A reference assembly for the legume cover crop hairy vetch (Vicia villosa).

Vicia villosa is an incompletely domesticated annual legume of the Fabaceae family native to Europe and Western Asia. V. villosa is widely used as a cover crop and forage due to its ability to withstand harsh winters. Here, we generated a reference-quality genome assembly (Vvill1.0) from low error-rate long-sequence reads to improve the genetic-based trait selection of this species. Our Vvill1.0 assembly includes seven scaffolds corresponding to the seven estimated linkage groups and comprising approximately 68% of the total genome size of 2.03 Gbp. This assembly is expected to be a useful resource for genetically improving this emerging cover crop species and provide useful insights into legume genomics and plant genome evolution.

Intraspecific Variation for Leaf Physiological and Root Morphological Adaptation to Drought Stress in Alfalfa (Medicago sativa L.).

Drought stress reduces crop biomass yield and the profitability of rainfed agricultural systems. Evaluation of populations or accessions adapted to diverse geographical and agro-climatic environments sheds light on beneficial plant responses to enhance and optimize yield in resource-limited environments. This study used the morphological and physiological characteristics of leaves and roots from two different alfalfa subspecies during progressive drought stress imposed on controlled and field conditions. Two different soils (Experiments 1 and 2) imposed water stress at different stress intensities and crop stages in the controlled environment. Algorithm-based image analysis of leaves and root systems revealed key morphological and physiological traits associated with biomass yield under stress. The Medicago sativa subspecies (ssp.) sativa population, PI478573, had smaller leaves and maintained higher chlorophyll content (CC), leaf water potential, and osmotic potential under water stress. In contrast, M. sativa ssp. varia, PI502521, had larger leaves, a robust root system, and more biomass yield. In the field study, an unmanned aerial vehicle survey revealed PI502521 to have a higher normalized difference vegetation index (vegetation cover and plant health characteristics) throughout the cropping season, whereas PI478573 values were low during the hot summer and yielded low biomass in both irrigated and rainfed treatments. RhizoVision Explorer image analysis of excavated roots revealed a smaller diameter and a narrow root angle as target traits to increase alfalfa biomass yield irrespective of water availability. Root architectural traits such as network area, solidity, volume, surface area, and maximum radius exhibited significant variation at the genotype level only under limited water availability. Different drought-adaptive strategies identified across subspecies populations will benefit the plant under varying levels of water limitation and facilitate the development of alfalfa cultivars suitable across a broad range of growing conditions. The alleles from both subspecies will enable the development of drought-tolerant alfalfa with enhanced productivity under limited water availability.

Genotype- and tissue-specific miRNA profiles and their targets in three alfalfa (Medicago sativa L) genotypes.

BACKGROUND: Alfalfa (Medicago sativa L.) is a forage legume with significant agricultural value worldwide. MicroRNAs (miRNAs) are key components of post-transcriptional gene regulation and essentially regulate many aspects of plant growth and development. Although miRNAs were reported in alfalfa, their expression profiles in different tissues and the discovery of novel miRNAs as well as their targets have not been described in this plant species. RESULTS: To identify tissue-specific miRNA profiles in whole plants, shoots and roots of three different alfalfa genotypes (Altet-4, NECS-141and NF08ALF06) were used. Small RNA libraries were generated and sequenced using a high-throughput sequencing platform. Analysis of these libraries enabled identification of100 miRNA families; 21 of them belong to the highly conserved families while the remaining 79 families are conserved at the minimum between M. sativa and the model legume and close relative, M. truncatula. The profiles of the six abundantly expressed miRNA families (miR156, miR159, miR166, miR319, miR396 and miR398) were relatively similar between the whole plants, roots and shoots of these three alfalfa genotypes. In contrast, robust differences between shoots and roots for miR160 and miR408 levels were evident, and their expression was more abundant in the shoots. Additionally, 17 novel miRNAs were identified and the relative abundance of some of these differed between tissue types. Further, the generation and analysis of degradome libraries from the three alfalfa genotypes enabled confirmation of 69 genes as targets for 31 miRNA families in alfalfa. CONCLUSIONS: The miRNA profiles revealed both similarities and differences in the expression profiles between tissues within a genotype as well as between the genotypes. Among the highly conserved miRNA families, miR166 was the most abundantly expressed in almost all tissues from the three genotypes. The identification of conserved and novel miRNAs as well as their targets in different tissues of multiple genotypes increased our understanding of miRNA-mediated gene regulation in alfalfa and could provide valuable insights for practical research and plant improvement applications in alfalfa and related legume species.

Alfalfa (Medicago sativa L.).

Alfalfa (Medicago sativa L.) is a high-quality forage crop widely grown throughout the world. This chapter describes an efficient protocol that allows for the generation of large number of transgenic alfalfa plants by sonication-assisted Agrobacterium-mediated transformation. Binary vectors carrying different selectable marker genes that confer resistance to phosphinothricin (bar), kanamycin (npt II), or hygromycin (hph) were used to generate transgenic alfalfa plants. Intact trifoliates collected from clonally propagated plants in the greenhouse were sterilized with bleach and then inoculated with Agrobacterium strain EHA105. More than 80 % of infected leaf pieces could produce rooted transgenic plants in 4-5 months after Agrobacterium-mediated transformation.

Early lignin pathway enzymes and routes to chlorogenic acid in switchgrass (Panicum virgatum L.).

Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties.

The down-regulation of enzymes of the monolignol pathway results in reduced recalcitrance of biomass for lignocellulosic ethanol production. Cinnamoyl CoA reductase (CCR) catalyzes the first step of the phenylpropanoid pathway specifically dedicated to monolignol biosynthesis. However, plants contain multiple CCR-like genes, complicating the selection of lignin-specific targets. This study was undertaken to understand the complexity of the CCR gene family in tetraploid switchgrass (Panicum virgatum) and to determine the biochemical properties of the encoded proteins. Four switchgrass cDNAs (most with multiple variants) encoding putative CCRs were identified by phylogenetic analysis, heterologously expressed in Escherichia coli, and the corresponding enzymes were characterized biochemically. Two cDNAs, PvCCR1 and PvCCR2, encoded enzymes with CCR activity. They are phylogenetically distinct, differentially expressed, and the corresponding enzymes exhibited different biochemical properties with regard to substrate preference. PvCCR1 has higher specific activity and prefers feruloyl CoA as substrate, whereas PvCCR2 prefers caffeoyl and 4-coumaroyl CoAs. Allelic variants of each cDNA were detected, but the two most diverse variants of PvCCR1 encoded enzymes with similar catalytic activity. Based on its properties and expression pattern, PvCCR1 is probably associated with lignin biosynthesis during plant development (and is therefore a target for the engineering of improved biomass), whereas PvCCR2 may function in defense.