Microsatellite (simple sequence repeats, SSRs) marker is one of the most widely used markers in marker-assisted breeding. As one type of functional markers, MicroRNA-based SSR (miRNA-SSR) markers have been exploited mainly in animals, but the development and characterization of miRNA-SSR markers in plants are still limited. In the present study, miRNA-SSR markers for Medicago truncatula (M. truncatula) were developed and their cross-species transferability in six leguminous species was evaluated. A total of 169 primer pairs were successfully designed from 130 M. truncatula miRNA genes, the majority of which were mononucleotide repeats (70.41%), followed by dinucleotide repeats (14.20%), compound repeats (11.24%) and trinucleotide repeats (4.14%). Functional classification of SSR-containing miRNA genes showed that all targets could be grouped into three Gene Ontology (GO) categories: 17 in biological process, 11 in molecular function, and 14 in cellular component. The miRNA-SSR markers showed high transferability in other six leguminous species, ranged from 74.56% to 90.53%. Furthermore, 25 Mt-miRNA-SSR markers were used to evaluate polymorphisms in 20 alfalfa accessions, and the polymorphism information content (PIC) values ranged from 0.39 to 0.89 with an average of 0.71, the allele number per marker varied from 3 to 18 with an average of 7.88, indicating a high level of informativeness. The present study is the first time developed and characterized of M. truncatula miRNA-SSRs and demonstrated their utility in transferability, these novel markers will be valuable for genetic diversity analysis, marker-assisted selection and genotyping in leguminous species.
Galactinol synthase (GolS, EC 188.8.131.52) catalyzes formation of galactinol and the subsequent synthesis of raffinose family oligosaccharides (RFOs). The relationship of GolS to drought and salt tolerance has been well documented, however, little information is available about the role of GolS gene in cold tolerance. A coding sequence of MfGolS1 cDNA was cloned from Medicago sativa subsp. falcata (i.e. M. falcata), a species that exhibits greater cold tolerance than alfalfa (Medicago sativa). MfGolS1 transcript was not detected in untreated vegetative tissues using RNA blot hybridization; however, it was greatly induced in leaves, but not in stem and petiole, after cold treatment. Higher levels of MfGolS1 transcript was induced and maintained in M. falcata than in M. sativa during cold acclimation. Accordingly, more sugars including sucrose, galactinol, raffinose and stachyose were accumulated in M. falcata than in M. sativa. The data indicated that MfGolS1 transcript and its resultant sugar accumulation were associated with the differential cold tolerance between M. falcata and M. sativa. MfGolS1 transcript was weakly induced by dehydration and salt stresses, but not responsive to abscisic acid (ABA). MfGolS1 could be induced by myo-inositol, which is proposed to participate in cold-induced MfGolS1 expression. Overexpression of MfGolS1 in tobacco resulted in elevated tolerance to freezing and chilling in transgenic plants as a result of enhanced levels of galactinol, raffinose, and stachyose. Tolerance to drought and salt stresses was also increased in the transgenic tobacco plants. It is suggested that MfGolS1 plays an important role in plant tolerance to abiotic stresses.
Pentachloronitrobenzene (PCNB) is a fungicide belonging to the organochlorine family and used extensively in agriculture for crop production. Many studies have implied that PCNB has become an environmental concern due to its widespread contamination in eco-systems. However, whether PCNB is bioaccumulated, degraded and phytotoxic in plants is poorly understood. In this study, several alfalfa (Medicago sativa) cultivars were grown in soil with PCNB to investigate their absorption and catabolism, including PCNB residues in the soil and PCNB-induced toxic responses in plants. Alfalfa plants varied widely in their ability to accumulate and degrade PCNB. The degradation rate of PCNB was 66.26-77.68% after alfalfa growth in the soils for 20 d, while the rates in the control (soil without alfalfa) were only 48.42%. Moreover, concentrations of PCNB residues in the rhizosphere soil were significantly higher than those in the non-rhizosphere soils. Alfalfa exposed to 10 mg kg(-1) PCNB showed inhibited growth and oxidative damage, but the effects of PCNB on the cultivars differed significantly, indicating that the alfalfa cultivars have different tolerance to PCNB. Activities of invertase (INV), urease (URE), polyphenol oxidase (PPO), alkaline phosphatase (ALP) and acid phosphatase (ACP) were assayed in the treated soils and showed that the enzyme activities were altered after PCNB exposure. The URE, PPO, ALP and ACP activities were increased in soil following the planting of alfalfa. The objective of the study was to analyze the potential of different cultivars of alfalfa to accumulate and degrade PCNB from the contaminated soil.
