Alfalfa Germplasm Research Articles

Identification of salt tolerance in alfalfa (Medicago sativa L.) germplasm based on two-way stress tolerance index

Identification of salt tolerance in alfalfa (Medicago sativa L.) germplasm based on two-way stress tolerance index

Serological detection of a novel ipomo-like virus infecting alfalfa in the U.S.

This study describes production of polyclonal antibodies against recently reported novel potyvirid infecting alfalfa (Medicago sativa L.). The virus was first found in alfalfa seed material and later identified in plant samples collected from commercial alfalfa fields in Arizona, USA. It was classified as a novel species related to the members of the genus Ipomovirus and potentially representing a new genus in the family Potyviridae (Nemchinov et al., 2023b). Polyclonal antibodies were produced against the predicted viral coat protein expressed in bacterial cells and used in different types of immunoassays for specific detection of this emerging virus. They could be helpful in plant virus certification programs, screening of alfalfa germplasm, research on pathogenicity, biology, and geographic distribution of this emerging virus.

Genetic diversity studies in local collections and advanced generations of lucerne (Medicago sativa L.) genotypes for fodder yield and yield-attributing traits

Precise information about genetic divergence is vital for a productive breeding program, as genetically diverse parents produce high heterotic effects, producing desirable segregants with higher yield levels. This study assessed seventy genotypes and four checks to estimate the extent of genetic diversity based on ten morphological traits attributed to yield. The genotypes showed significant differences for all the traits considered. Based on the Mahalanobis D2 statistics, the genotypes were grouped into ten clusters, with cluster I having the maximum number of genotypes, whereas cluster IX had a single genotype. The average inter-cluster distances revealed that the genotypes in clusters VI and VII were more diverse. In contrast, clusters I and III genotypes had the shortest inter-cluster distances, indicating genetic closeness. The study revealed a broad genotypic diversity within and between the alfalfa germplasm collections. The genotypes in clusters I, III, and V provide excellent genetic material for alfalfa yield improvement. They offer a possible way to exploit the existing variability to develop superior populations or composites

Alfalfa Responses to Intensive Soil Compaction: Effects on Plant and Root Growth, Phytohormones and Internal Gene Expression.

The perennial legume alfalfa (Medicago sativa L.) is of high value in providing cheap and high-nutritive forages. Due to a lack of tillage during the production period, the soil in which alfalfa grows prunes to become compacted through highly mechanized agriculture. Compaction deteriorates the soil's structure and fertility, leading to compromised alfalfa development and productivity. However, the way alfalfa responses to different levels of soil compaction and the underlying molecular mechanism are still unclear. In this study, we systematically evaluated the effects of gradient compacted soil on the growth of different cultivars of alfalfa, especially the root system architecture, phytohormones and internal gene expression profile alterations. The results showed that alfalfa growth was facilitated by moderate soil compaction, but drastically inhibited when compaction was intensified. The inhibition effect was universal across different cultivars, but with different severity. Transcriptomic and physiological studies revealed that the expression of a set of genes regulating the biosynthesis of lignin and flavonoids was significantly repressed in compaction treated alfalfa roots, and this might have resulted in a modified secondary cell wall and xylem vessel formation. Phytohormones, like ABA, are supposed to play pivotal roles in the regulation of the overall responses. These findings provide directions for the improvement of field soil management in alfalfa production and the molecular breeding of alfalfa germplasm with better soil compaction resilience.

