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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Research Project #424531

Research Project: Regulation of Gene Expression in Alfalfa Development and Stress Tolerance

Location: Molecular Plant Pathology Laboratory

2014 Annual Report

1. Improve the efficiency of developing alfalfa with greater tolerance to biotic and abiotic stresses by characterizing gene-stress responses and pathways. Biotic and abiotic stresses cause significant yield losses in alfalfa and greatly reduce the crop’s productivity. Understanding the molecular mechanisms of stress tolerance and the ways by which stress-responsive genes are regulated is essential for improvement of alfalfa adaptability and breeding programs. 2. Aid plant breeders in improving alfalfa productivity and adaptability by implementing genetic and genomic approaches to improve traits related to biotic and abiotic stress tolerance, including, but not limited to, root-knot nematodes and salinity tolerance. Data on stress-responsive genes obtained in this study and other information on alfalfa genomics will be used to identify molecular markers associated with resistance and adaptation to abiotic and biotic stresses in alfalfa.

The research project will identify stress-responsive gene-candidates in alfalfa and assign them to cognate functional groups related to specific stress responses. It will quantify and confirm roles of the selected genes in adaptation to abiotic and biotic stresses and in regulation of stress responses. Sequence polymorphism in genes underlying stress tolerance will be delineated and molecular markers associated with resistance and adaptation of alfalfa to biotic and abiotic stresses developed. Markers will be validated through cooperative research collaborations.

Progress Report
Nematodes are one of the major limiting factors in alfalfa production. Widely distributed root knot nematode (Meloidogyne spp) may inflict significant damage to alfalfa fields. Very little information is available about molecular mechanisms that contribute to defense responses in alfalfa against Meloidugyne spp. Next generation sequencing (NGS) was performed to identify genes that defend alfalfa from damage caused by root knot nematode and alfalfa transcriptomes are being analyzed. There is need for alfalfa that is tolerant to stresses, including stress due to soil conditions of salinity (high salt). We are working toward identifying genes that underlie salt tolerance and associated molecular markers that would indicate the presence of those genes. NGS was performed on alfalfa varieties contrasting in salt tolerance. Transcriptome profiling identified multiple genes with putative roles in adaptation to salt stress. Data mining enabled prediction of nearly a thousand alfalfa transcription factors (TF), proteins that govern organismal development and response to the environment by regulating gene expression. In addition, transcriptome analysis revealed several hundred polymorphic simple sequence repeats (SSRs) associated with salt tolerance. Long non-coding (lnc) RNAs candidates in alfalfa were computationally predicted and their roles are being assessed experimentally. NGS data and bioinformatics analysis are producing genomic resources for integration into breeding programs aimed at the development of new improved cultivars and economic viability of alfalfa germplasm. Progress is directly related to the Objective 1 of the Project Plan: Improve the efficiency of developing alfalfa with greater tolerance to biotic and abiotic factors by characterizing gene-stress responses and pathways.

1. For the first time, alfalfa transcription factors, proteins that regulate gene expression in response to the environment, were systematized in a simple open-access database named AlfalfaTFDB ( The database provides users with an overview of all annotated alfalfa TF families, a reference to the description of each family, identified transcript and protein sequences and predicted ORFs (Open Reading Frames). Transcription factor datasets can be retrieved using a convenient search tool. Phylogenetic analysis of the largest TF families in alfalfa revealed that composition of the important ERF (Ethylene Responsive Factor) and NAC (Nam, ATAF, and CUC2 family of transcription factors) proteins that control diverse biological processes in alfalfa, including response to biotic and abiotic stresses, is quite distinct from other higher plants suggesting their specific biological roles in the development and evolution of alfalfa and legumes. GO (Gene Ontology) annotations, implemented to uncover specific functions of identified alfalfa TFs showed that significant portions of transcription factors are implicated in activities described by the GO categories developmental processes, response to stress and response to abiotic and biotic stimulus. These results suggest that regulation of genes involved in alfalfa development and responses to environmental factors is essential for the plant’s high value as a forage crop and its wide adaptability. In the absence of M. sativa’s genomic sequence and limited information on individual transcription factors available through random published studies, our data represent an essential resource addressing key proteins in the gene regulatory network of alfalfa.

2. Experimental real-time PCR assessment of alfalfa’s long non-coding RNAs, previously identified using in silico analysis of NGS data, revealed transcripts with potential roles in salinity tolerance. This project explores new avenues of research that were first identified in the predecessor project #1245-21000-265-00D on the development of molecular markers in alfalfa.

Review Publications
Postnikova, O., Shao, J.Y., Nemchinov, L.G. 2014. In silico identification of transcription factors in medicago sativa using available transcriptomic resources. Molecular Genetics and Genomics. 289:457-468.