Manjusha Verma

Development of EST-SSRs in finger millet (Eleusine coracana ssp. coracana) and their transferability to pearl millet (Pennisetum glaucum).

EST-SSR markers were developed using sequence information from 1740 expressed sequence tags (ESTs) of finger millet available in the public domain. A set of 31 SSR markers were synthesized based on di, tri, tetra and penta-nucleotide repeat sequences. These were used for PCR analysis of 11 elite germplasm lines of finger millet of Indian and African origin. Out of 31 SSR markers, amplification products were obtained for 17 primer pairs. Of these nine were found polymorphic with two alleles per locus. These 17 SSR primer pairs were also tested for amplification in three varieties of pearl millet (Pennisetum glaucum) and 11 could be transferred to pearl millet. The informative EST SSR markers developed, can be used in finger millet as well as pearl millet genetic improvement projects.

Genetic and Genomic Resources of Small Millets

ABSTRACT Small millets are very promising agricultural entity to ensure global food security. They gained remarkable importance in agriculture due to their resilience to climatic changes and increasing demand for nutritious food and feed. The genetic variability in the core and mini-core germplasm of small millets was characterized for nutritional composition and capacity to tolerate abiotic stresses that can be infused in breeding programs. Other than the foxtail millet, availability of genomic information in small millets is far below the mark for use in marker-assisted breeding and other genetic improvement programs. The genome sequence of foxtail millet has recently triggered a plethora of post-genomic analysis and envisaged foxtail millet as a model organism for the C4 grasses and bioenergy research. Recent developments in the next-generation sequencing technologies enabled us, with the simultaneous discovery of high-throughput markers and multiplexed genotyping of germplasm, to speedup marker-assisted breeding. In this context, an in-depth analysis of the wealth of diverse germplasm resources and future perspectives of integrating genomics in genome-wide marker-trait association and breeding in small millets is worthy.

Screening of Luffa cylindrica Roem. for resistance against Tomato Leaf Curl New Delhi Virus, inheritance of resistance, and identification of SRAP markers linked to the single dominant resistance gene

Summary Sponge gourd (Luffa cylindrica Roem.) is a popular cucurbitaceous vegetable on the Indian sub-continent. The crop is severely affected by Tomato Leaf Curl New Delhi Virus (ToLCNDV) a Geminivirus causing a yellow mosaic disease which results in 100% crop loss. Natural epiphytotic and challenge inoculation experiments revealed that DSG-6, an advanced inbred breeding line of L. cylindrica Roem., was resistant to ToLCNDV during both the Spring-Summer and rainy seasons of 2006 and 2007. Resistance was confirmed by PCR amplification with viral coat protein gene-specific primers that revealed a gene-specific band of 750 bp in susceptible (positive control) plants, but no 750 bp band in resistant plants. The inheritance of ToLCNDV resistance was studied through challenge inoculations in F2 and backcross (B1 and B2) populations of a cross between a resistant female (DSG-6) and a susceptible male parent (‘Pusa Sneha’). This revealed the dominant monogenic inheritance of the resistance gene. Two sequence-related amplified polymorphism (SRAP) markers were closely-linked to the ToLCNDV-susceptible gene in the susceptible parent and in a susceptible bulk population, while two SRAP markers were closely-linked to the resistance gene in the resistant parent and in a resistant bulk population. The SRAP markers thus identified can be used for early seedling selection in a large number of genotypes and should speed-up the development of Luffa cultivars that are resistant to ToLCNDV, thereby decreasing the incidence of this disease in this crop.

Development of EST-SSRs in watermelon (Citrullus lanatus var. lanatus) and their transferability to Cucumis spp.

