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.

Genotyping-by-sequencing and multilocation evaluation of two interspecific backcross populations identify QTLs for yield-related traits in pigeonpea.

This study has identified single-nucleotide polymorphism (SNP) markers associated with nine yield-related traits in pigeonpea by using two backcross populations (BP) developed through interspecific crosses and evaluating them at two locations and 3 years. In both the populations, markers have shown strong segregation distortion; therefore, a quantitative trait locus (QTL) mapping mixed model was used. A total of 86 QTLs explaining 12-21% phenotypic variation were detected in BP-1. On the other hand, 107 QTLs explaining 11-29% phenotypic variation were detected in BP-2. Although most QTLs were environment and trait specific, few stable and consistent QTLs were also detected. Interestingly, 11 QTLs in BP-2 were associated with more than one trait. Among these QTLs, eight QTLs associated with days to 50% flowering and days to 75% maturity were located on CcLG07. One SNP "S7_14185076" marker in BP-2 population has been found associated with four traits, namely days to 50% flowering, days to 75% maturity, primary branches per plant and secondary branches per plant with positive additive effect. Hence, the present study has not only identified QTLs for yield-related traits, but also discovered novel alleles from wild species, which can be used for improvement of traits through genomics-assisted breeding.

Identification of QTLs and possible candidate genes conferring sheath blight resistance in rice (Oryza sativa L.).

Sheath blight, caused by the pathogenic fungus Rhizoctonia solani Kühn, is one of the most devastating diseases in rice. Breeders have always faced challenges in acquiring reliable and absolute resistance to this disease in existing rice germplasm. In this context, 40 rice germplasm including eight wild, four landraces, twenty- six cultivated and two advanced breeding lines were screened utilizing the colonized bits of typha. Except Tetep and ARC10531 which expressed moderate level of resistance to the disease, none could be found to be authentically resistant. In order to map the quantitative trait loci (QTLs) governing the sheath blight resistance, two mapping populations (F2 and BC1F2) were developed from the cross BPT-5204/ARC10531. Utilizing composite interval mapping analysis, 9 QTLs mapped to five different chromosomes were identified with phenotypic variance ranging from 8.40 to 21.76%. Two SSR markers namely RM336 and RM205 were found to be closely associated with the major QTLs qshb7.3 and qshb9.2 respectively and were attested as well in BC1F2 population by bulk segregant analysis approach. A hypothetical ß 1-3 glucanase with other 31 candidate genes were identified in silico utilizing rice database RAP-DB within the identified QTL region qshb9.2. A detailed insight into these candidate genes will facilitate at molecular level the intricate nature of sheath blight, a step forward towards functional genomics.

Expression of sunflower cytoplasmic male sterility-associated open reading frame, orfH522 induces male sterility in transgenic tobacco plants.

Sterility in the universally exploited PET1-CMS system of sunflower is associated with the expression of orfH522, a novel mitochondrial gene. Definitive evidence that ORFH522 is directly responsible for male sterility is lacking. To test the hypothesis that ORFH522 is sufficient to induce male sterility, a set of chimeric constructs were developed. The cDNA of orfH522 was cloned in-frame with yeast coxIV pre-sequence, and was expressed under tapetum-specific promoter TA29 (construct designated as TCON). For developing control vectors, orfH522 was cloned without the transit peptide under TA29 promoter (TON) or orfH522 was cloned with or without transit peptide under the constitutive CaMV35S promoter (SCOP and SOP). Among several independent transformants obtained with each of the gene cassettes, one third of the transgenics (6/17) with TCON were completely male sterile while more than 10 independent transformants obtained with each of the control vectors were fertile. The male sterile plants were morphologically similar to fertile plants, but had anthers that remained below the stigmatic surface at anthesis. RT-PCR analysis of the sterile plants confirmed the anther-specific expression of orfH522 and bright-field microscopy demonstrated ablation of the tapetal cell layer. Premature DNA fragmentation and programmed cell death was observed at meiosis stage in the anthers of sterile plants. Stable transmission of induced male sterility trait was confirmed in test cross progeny. This constitutes the first report at demonstrating the induction of male sterility by introducing orfH522 gene that could be useful for genetic engineering of male sterility.