Mining the Cicer arietinum genome for the mildew locus O (Mlo) gene family and comparative evolutionary analysis of the Mlo genes from Medicago truncatula and some other plant species.

The mildew locus O (Mlo) gene family is ubiquitous in land plants. Some members of this gene family are involved in negative regulation of powdery mildew resistance, while others are involved in several other biological functions. Mlo proteins have characteristic seven transmembrane domains and a calmodulin-binding domain at their C-termini, and are associated with plasma membrane. The Mlo gene family has been studied in several economically important cereals, but little information is available on this gene family in the important legumes, Medicago truncatula Gaertn. and Cicer arietinum L. We carried out a comprehensive and comparative investigation of the Mlo gene family in these two species using the genome sequences available at the M. truncatula genome database (Mt v4.0) and NCBI (C. arietinum). A genome-wide homology-based search using Arabidopsis Mlo proteins as query identified 16 MtMlo (M. truncatula Mlo) and 14 CarMlo (C. arietinum Mlo) genes. The MtMlo and CarMlo genes had comparable gene structure, protein sequence and topology. Their chromosomal locations indicated the occurrence of extensive reorganization in the genomes of the two species after their divergence from the common ancestor. A multiple sequence alignment of 53 Mlo proteins from these two and several other species showed a highly conserved sequence block of seven amino acids, viz., L-ETPTW, towards their N-termini. The evolutionary phylogenetic analysis grouped the MtMlo and CarMlo members into four clusters, and most of the MtMlo and CarMlo members formed one-to-one ortholog pairs. The ka/ks analyses indicated that the MtMlo and CarMlo genes are subjected to intense purifying selection.

Validation of SSR markers associated with rust (Uromyces fabae) resistance in pea (Pisum sativum L.).

Pea rust is a devastating disease of peas especially in the sub-tropical regions of the world and greatly influenced by the environmental conditions during disease development. Molecular markers associated with pea rust resistance would be useful in marker assisted selection (MAS). Utility of molecular markers associated with the pea rust resistance were evaluated in 30 diverse pea genotypes using four SSR markers (AA446 and AA505 flanking the major QTL Qruf; AD146 and AA416 flanking the minor QTL, Qruf1). QTL, Qruf flanking markers were able to identify all the resistant genotypes when used together, except Pant P 31. While, SSR markers AD146 and AA416 flanking the minor QTL, Qruf1 were able to identify all the pea resistant genotypes used for validation, except for HUDP-11 by AD146 and Pant P 31 by AA416. Similarly, SSR markers AA446 and AA505 were able to identify all the susceptible pea genotypes, except IPFD 99-13, HFP 9415 and S- 143. SSR markers AD146 and AA416 were together able to identify all the pea susceptible genotypes used for validation, except KPMR 526, KPMR 632 and IPFD 99-13. On the basis of marker allele analysis it may be concluded that SSR markers (AA446, AA505, AD146 and AA416) can be used in MAS of pea rust resistance.

Molecular mapping for resistance to pea rust caused by Uromyces fabae (Pers.) de-Bary.

Pea rust caused by Uromyces fabae (Pers.) de-Bary is a major problem in warm humid regions causing huge economic losses. A mapping population of 136 F(6:7) recombinant inbred lines (RILs) derived from the cross between pea genotypes, HUVP 1 (susceptible) and FC 1 (resistant) was evaluated in polyhouse as well as under field conditions during two consecutive years. Infection frequency (IF) and area under disease progress curve (AUDPC) were used for evaluation of rust reaction of the RILs. A linkage map was constructed with 57 polymorphic loci selected from 148 simple sequence repeats (SSRs), 3 sequence tagged sites (STS), and 2 random amplified polymorphic (RAPD) markers covering 634 cM of genetic distance on the seven linkage groups of pea with an average interval length of 11.3 cM. Composite interval mapping (CIM) revealed one major (Qruf) and one minor (Qruf1) QTL for rust resistance on LGVII. The LOD (5.2-15.8) peak for Qruf was flanked by SSR markers, AA505 and AA446 (10.8 cM), explaining 22.2-42.4% and 23.5-58.8% of the total phenotypic variation for IF and AUDPC, respectively. The minor QTL was environment-specific, and it was detected only in the polyhouse (LOD values 4.2 and 4.8). It was flanked by SSR markers, AD146 and AA416 (7.3 cM), and explained 11.2-12.4% of the total phenotypic variation. The major QTL Qruf was consistently identified across all the four environments. Therefore, the SSR markers flanking Qruf would be useful for marker-assisted selection for pea rust (U. fabae) resistance.

Effect of qGN4.1 QTL for Grain Number per Panicle in Genetic Backgrounds of Twelve Different Mega Varieties of Rice.

