Molecular Mapping of QTLs Associated with Lodging Resistance in Dry Direct-Seeded Rice (Oryza sativa L.).

Dry direct-seeded rice (DSR) is an alternative crop establishment method with less water and labor requirement through mechanization. It provides better opportunities for a second crop during the cropping season and therefore, a feasible alternative system to transplanted lowland rice. However, lodging is one of the major constraints in attaining high yield in DSR. Identification of QTLs for lodging resistance and their subsequent use in improving varieties under DSR will be an efficient breeding strategy to address the problem. In order to map the QTLs associated with lodging resistance, a set of 253 BC3F4 lines derived from a backcross between Swarna and Moroberekan were evaluated in two consecutive years. A total of 12 QTLs associated with lodging resistance traits [culm length (qCL), culm diameter (qCD), and culm strength (qCS)] were mapped on chromosomes 1, 2, 6, and 7 using 193 polymorphic SNP markers. Two major and consistent effect QTLs, namely qCD1.1 (with R2 of 10%) and qCS1.1 (with R2 of 14%) on chromosome 1 with id1003559 being the peak SNP marker (flanking markers; id1001973-id1006772) were identified as a common genomic region associated with important lodging resistance traits. In silico analysis revealed the presence of Gibberellic Acid 3 beta-hydroxylase along with 34 other putative candidate genes in the marker interval region of id1001973-id1006772. The positive alleles for culm length, culm diameter, and culm strength were contributed by the upland adaptive parent Moroberekan. Our results identified significant positive correlation between lodging related traits (culm length diameter and strength) and grain yield under DSR, indicating the role of lodging resistant traits in grain yield improvement under DSR. Deployment of the identified alleles influencing the culm strength and culm diameter in marker assisted introgression program may facilitate the lodging resistance under DSR.

Molecular characterization of EcCIPK24 gene of finger millet (Eleusine coracana) for investigating its regulatory role in calcium transport.

Finger millet grains contain exceptionally high levels of calcium which is much higher compared to other cereals and millets. Since calcium is an important macronutrient in human diet, it is necessary to explore the molecular basis of calcium accumulation in the seeds of finger millet. CIPK is a calcium sensor gene, having role in activating Ca2+ exchanger protein by interaction with CBL proteins. To know the role of EcCIPK24 gene in seed Ca2+ accumulation, sequence is retrieved from the transcriptome data of two finger millet genotypes GP1 (low Ca2+) and GP45 (high Ca2+), and the expression was determined through qRT-PCR. The higher expression was found in root, shoot, leaf and developing spike tissue of GP45 compared to GP1; structural analysis showed difference of nine SNPs and one extra beta sheet domain as well as differences in vacuolar localization was predicted; besides, the variation in amino acid composition among both the genotypes was also investigated. Molecular modeling and docking studies revealed that both EcCBL4 and EcCBL10 showed strong binding affinity with EcCIPK24 (GP1) compared to EcCIPK24 (GP45). It indicates a genotypic structural variation, which not only affects the affinity but also calcium transport efficiency after interaction of CIPK-CBL with calcium exchanger (EcCAX1b) to pull calcium in the vacuole. Based on the expression and in silico study, it can be suggested that by activating EcCAX1b protein, EcCIPK24 plays an important role in high seed Ca2+ accumulation.

QTL Hotspots for Early Vigor and Related Traits under Dry Direct-Seeded System in Rice (Oryza sativa L.).

