B. Kalyana Babu

Comparative Genomics and Association Mapping Approaches for Blast Resistant Genes in Finger Millet Using SSRs

The major limiting factor for production and productivity of finger millet crop is blast disease caused by Magnaporthe grisea. Since, the genome sequence information available in finger millet crop is scarce, comparative genomics plays a very important role in identification of genes/QTLs linked to the blast resistance genes using SSR markers. In the present study, a total of 58 genic SSRs were developed for use in genetic analysis of a global collection of 190 finger millet genotypes. The 58 SSRs yielded ninety five scorable alleles and the polymorphism information content varied from 0.186 to 0.677 at an average of 0.385. The gene diversity was in the range of 0.208 to 0.726 with an average of 0.487. Association mapping for blast resistance was done using 104 SSR markers which identified four QTLs for finger blast and one QTL for neck blast resistance. The genomic marker RM262 and genic marker FMBLEST32 were linked to finger blast disease at a P value of 0.007 and explained phenotypic variance (R2) of 10% and 8% respectively. The genomic marker UGEP81 was associated to finger blast at a P value of 0.009 and explained 7.5% of R2. The QTLs for neck blast was associated with the genomic SSR marker UGEP18 at a P value of 0.01, which explained 11% of R2. Three QTLs for blast resistance were found common by using both GLM and MLM approaches. The resistant alleles were found to be present mostly in the exotic genotypes. Among the genotypes of NW Himalayan region of India, VHC3997, VHC3996 and VHC3930 were found highly resistant, which may be effectively used as parents for developing blast resistant cultivars in the NW Himalayan region of India. The markers linked to the QTLs for blast resistance in the present study can be further used for cloning of the full length gene, fine mapping and their further use in the marker assisted breeding programmes for introgression of blast resistant alleles into locally adapted cultivars.

In-silico mining, type and frequency analysis of genic microsatellites of finger millet (Eleusine coracana (L.) Gaertn.): a comparative genomic analysis of NBS-LRR regions of finger millet with rice.

In recent years, the increased availability of the DNA sequences has given the possibility to develop and explore the expressed sequence tags (ESTs) derived SSR markers. In the present study, a total of 1956 ESTs of finger millet were used to find the microsatellite type, distribution, frequency and developed a total of 545 primer pairs from the ESTs of finger millet. Thirty-two EST sequences had more than two microsatellites and 1357 sequences did not have any SSR repeats. The most frequent type of repeats was trimeric motif, however the second place was occupied by dimeric motif followed by tetra-, hexa- and penta repeat motifs. The most common dimer repeat motif was GA and in case of trimeric SSRs, it was CGG. The EST sequences of NBS-LRR region of finger millet and rice showed higher synteny and were found on nearly same positions on the rice chromosome map. A total of eight, out of 15 EST based SSR primers were polymorphic among the selected resistant and susceptible finger millet genotypes. The primer FMBLEST5 could able to differentiate them into resistant and susceptible genotypes. The alleles specific to the resistant and susceptible genotypes were sequenced using the ABI 3130XL genetic analyzer and found similarity to NBS–LRR regions of rice and finger millet and contained the characteristic kinase-2 and kinase 3a motifs of plant R-genes belonged to NBS–LRR region. The In-silico and comparative analysis showed that the genes responsible for blast resistance can be identified, mapped and further introgressed through molecular breeding approaches for enhancing the blast resistance in finger millet.

Molecular and Biochemical Characterization of Short Duration Quality Protein Maize

Fifty microsatellite or simple sequence repeat (SSR) markers, spread across the maize genome were used for analyzing a set of 19 elite Quality Protein Maize (QPM) lines, including seventeen lines developed in India and two at CIMMYT, Mexico. Polymorphic profiles for 47 SSR loci have aided in differentiating the QPM inbred lines. The polymorphism information content (PIC) values among the inbreds ranged from 0.06 (umc2229) to 0.70 (umc1071) with an average of 0.45 per primer-pair. The genetic relationships as indicated by the cluster analysis of SSR data were largely in congruence with the known pedigree of the QPM lines. The study resulted in identification of two SSR markers, umc1071 and umc1063 with higher PIC values of 0.70 and 0.64, respectively. The tryptophan content among the genotypes was found to vary considerably. Two genotypes viz., VOL 2 and VOL 8 were found to differ significantly for tryptophan content (0.51% and 0.94%, respectively). Both these QPM genotypes being derived from the same non-QPM parent CM 145, makes them ideal for mapping of modifiers for tryptophan content.

