Genotyping-by-sequencing and functional genomics approaches for identification and validation of SNP markers/genes associated with grain calcium trait in finger millet
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Date
2018-07
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Finger millet (Eleusine coracana (L.) Gaertn), holds immense agricultural and economic importance due to its richness in calcium and its proteins being good source of essential amino acids. Hence, research towards such indigenous, mineral nutrient rich crop will surely help in accomplishing the challenges of mineral malnutrition. Markertrait studies in finger millet could be useful in the detection of markers associated with high grain calcium and, eventually, in selection of superior genotypes. With an aim to identify markers linked to grain yeild or its component traits and calcium content, initially phenotyping was done for fourteen agro-morphological traits and grain calcium content amongst finger millet global germplasm collection at two different environments viz. Almora and Pantnagar. Analysis of variance for both the environments resulted in highly significant differences among accessions for most of the traits, which showed the existence of sufficient variability. Also the wide range of variation in the agronomic performance of the accessions suggested that these traits could be considered as good candidates for marker-trait associations. The calcium content varied from 53mg/100g seed to 454mg/100g seed with an average of 277mg/ 100 g seed at E1 and from 42mg/100g seed to 466mg/100g seed with an average of 274mg/ 100 g seed at E2, which indicates presence of sufficient variability for grain calcium trait amongst the selected finger millet genotypes. Further, to study the genetic makeup of 202 diverse finger millet accessions Genotyping by Sequencing (GBS) analysis was done, which resulted in a genome wide set of
several thousand SNPs identified within every accession. Genetic diversity and population structure analysis was conducted on a natural collection of 202 finger millet genotypes. Neighbour joining cluster analysis grouped the population into 3 clusters ‘A’, ‘B’ and ‘C’ based on their geographical location. The major cluster A consisted of broadly all the Indian genotypes whereas cluster B and C comprised of exotic genotypes. A majority of accessions of Asian origin showed their tendency to cluster together with accessions of African lowland countries, which fits the hypothesis that finger millet was first domesticated in African highlands and from there it was introduced to India, thereby proving the origin of finger millet. There was good congruence between the phylogenetic tree and the population structure identified using STRUCTURE software. SNPs through GBS were used for association mapping to identify reliable marker(s) linked to grain yield or its component traits and calcium content. Five SNP markers showed homology to candidate genes of Oryza sativa (Rice) and Setaria italica, which play an important role in flowering, maturity and grain yield. Two potential SNP markers were found to be significantly associated to grain calcium content. The orthologous regions of the two identified SNP markers were found in cereal crops like Setaria italica encoding genes for Calmodulin Binding Protein (CBP) and CBL-Interacting Protein Kinase 7(CIPK7), which might play important role in grain calcium accumulation process. Promoter analysis of these genes revealed that they contain many stress responsive elements. Since, finger millet is a drought tolerant crop and also accumulate high amounts of calcium, the promoter region of these genes contain stress responsive elements, thus imparting stress tolerance to finger millet along with high grain calcium content as calcium ions play a central role in stress signaling. Differential expression analysis by qPCR showed that the two genes (EcCBP and EcCIPK7) were highly expressed in high calcium genotypes as compared to medium and low calcium genotypes. EcCIPK7 gene identified in the present study might be involved in regulating the activity of CaX exchanger and consequently increases the calcium storage in vacuoles, thus forming the basis of high grain calcium accumulation in finger millet. Since calmodulin (CaM) expresses abundantly in developing spikes and thus regulates the Ca2+ ATpase transporter located in vacuolar membrane, therefore, higher expression of EcCBP gene in later stages of spike development might be responsible for its interaction with CaM and might be responsible for higher accumulation of calcium in finger millet grains. Therefore, these genes could be considered as strong candidates for higher grain yield, Ca accumulation and its further use for Ca bio-fortification. However, exploration of its spatial distribution within seed, over expression and
knockout studies will help in understanding the exact role of these genes in high seed calcium accumulation. Since, there is very limited information on genetic analysis of calcium content in finger millet grains, results from the present study would provide a fundamental basis for future research on genetic improvement of calcium content in finger millet and other crops.
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