Strong seedling vigor is desirable trait in dry direct-seeded rice (DSR) for enhancing crop establishment and the ability to compete against weeds. A set of 253 BC3F4 lines derived from cross between Swarna and Moroberekan was phenotyped for early vigor (EV) and 8 related traits viz., early uniform emergence (EUE), shoot length (SHL), stem length (SL), shoot fresh weight (SFW), total fresh weight (TFW), shoot dry weight (SDW), total dry weight (TDW), and root dry weight (RDW). Composite interval mapping analysis using genotypic data from 194 SNP markers identified six genomic regions associated with traits on chromosomes 3, 4, 5, and 6 with phenotypic variance ranging from 2.5 to 18.6%. Among them 2 QTL regions; one on chr3 (id3001701-id300833) and the other on chr5 (wd5002636-id5001470) were identified as QTL hotspots A and B respectively and expressed consistently in field as well as glasshouse condition. The majority of QTLs identified for early vigor, and related traits were clustered in the QTL hotspots A (qEV3.1, qEUE3.1, qSHL3.1, qSL3.1, qSFW3.1, qTFW3.1, qRDW3.1 ) and QTL hotspot B (qEV5.1, qEUE5.1, qSHL5.1, qSL5.1, qSFW5.1, qSDW5.1, qTDW5.1 ). Ten putative candidate genes viz., 1-alpha-amylase precursor, 2-glutamate decarboxylase, 1-ethylene-insensitive 3, 3-expansin precursor, and 3-phenylalanine ammonia-lyase associated with the target traits were identified in the selected QTL regions. Mutations were identified in the coding region of alpha-amylase precursor and ethylene-insensitive 3 gene between the parents which can be utilized in marker assisted breeding. Trait relationships among the agro-physiological traits were examined to select the best genotypes for the given traits for use in future breeding programs.

Calcium Biofortification: Three Pronged Molecular Approaches for Dissecting Complex Trait of Calcium Nutrition in Finger Millet (Eleusine coracana) for Devising Strategies of Enrichment of Food Crops.

Calcium is an essential macronutrient for plants and animals and plays an indispensable role in structure and signaling. Low dietary intake of calcium in humans has been epidemiologically linked to various diseases which can have serious health consequences over time. Major staple food-grains are poor source of calcium, however, finger millet [Eleusine coracana (L.) Gaertn.], an orphan crop has an immense potential as a nutritional security crop due to its exceptionally high calcium content. Understanding the existing genetic variation as well as molecular mechanisms underlying the uptake, transport, accumulation of calcium ions (Ca2+) in grains is of utmost importance for development of calcium bio-fortified crops. In this review, we have discussed molecular mechanisms involved in calcium accumulation and transport thoroughly, emphasized the role of molecular breeding, functional genomics and transgenic approaches to understand the intricate mechanism of calcium nutrition in finger millet. The objective is to provide a comprehensive up to date account of molecular mechanisms regulating calcium nutrition and highlight the significance of bio-fortification through identification of potential candidate genes and regulatory elements from finger millet to alleviate calcium malnutrition. Hence, finger millet could be used as a model system for explaining the mechanism of elevated calcium (Ca2+) accumulation in its grains and could pave way for development of nutraceuticals or designer crops.

Identification and characterization of calcium transporter gene family in finger millet in relation to grain calcium content.

Calcium (Ca) is an essential mineral for proper growth and development of plants as well as animals. In plants including cereals, calcium is deposited in seed during its development which is mediated by specialized Ca transporters. Common cereal seeds contain very low amounts of Ca while the finger millet (Eleusine coracana) contains exceptionally high amounts of Ca in seed. In order to understand the role of Ca transporters in grain Ca accumulation, developing seed transcriptome of two finger millet genotypes (GP-1, low Ca and GP-45 high Ca) differing in seed Ca content was sequenced using Illumina paired-end sequencing technology and members of Ca transporter gene family were identified. Out of 109,218 and 120,130 contigs, 86 and 81 contigs encoding Ca transporters were identified in GP-1 and GP-45, respectively. After removal of redundant sequences, a total of 19 sequences were confirmed as Ca transporter genes, which includes 11 Ca(2+) ATPases, 07 Ca(2+)/cation exchangers and 01 Ca(2+) channel. The differential expressions of all genes were analyzed from transcriptome data and it was observed that 9 and 3 genes were highly expressed in GP-45 and GP-1 genotypes respectively. Validation of transcriptome expression data of selected Ca transporter genes was performed on different stages of developing spikes of both genotypes grown under different concentrations of exogenous Ca. In both genotypes, significant correlation was observed between the expression of these genes, especially EcCaX3, and on the amount of Ca accumulated in seed. The positive correlation of seed mass with the amount of Ca concentration was also observed. The efficient Ca transport property and responsiveness of EcCAX3 towards exogenous Ca could be utilized in future biofortification program.