Assessment of Genetic Diversity among the Elite Maize (Zea mays L) Genotypes Adapted to North-Western Himalayan Region of India using Microsatellite Markers

Maize is an important crop in the North-Western Himalayan states of India for food, feed and nutritional security of human population. Hybrid maize constitutes the major part of the maize area. Twenty four maize lines including the indigenous and exotic inbreds were amplified using 68 SSR primers, spread over the whole genome. The number of alleles across the primers ranged from two to eleven. The genotypes were grouped into different clusters using NTSYSpc2.11 programme. The clusters were well correlated with agronomic traits and resistance against turcicum blight. The PIC value was found to be highest for the primer bnlg1267 (0.84) while the lowest value was for the primer dupssr14 (0.09) with the mean value of 0.60. From this study we concluded that inbred V 359 is expected to give better combinations with CM 128, CM 129, V 340, V 357 and CM 212 for the development of hybrids suitable for the sub-tropical hill regions of India and elsewhere.

Development, identification and validation of CAPS marker for SHELL trait which governs dura, pisifera and tenera fruit forms in oil palm (Elaeis guineensis Jacq.)

The oil palm fruit forms (dura, pisifera and tenera) governed by the shell thickness gene (Sh) plays a major role in identification of fruit type and also influences palm oil yield. Identification of desired fruit type is a major asset to the breeders and oil palm workers for applications in breeding, seed certification and to reduce time, space and money spent on identification of fruit form. In the present study, we developed Sh gene specific primer pairs and bulk segregant analysis was done using 300 genomic and 8 genic SSR markers. We identified one cleaved amplified polymorphic site (CAPS) marker for differentiation of oil palm fruit type which produced two alleles (280 and 250bp) in dura genotypes, three alleles in tenera genotypes (550, 280, and 250bp) and one allele in pisifera genotypes (550bp). The shell allele sequencing results showed that two SNPs were present, of which SNP2 contributed for variation of fruit forms. The nucleotide ‘A’ was present in only dura genotypes, where as ‘T’ was present only in pisifera genotypes, which in turn led to the change of amino acid lysine to aspargine. The identified CAPS marker was validated on 300 dura, 25 pisifera and 80 tenera genotypes, 80 dura/ pisifera cross progenies and 60 lines of tenera/ tenera cross progeny. Association mapping of marker data with phenotypic data of eight oil yield related traits resulted in identification of seven significant QTLs by GLM approach, four by MLM approach at a significant threshold (P) level of 0.001. Significant QTLs were identified for fruit to bunch and oil to bunch traits, which explained R2 of 12.9% and 11.5% respectively. The CAPS marker used in the present study facilitate selection and timely distribution of desirable high yielding tenera sprouts to the farmers instead of waiting for 4–5 years. This saves a lot of land, time and money which will be a major breakthrough to the oil palm community.

Identification of microsatellite markers for finger millet genomics application through cross transferability of rice genomic SSR markers

In the present investigation, 345 rice genomic SSR markers were used for finding the cross transferability in twelve finger millet accessions. Out of which, 202 SSRs (58.6%) showed transferability among the finger millet genotypes and only 26 (13%) were found to be polymorphic. Thirteen markers were polymorphic between two finger millet genotypes, VR708 and GPU48, whereas five markers were between GE86 and PRM801. These markers can be effectively used in mapping populations for construction of linkage maps. Few putative orthologous regions for grain yield and its components like 1000 grain weight, leaf characteristics and root traits between rice and finger millet were detected. Among the biotic stresses, blast and brown plant hopper (BPH) resistance loci were found to be highly conserved. The PIC values of all the polymorphic loci varied from 0.15 to 0.55. Power Marker grouped the finger millet genotypes into two major clusters based on the races. The average gene diversity existing among the genotypes was relatively high (41%) indicating the usefulness of cross transferability in millet

Allele mining for resistance gene analogs (RGAs) in crop plants: A special emphasis on blast resistance in finger millet (Eleusine coracana L.)

Finger millet a nutritionally rich underutilized crop requires more attention of research community. One of the major limitations of finger millet for wider agronomic acceptability is because of its susceptibility to blast fungus Magnaporthe grisea, which is also the causative agent of blast in rice. A large amount of sequence data available in the public domain has facilitated identification and isolation of novel genes for blast resistance and other agronomically important traits. Availability of such large genomic data has made allele mining a viable approach for detecting novel alleles for blast resistance in finger millet. However, very scarce genomic information is available in finger millet, being the major hurdle for such approaches. In the present review, we have summarized different strategic approaches suitable for allele mining of resistance gene analogs (RGAs) in finger millet by utilizing the large sequence data available for rice through comparative genomics. This paves the way for transfer of blast alleles into high yielding, blast susceptible and locally well adapted germplasm through molecular breeding and genetic engineering approaches.