Dairy cattle often make poor use of protein when offered diets comprising high proportions of alfalfa (Medicago sativa L.) hay or silage because nonprotein N formed during forage conservation and ruminal fermentation exceeds requirements for rumen microbial protein synthesis; however, condensed tannins (CT) may reduce proteolysis in the silo and in the rumen, thereby potentially improving the efficiency of crude protein (CP) use in ruminant diets. Two harvests, yielding 12 hays and 12 silages made from alfalfa and birdsfoot trefoil (Lotus corniculatus L.) that varied in concentrations of CT, were evaluated for in situ disappearance kinetics of CP in 6 ruminally cannulated lactating Holstein dairy cows (627 ± 56.3 kg). Prior to conservation, alfalfa contained no detectable CT, whereas CT in fresh lyophilized birdsfoot trefoil ranged from 1.16 to 2.77% of dry matter, as determined by a modified acetone-butanol-HCl assay. Percentages of CP remaining at each incubation time were fitted to nonlinear regression models with or without a discrete lag time. Effective ruminal disappearance of CP (rumen-degradable protein, RDP) was calculated by 3 procedures that included (1) no discrete lag (RDPNL), (2) discrete lag (RDPL), and (3) discrete lag with a lag adjustment (RDPLADJ). Regardless of the calculation method, RDP declined linearly with increasing CT concentrations (R(2) = 0.62 to 0.97). Generally, tests of homogeneity showed that conservation type (hay or silage) or harvest (silage only) affected intercepts, but not slopes in regressions of RDP on CT. A positive relationship between lag time and CT suggests that the RDPLADJ approach may be most appropriate for calculating RDP for legumes containing tannins. With this approach, regression intercepts were mainly affected by conservation method, and RDPLADJ averaged 77.5 and 88.7% of CP for hay and silage, respectively, when no CT was present. Greater estimates of RDP for silages were related to extensive proteolysis in laboratory silos resulting in conversions of protein into nonprotein N forms, which readily washed out of Dacron bags. When RDPLADJ and CT were expressed on a CP basis, regression slopes indicated that each unit of CT protected 0.61 U of CP from ruminal degradation in hays and silages. Applying this relationship to a typical mid-maturity forage legume containing 21% CP suggests that a CT concentration of 3.8% of DM would be required to reduce RDP from 81% to a 70% target considered optimal for improving protein utilization and milk yields by dairy cattle.
The potential environmental risks of transgene exposure are not clear for alfalfa (Medicago sativa subsp. sativa), a perennial crop that is cross-pollinated by insects. We gathered data on feral alfalfa in major alfalfa seed-production areas in the western United States to (1) evaluate evidence that feral transgenic plants spread transgenes and (2) determine environmental and agricultural production factors influencing the location of feral alfalfa, especially transgenic plants. Road verges in Fresno, California; Canyon, Idaho; and Walla Walla, Washington were surveyed in 2011 and 2012 for feral plants, and samples were tested for the CP4 EPSPS protein that conveys resistance to glyphosate. Of 4580 sites surveyed, feral plants were observed at 404 sites. Twenty-seven percent of these sites had transgenic plants. The frequency of sites having transgenic feral plants varied among our study areas. Transgenic plants were found in 32.7%, 21.4.7% and 8.3% of feral plant sites in Fresno, Canyon and Walla Walla, respectively. Spatial analysis suggested that feral populations started independently and tended to cluster in seed and hay production areas, places where seed tended to drop. Significant but low spatial auto correlation suggested that in some instances, plants colonized nearby locations. Neighboring feral plants were frequently within pollinator foraging range; however, further research is needed to confirm transgene flow. Locations of feral plant clusters were not well predicted by environmental and production variables. However, the likelihood of seed spillage during production and transport had predictive value in explaining the occurrence of transgenic feral populations. Our study confirms that genetically engineered alfalfa has dispersed into the environment, and suggests that minimizing seed spillage and eradicating feral alfalfa along road sides would be effective strategies to minimize transgene dispersal.
Abstract Legumes (including alfalfa, clover, lupins, green beans and peas, peanuts, soybeans, dry beans, broad beans, dry peas, chickpeas, and lentils) represent an important component of the human diet in several areas of the world, especially in the developing countries, where they complement the lack of proteins from cereals, roots, and tubers. In some regions of the world, legume seeds are the only protein supply in the diet. The health benefits of legume consumption have received rising interest from researchers, and their consumption and production extends worldwide. Among European countries, higher legume consumption is observed around the Mediterranean, with per capita daily consumption between 8 and 23 g, while in Northern Europe, the daily consumption is less than 5 g per capita. The physiological effects of different legumes vary significantly. These differences may result from the polysaccharides composition, in particular, the quantity and variety of dietary fibers and starch, protein make-up, and variability in phytochemical content. The majority of legumes contain phytochemicals: bioactive compounds, including enzyme inhibitors, phytohemagglutinins (lectins), phytoestrogens, oligosaccharides, saponins, and phenolic compounds, which play metabolic roles in humans who frequently consume these foods. Dietary intake of phytochemicals may provide health benefits, protecting against numerous diseases or disorders, such as coronary heart disease, diabetes, high blood pressure and inflammation. The synergistic or antagonistic effects of these phytochemical mixtures from food legumes, their interaction with other components of the diet, and the mechanism of their action have remained a challenge with regard to understanding the role of phytochemicals in health and diseases. Their mitigating effects and the mechanism of their action need to be further addressed if we are to understand the role of phytochemicals in health and diseases. This review provides an overview of the nutritional quality of legumes and their potential contribution in cardiometabolic risk prevention.