Transcriptome wide identification and validation of calcium sensor gene family in the developing spikes of finger millet genotypes for elucidating its role in grain calcium accumulation.

BACKGROUND: In finger millet, calcium is one of the important and abundant mineral elements. The molecular mechanisms involved in calcium accumulation in plants remains poorly understood. Transcriptome sequencing of genetically diverse genotypes of finger millet differing in grain calcium content will help in understanding the trait. PRINCIPAL FINDING: In this study, the transcriptome sequencing of spike tissues of two genotypes of finger millet differing in their grain calcium content, were performed for the first time. Out of 109,218 contigs, 78 contigs in case of GP-1 (Low Ca genotype) and out of 120,130 contigs 76 contigs in case of GP-45 (High Ca genotype), were identified as calcium sensor genes. Through in silico analysis all 82 unique calcium sensor genes were classified into eight calcium sensor gene family viz., CaM & CaMLs, CBLs, CIPKs, CRKs, PEPRKs, CDPKs, CaMKs and CCaMK. Out of 82 genes, 12 were found diverse from the rice orthologs. The differential expression analysis on the basis of FPKM value resulted in 24 genes highly expressed in GP-45 and 11 genes highly expressed in GP-1. Ten of the 35 differentially expressed genes could be assigned to three documented pathways involved mainly in stress responses. Furthermore, validation of selected calcium sensor responder genes was also performed by qPCR, in developing spikes of both genotypes grown on different concentration of exogenous calcium. CONCLUSION: Through de novo transcriptome data assembly and analysis, we reported the comprehensive identification and functional characterization of calcium sensor gene family. The calcium sensor gene family identified and characterized in this study will facilitate in understanding the molecular basis of calcium accumulation and development of calcium biofortified crops. Moreover, this study also supported that identification and characterization of gene family through Illumina paired-end sequencing is a potential tool for generating the genomic information of gene family in non-model species.

Universal plant DNA barcode loci may not work in complex groups: a case study with Indian berberis species.

BACKGROUND: The concept of DNA barcoding for species identification has gained considerable momentum in animals because of fairly successful species identification using cytochrome oxidase I (COI). In plants, matK and rbcL have been proposed as standard barcodes. However, barcoding in complex genera is a challenging task. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the species discriminatory power of four reportedly most promising plant DNA barcoding loci (one from nuclear genome--ITS, and three from plastid genome--trnH-psbA, rbcL and matK) in species of Indian Berberis L. (Berberidaceae) and two other genera, Ficus L. (Moraceae) and Gossypium L. (Malvaceae). Berberis species were delineated using morphological characters. These characters resulted in a well resolved species tree. Applying both nucleotide distance and nucleotide character-based approaches, we found that none of the loci, either singly or in combinations, could discriminate the species of Berberis. ITS resolved all the tested species of Ficus and Gossypium and trnH-psbA resolved 82% of the tested species in Ficus. The highly regarded matK and rbcL could not resolve all the species. Finally, we employed amplified fragment length polymorphism test in species of Berberis to determine their relationships. Using ten primer pair combinations in AFLP, the data demonstrated incomplete species resolution. Further, AFLP analysis showed that there was a tendency of the Berberis accessions to cluster according to their geographic origin rather than species affiliation. CONCLUSIONS/SIGNIFICANCE: We reconfirm the earlier reports that the concept of universal barcode in plants may not work in a number of genera. Our results also suggest that the matK and rbcL, recommended as universal barcode loci for plants, may not work in all the genera of land plants. Morphological, geographical and molecular data analyses of Indian species of Berberis suggest probable reticulate evolution and thus barcode markers may not work in this case.