Omics of Model Plants

The multiple omics tools and strategies like high-throughput genome-scale genotyping platforms such as whole-genome re-sequencing, proteomics, and metabolomics provide greater opportunities to dissect molecular mechanisms and the discovery of key genes in developing ideal genotypes in the changing climate scenario. The last decade has seen rapid advances in functional genomic research globally. Most of the efforts involve construction of technological and resource platforms for high-throughput DNA sequencing, gene identification, and physical and genetic mapping; functional analysis of genomes for agronomic traits and biological processes; and identification and isolation of functional genes. The functional genomic research aims to understand how the genome functions at the whole-genome level, whereas proteomics looks for the systematic analysis of the protein population in a tissue, cell, or subcellular compartment. Metabolites are the end products of cellular process, and they show the response of biological systems to environmental changes. The current trend in metabolomic studies is to define the cellular status at a particular time point of development or physiological status. These techniques complement other techniques such as transcriptomics and proteomics and depict precise pictures of the whole cellular process. The growing number of sequenced plant genomes has opened up immense opportunities to study biological processes related to physiology, growth and development, and tolerance to biotic and abiotic stresses at the cellular and whole plant level using a novel systems-level approach. The “omics” approach integrates genome, proteome, transcriptome, and metabolome data into a single data set and can lead to the identification of unknown genes and their regulatory networks involved in metabolic pathways of interest. This will also help in understanding the genotype–phenotype relationship and consequently help to improve the quality and productivity of crop plants for the food and nutritional security of millions of human populations.

Comparative genetic analysis of wild finger millet accessions using finger millet and maize microsatellite markers

The genomic information available for finger millet is very scarce though the plant is a rich source of highly digestible proteins and dietary fibre with good amounts of soluble and insoluble fractions. In present study, 64 maize and finger millet genomic SSRs were used for cross transferability, identification of polymorphic markers and genetic diversity of finger millet, both cultivated and wild species. Out of 64 SSRs, only 43 (67%) were amplified across the finger millet genotypes. The PIC values of all the polymorphic loci across the 23 finger millet genotypes varied from 0.04 to 0.47 (average value 0.17). Based on the parameters of PIC values ≥ 0.26, gene diversity ≥0.43, inbreeding coefficient ≥ 0.60, the SSR loci UGEP33, UMC1858, UMC1805, and UMC2163 were found highly polymorphic. Comparison of SSR polymorphism in finger millet genotypes revealed that microsatellites of maize were more polymorphic and were able to identify more diversity in finger millet genotypes than the finger millet SSRs. Good correlations were found between genetic diversity analysis in differentiation of finger millet genotypes using finger millet and maize microsatellite markers. Dendrogram generated through UPGMA analysis grouped all the 23 genotypes clustered them into two major groups A and B with maximum similarity found between genotype pairs T552, T622 and T507, T74. The genotype pairs T671 and T760; T187 and T 631, T824 showed lowest similarity in finger millet which could be selected for hybrid plant production. The present study enriched the finger millet genomics by identifying suitable polymorphic markers of maize and finger millet, which can be used for diversity analysis, cultivar identification and QTL mapping studies.

Molecular marker technology for finger millet crop Improvement – An under-utilized, food and nutritional security crop

Finger millet, is a highly self-pollinated, allo-tetraploid crop believed to have been domesticated in East Africa, probably in the highland region that extends from Ethiopia to Uganda. It is known to save the lives of poor farmers from starvation at times of extreme drought. One of the major yield limiting factors is blast disease caused by Magnaporthe grisea. It is more nutritious than most cereal grains with respect to minerals, dietary fibre and amino acids. With the advancement of molecular marker technology, a lot of progress has been made in majority of the crops except neglected crops like small millets, such as finger millet, though it is highly nutritious crop. Aconsiderable amount of work has been done for assessing the genetic diversity among finger millet germplasm using microsatellite and EST based microsatellite markers. The first genetic map of finger millet was also available using 82 SSR markers. The present review describes the application, status and future perspectives of molecular marker technology for finger millet crop improvement.