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United States Department of Agriculture

Agricultural Research Service

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

Location: Molecular Plant Pathology

2013 Annual Report


1a.Objectives (from AD-416):
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 providing approaches for using genetic data to increase desired traits including improved biotic and abiotic stress tolerance. Observations gathered through research on reference genomes may not always be applicable to alfalfa. Under Objective 2, the species-specific 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.


1b.Approach (from AD-416):
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.


3.Progress Report:
There is need for alfalfa that is tolerant to stresses, including stress due to soil conditions of salinity (high salt). Currently, programs in breeding alfalfa for tolerance to salinity are based on recurrent phenotypic selection. Tolerance to salinity is complex, in that it is determined by multiple genes acting in concert. This feature makes it difficult to achieve salt-tolerant alfalfa varieties through conventional breeding. We are working toward identifying genes that underlie salt tolerance and associated molecular markers that would indicate the presence of those genes.

We are continuing in-depth bioinformatics analysis of next generation sequencing data obtained from alfalfa varieties contrasting in salt tolerance. Several de novo assemblies of the alfalfa root transcriptome in response to salt stress, were generated using various k-mer values (overlap length between two sequence reads required to consider them as contiguous). As a result, multiple differentially expressed genes were identified, including genes with putative roles in adaptation to salt stress. Data mining revealed several hundred polymorphic simple sequence repeats (SSRs), molecular markers associated with salt tolerance. Our RNA sequencing and bioinformatics analysis are thus 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.


4.Accomplishments
1. Elevated salinity of soils greatly limits alfalfa production. Information is lacking on the many genes controlling salt tolerance in alfalfa. Identifying genes that control complex trait of salt tolerance in alfalfa will accelerate conventional breeding programs, increase production efficiency, sustainability and value of this most widely grown forage crop in the United States. We performed next generation sequencing to determine the activities (expression) of genes in alfalfa cultivars that contrasted in salt tolerance. The findings revealed a broad spectrum of genes affected by salt stress. More than 60,000 tentative consensus sequences (sequence assemblies) were obtained and analyzed. Bioinformatics analysis showed that the expression of 1,165 genes was significantly altered under salt stress. About 40% of differentially expressed genes were assigned to known gene ontology categories that describe gene products in terms of their associated biological processes and molecular functions. We hypothesized that certain genes play significant roles in the adaptation to salinity. Knowledge on genes and global gene expression data obtained in this work will contribute to development of new salt-tolerant cultivars and the molecular markers identified will be used for genetic mapping of the salinity trait and for marker-assisted selection in breeding programs.


Review Publications
Postnikova, O.A., Shao, J.Y., Nemchinov, L.G. 2013. Analysis of alfalfa root transcriptome in response to salinity stress. Plant And Cell Physiology. 54(7):1041-1055.

Last Modified: 10/22/2014
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