P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling.
Microgreens, like sprouts, are relatively fast growing, and generally consumed raw. Moreover, as observed in sprouts, microbial contamination from preharvest sources could also be present in the production of microgreens. In this study, twoSalmonella entericaserovars (Hartford and Cubana) applied, at multiple inoculation levels, were evaluated for survival and growth on alfalfa sprouts and Swiss chard microgreens using the most probable number (MPN) method. Various abiotic factors were also examined for their effects onSalmonellasurvival and growth on sprouts and microgreens. Community level physiological profiles (CLPP) of sprout/microgreen rhizospheres with different levels ofS. entericainoculation at different growth stage were characterized with Biolog EcoPlates. In the seed contamination group, the ability ofS. entericato grow on sprouting alfalfa seeds was affected by both seed storage time and inoculation level, but not serovar. However, the growth ofS. entericaon Swiss chard microgreens was affected by serovar, and inoculation level. Seed storage time had little effect on the average level ofSalmonellapopulation in microgreens. In the irrigation water contamination group, the growth ofSalmonellain both alfalfa sprouts and microgreens was largely affected by inoculation level. Surprisingly, growing medium was found to play an important role inSalmonellasurvival and growth in microgreens. CLPP analysis showed significant changes in the microbial community metabolic diversity during sprouting for alfalfa sprouts, but few temporal changes were seen in microgreens. The data suggest that the change in rhizosphere bacterial functional diversity was host-dependent but independent ofSalmonellacontamination.IMPORTANCE:Sprouts and microgreens are considered “functional foods”, containing health promoting or disease preventing properties in addition to normal nutritional values. However, the microbial risk associated with microgreens has not been well-studied. Compared with sprouts, this study evaluatedSalmonellasurvival and growth on microgreens, and other abiotic factors that could affectSalmonellasurvival and growth on microgreens. This work provides baseline data to risk assessment of microbial contamination in sprouts and microgreens. Understanding the risks ofSalmonellacontamination and its effects on rhizosphere microbial communities enables a better understanding of host-pathogen dynamics in sprouts and microgreens. The data also contributes to innovative preventive control strategies forSalmonellacontamination in sprouts and microgreens.
The glyphosate-resistant gene, GR79Ms, was successfully introduced into the genome of alfalfa. The transgenic events may serve as novel germplasm resources in alfalfa breeding. Weed competition can reduce the alfalfa yield, generating new alfalfa germplasm with herbicide resistance is essential. To obtain transgenic alfalfa lines with glyphosate resistance, a new synthetic glyphosate-resistant gene GR79Ms encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was introduced into alfalfa germplasm by Agrobacterium tumefaciens-mediated transformation. In total, 67 transformants were obtained. PCR and Southern blot analyses confirmed that GR79Ms was successfully inserted into the genome of alfalfa. Reverse transcription-PCR and western blot analyses further demonstrated the expression of GR79Ms and its product, GR79Ms EPSPS. Moreover, two homozygous transgenic lines were developed in the T2 generation by means of molecular-assisted selection. Herbicide tolerance spray tests showed that the transgenic plants T0-GR1, T0-GR2, T0-GR3 and two homozygous lines were able to tolerate fourfold higher commercial usage of glyphosate than non-transgenic plants.
Alfalfa, usually known as the “Queen of Forages”, is the main source of vegetable protein to meat and milk production systems worldwide. This legume is extremely rich in proteins due to its highly efficient symbiotic association with nitrogen-fixing strains. In the last years, alfalfa culture has been displaced to saline environments by other important crops, including major cereals, a fact that has reduced its biomass production and symbiotic nitrogen fixation. In this short communication, we report the high forage production and nutrient quality of alfalfa under saline conditions by alfalfa transformation with the AtNHX1 Na+/H+ antiporter and inoculation with the stress-resistant nitrogen-fixing strain Sinorhizobium meliloti B401. Therefore, the incorporation of transgenic traits into salt-sensitive legumes in association with the inoculation with natural stress-resistant isolates could be a robust approach to improve the productivity and quality of these important nitrogen-fixing crops.