Summary Watermelon, an important fruit crop in the family Cucurbitaceae, currently has few available Simple Sequence Repeat (SSR) markers. This study developed Expressed Sequence Tag-Simple Sequence Repeat (EST-SSRs) markers from watermelon and examined their transferability to Cucumis spp. We mined and characterised EST-SSRs from 1,762 watermelon Expressed Sequence Tags (ESTs) retrieved from the National Centre for Biotechnology Information (NCBI) database. Two hundred and thirty-two SSRs were detected following an analysis of the EST sequences and primer pairs were designed for these sequences using the “SSR Primer” programme. A subset of 40 new primer pairs flanking di-, tri-, tetra-, and penta-nucleotide repeat sequences were synthesised. These primer pairs were evaluated for amplification and polymorphism using DNA from seven watermelon varieties from India. Clear amplification products were obtained for 31 primer pairs, of which seven were found to be polymorphic with a mean allele number of two per locus. In addition, 14 and 20 EST-SSR markers could also be transferred to muskmelon and cucumber, respectively. Thus, this study has provided a new set of EST-SSR markers for watermelon, muskmelon, and cucumber, which can be used for genetic diversity analysis, varietal identification, and genetic improvement of watermelon.

Genetic polymorphisms among and between blast disease resistant and susceptible finger millet, Eleusine coracana (L.) Gaertn.

Fungal blast disease is one of the major constraints in finger millet production. Breeding for disease resistance in finger millet, needs characterization of genetic polymorphism among and between the resistant and susceptible genotypes. In total, 67 finger millet genotypes, which are resistant or susceptible to fungal blast disease, were analysed using sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers to assess genetic variations and select diverse parents. Twelve each of SRAP and SSR primers produced 95.1 and 93.1% polymorphic bands and grouped them into unweighted pair-group method with arithmetic average clusters. Two of the finger millet genotypes, IE 4709 (blast resistant) and INDAF 7 (susceptible) were distinguished as most diverse genotypes as parents. Several genotype-specific bands observed with SSR primers are potential in developing genotype-specific markers. A high genetic diversity within the resistant and susceptible genotypes, rather than between them, was revealed through Neis gene diversity (h) index and analysis of molecular variance. The finding helps us to understand the extent of genetic polymorphism between blast disease resistant and susceptible finger millet genotypes to exploit in resistance breeding programs.

Genome wide identification of calcium dependent protein kinase and related kinase gene families in Solanum melongena L.

In plants, Calcium-Dependent Protein Kinases (CDPKs) play multiple roles with a variety of functions inphysiological processes, biotic and abiotic stress together with plant hormone signalling. We conducted agenome-wide analysis of CDPK gene family in eggplant. Total 28 CDPKs and 2 CDPK-related kinases (CRKs)genes were identified in eggplant genome. Structural organisation of Solanum melongena CDPKs and CRK genes as well as different conserved domains and motifs were studied. Both SmeCDPKs and SmeCRK proteinsharbour STKc_CAMK type protein kinase domain, while only SmeCDPKs contained EF-hand type Ca2+ bindingdomain(s). Phylogenetic analysis suggested the conserved basic structure of SmeCDPKs and classified into four clades. The amino acid residue patterns of sub-domains including the conserved EF hands were identifiedfor SmeCDPKs. An elaborate search of eggplant ESTs available in public domain shows presence of SmeCDPK in different tissue specific libraries and most of them were expressed in mixture of flower bud, young fruit, leafand ovary tissues. SmeCDPK 28/18 and 34 were specific to ovary and stamen libraries, respectively. Further, expression of SmeCDPKs was checked making use of eggplant transcriptome available in public domain. Theinformation generated will significantly imparts the basic step for further functional study of CDPKs gene familyin eggplant.

Transferability of sponge gourd EST-SSR markers for genetic diversity assessment of Luffa species

Genetic diversity was studied in 47 Luffa genotypes with 17 EST-SSR primers, which generated 34 alleles, ranging 1–2 loci per primer. Forty seven genotypes were broadly classified into two different clusters. ClusterI comprised of genotypes DRG-98, Utkal Tripti, DRG-6, Sel-102, Pusa Nasdar, Pusa Nutan, Arka Sujat, DRG-73, DRG-61, DRG-42 and DRG-50, while Cluster II consisted of 36 genotypes, respectively. EST-SSR primer C90830_G3was found to be highly informative with PIC value of 0.3750. The variability in the species could be creditedto introgression and selection as a result of long history of cultivation under varied climatic conditions. Thepresent data provide adequate evidence of the applicability of EST-SSR markers for diversity analyses, cultivaridentification and characterization of the Luffa germplasm.