BACKGROUND: Rice is a major source of food, particularly for the growing Asian population; hence, the utilization of genes for enhancing its yield potential is important for ensuring food security. Earlier, we have mapped a major quantitative trait loci (QTL) for the grain number per panicle, qGN4.1, using biparental recombinant inbred line (RIL) populations involving a new plant type Indica rice genotype Pusa 1266. Later, three independent studies have confirmed the presence of a major QTL for spikelet number by three different names (SPIKE, GPS and LSCHL4) in the same chromosomal region, and have implicated the overexpression of Nal1 gene as the causal factor for high spikelet number. However, the effect of qGN4.1 in different rice genetic backgrounds and expression levels of the underlying candidate genes is not known. RESULTS: Here, we report the effect of qGN4.1 QTL in the genetic backgrounds of 12 different high-yielding mega varieties of rice, introgressed by marker assisted-backcross breeding (MABB) using two QTL positive markers for foreground selection and two QTL negative flanking markers for recombinant selection together with phenotypic selection for the recovery of recipient parent genetic background. Analysis of the performance of BC2F3 plants showed a significant increase in the average number of well-filled grains per panicle in all the backgrounds, ranging from 21.6 in CSR 30-GN4.1 to 147.6 in Samba Mahsuri-GN4.1. Furthermore, qGN4.1 caused a significant increase in flag leaf width and panicle branching in most backgrounds. We identified BC3F3 qGN4.1 near-isogenic lines (NILs) with 92.0-98.0% similarity to the respective recipient parent by background analysis using a 50 K rice SNP genotyping chip. Three of the NILs, namely Pusa Basmati 1121-GN4.1, Samba Mahsuri-GN4.1 and Swarna-GN4.1, showed a significant yield superiority to their recipient parents. Analysis of differential gene expression revealed that high grain number in these QTL-NILs was unlikely due to the overexpression of Nal1 gene (LOC_Os04g52479). Instead, another tightly linked gene (LOC_Os04g52590) coding for a protein kinase domain-containing protein was consistently overexpressed in the high grain number NILs. CONCLUSION: We have successfully introgressed the qGN4.1 QTL for high grain number per panicle into 12 different mega varieties of rice using marker-assisted backcross breeding. The advanced near-isogenic lines are promising for the development of even higher yielding versions of these high-yielding mega varieties of rice.

Marker-Assisted Introgression of Saltol QTL Enhances Seedling Stage Salt Tolerance in the Rice Variety "Pusa Basmati 1".

Marker-assisted selection is an unequivocal translational research tool for crop improvement in the genomics era. Pusa Basmati 1 (PB1) is an elite Indian Basmati rice cultivar sensitive to salinity. Here, we report enhanced seedling stage salt tolerance in improved PB1 genotypes developed through marker-assisted transfer of a major QTL, Saltol. A highly salt tolerant line, FL478, was used as the Saltol donor. Parental polymorphism survey using 456 microsatellite (SSR)/QTL-linked markers revealed 14.3% polymorphism between PB1 and FL478. Foreground selection was carried out using three Saltol-linked polymorphic SSR markers RM8094, RM493, and RM10793 and background selection by 62 genome-wide polymorphic SSR markers. In every backcross generation, foreground selection was restricted to the triple heterozygotes of foreground markers, which was followed by phenotypic and background selections. Twenty-four near isogenic lines (NILs), with recurrent parent genome recovery of 96.0-98.4%, were selected after two backcrosses followed by three selfing generations. NILs exhibited agronomic traits similar to those of PB1 and additional improvement in the seedling stage salt tolerance. They are being tested for per se performance under salt-affected locations for release as commercial varieties. These NILs appear promising for enhancing rice production in salinity-affected pockets of Basmati Geographical Indication (GI) areas of India.

Distinct role of core promoter architecture in regulation of light-mediated responses in plant genes.

In the present study, we selected four distinct classes of light-regulated promoters. The light-regulated promoters can be distinctly grouped into either TATA-box-containing or TATA-less (initiator-containing) promoters. Further, using either native promoters or their swapped versions of core promoter elements, we established that TATA-box and Inr (Initiator) elements have distinct mechanisms which are involved in light-mediated regulation, and these elements are not swappable. We identified that mutations in either functional TATA-box or Inr elements lead to the formation of nucleosomal structure. The nucleotide diversity in either the TATA-box or Inr element in Arabidopsis ecotypes proposes that the nucleotide variation in core promoters can alter the gene expression. We show that motif overrepresentation in light-activated promoters encompasses different specific regulatory motifs present downstream of TSS (transcription start site), and this might serve as a key factor in regulating light promoters which are parallel with these elements. Finally, we conclude that the TATA-box or Inr element does not act in isolation, but our results clearly suggests the probable involvement of other distinct core promoter elements in concurrence with the TATA-box or Inr element to impart selectivity to light-mediated transcription.

Shoot bud regeneration from different explants of Bacopa monniera (L.) Wettst. by trimethoprim and bavistin.

A mass in vitro propagation system devoid of growth regulators for Bacopa monniera (L.) Wettst., a traditional Indian medicinal plant, has been developed. Direct shoot bud regeneration was induced by culturing internode and leaf explants on Murashige and Skoog's (MS) medium supplemented with an antibiotic (trimethoprim) or a fungicide (bavistin). Bavistin showed a marked cytokinin-like activity, as evident from high number of shoot buds induced in node, internode and leaf explants. Optimum adventitious shoot buds induction occurred at 300 mg/l bavistin from internode explants. In vitro regenerated shoots were elongated and rooted before transferred to field with 85% survival. The regeneration protocol developed in this study illustrates the usefulness of additives for mass propagation and germplasm conservation of B. monniera.