya and high-resolution mapping of a major QTL associated with resistance.

The cultivar Ajaya (IET 8585) exhibits durable broad-spectrum resistance to bacterial blight (BB) disease of rice and is widely used as a resistance donor. The present study was carried out to decipher the genetics of BB resistance in Ajaya and map the gene(s) conferring resistance. Genetic analysis in the F2 indicated a quantitative/additive nature of resistance governed by two loci with equal effects. Linked marker analysis and allelic tests revealed that one of the resistance genes is xa5. Sequence analysis of a 244 bp region of the second exon of the gene-encoding Transcription factor IIAγ (the candidate gene for xa5) confirmed the presence of xa5. Bulked-segregant analysis (BSA) revealed the putative location of the two quantitative trait loci (QTLs)/genes associated with resistance on chromosomes 5 and 8. Composite interval mapping located the first locus on Chr. 5S exactly in the genomic region spanned by xa5 and the second locus (qtl BBR 8.1) on Chr. 8L. Owing to its differential disease reaction with a set of seven hyper-virulent isolates of Xanthomonas oryzae, a map location on Chr. 8L, which was distinct from xa13 and data from allelism tests, the second resistance locus in Ajaya was determined to be novel and was designated as xaAj. A contig map spanning xaAj was constructed in silico and the genomic region was delimited to a 13.5 kb physical interval. In silico analysis of the genomic region spanning xaAj identified four putatively expressed candidate genes, one of which could be involved in imparting BB resistance in Ajaya along with xa5.

Bt rice evaluation and deployment strategies.

Bacillus thuringiensis (Bt), a gram positive soil bacteria was first identified and named by Japanese microbiologist Shigetane Ishiwata in 1901. During sporulation Bt produces proteinaceous parasporal crystal proteins called δ-endotoxins, or Cry proteins, which are insecticidal. Numerous Cry proteins have been isolated and characterized from different Bt strains with activity against insects, mites and nematodes. Sprayable formulations containing these Cry proteins as active ingredients have contributed significantly in the field of insect pest management. Since the first cloning of cry genes from Bt,1 scientists have successively demonstrated that plants could be genetically engineered to express these cry genes for the control of dreadful insect pests. Eventually, the first transgenic crop expressing Btcry1Ac gene in cotton was approved in 1996 for commercial cultivation in the USA to manage bollworms.

Allele mining in crops: prospects and potentials.

Enormous sequence information is available in public databases as a result of sequencing of diverse crop genomes. It is important to use this genomic information for the identification and isolation of novel and superior alleles of agronomically important genes from crop gene pools to suitably deploy for the development of improved cultivars. Allele mining is a promising approach to dissect naturally occurring allelic variation at candidate genes controlling key agronomic traits which has potential applications in crop improvement programs. It helps in tracing the evolution of alleles, identification of new haplotypes and development of allele-specific markers for use in marker-assisted selection. Realizing the immense potential of allele mining, concerted allele mining efforts are underway in many international crop research institutes. This review examines the concepts, approaches and applications of allele mining along with the challenges associated while emphasizing the need for more refined 'mining' strategies for accelerating the process of allele discovery and its utilization in molecular breeding.

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.