Overexpression of P5CDH from Cleistogenes songorica improves alfalfa growth performance under field drought conditions

Water stress affects the metabolic regulation and delays the growth and development of alfalfa, causing a reduction in biomass. New alfalfa germplasm was created with improved drought tolerance in greenhouse conditions by introducing the key gene P5CDH1 from C. songorica, a xerophytic grass. However, the field adaptability and response mechanism of new drought-tolerant alfalfa germplasms under water stress are still unclear. In the present study, the yield and quality traits of transgenic CsP5CDH1 alfalfa lines under water stress and normal irrigation conditions were measured and analyzed for two years. The genetic variance components of the tested traits were calculated from the data fitted by the mixed linear model. The plant height of all lines showed significant genotypic variation (σ2g) (P < 0.05), and the stem diameter, stem number, and dry weight of all lines had a significant genotype × environment interaction (σ2ge) (P < 0.05). The heritability (H) of plant height, stem diameter, stem number, dry weight and leaf-to-stem ratio of alfalfa lines were 0.87, 0.52, 0.59, 0.52 and 0.50, respectively. There were significant genotype × environment interactions (σ2ge) (P < 0.05) for the quality traits of all lines. The heritabilities (H) of acid detergent fiber and neutral detergent fiber were 0.65 and 0.64, respectively. The results of transcriptional expression analysis with RNA-seq showed that the genes MsProDH1, MsProDH4, MsProDH5, MsP5CDH1, MsP5CS5, MsP5CS9, and MsP5CR1, which are involved in the proline metabolism pathway, played an important role in the drought tolerance of innovative alfalfa germplasm. Under water stress, with the regulation of key genes in the proline metabolism pathway, the proline content of all alfalfa lines increased to varying degrees. Among them, the proline content in the shoots and roots of transgenic line L6 was 7.29 times and 12.22 times that under normal irrigation conditions, respectively. The present study helped to clarify that the new germplasm of alfalfa transformed with the CsP5CDH gene synthesized a large amount of proline under water stress, and effectively slowed leaf water loss, thus improving the drought resistance of alfalfa.

Transcriptome Analysis Reveals the Vital Role of ABA Plays in Drought Tolerance of the ABA-Insensitive Alfalfa (Medicago sativa L.)

Drought stress severely affects alfalfa (Medicago sativa L.) growth and production. It is particularly important to analyze the key networks of drought in alfalfa through physiological and molecular levels. However, how to quickly screen drought-tolerant alfalfa germplasm and how to elucidate the molecular pathways of alfalfa responding to drought are less studied. In this study, based on our previous research, we further verified the association between the heritability of ABA sensitivity during seed germination and drought tolerance of plants and identified the key pathways of drought tolerance differences between ABA-sensitivity (S1-0) and -insensitivity (S1-50) plants via RNA-seq and analysis. The results showed that the sensitivity to ABA in alfalfa seeds can be inherited and that plants that are insensitive to ABA during germination show stronger drought tolerance. An analysis of the differentially expressed genes (DEGs) revealed that ABA biosynthesis and signaling, amino acid metabolism, LEA, and wax synthesis-related pathways may be the key pathways that can be used for drought tolerance improvement in alfalfa. DEGs such as NCED, PYR/PYL, and PP2C may contribute to drought tolerance in the S1-50 plant. The study further confirms that screening with ABA at the seed germination stage can select alfalfa lines with good drought tolerance, which provides a new theoretical basis for alfalfa drought tolerance breeding. The expression of the key genes of alfalfa in response to drought stress was also tested.

Multi-omics integration analysis reveals the molecular mechanisms of drought adaptation in homologous tetraploid alfalfa(Medicago sativa 'Xinjiang-Daye').

Drought stress is a predominant abiotic factor leading to decreased alfalfa yield. Genomic ploidy differences contribute to varying adaptation mechanisms of different alfalfa cultivars to drought conditions. This study employed a multi-omics approach to characterize the molecular basis of drought tolerance in a tetraploid variant of alfalfa (Medicago sativa, Xinjiang-Daye). Under drought treatment, a total of 4446 genes, 859 proteins, and 524 metabolites showed significant differences in abundance. Integrative analysis of the multi-omics data revealed that regulatory modules involved in flavonoid biosynthesis, plant hormone signalling transduction, linoleic acid metabolism, and amino acid biosynthesis play crucial roles in alfalfa adaptation to drought stress. The severity of drought led to the substantial accumulation of flavonoids, plant hormones, free fatty acids, amino acids, and their derivatives in the leaves. Genes such as PAL, 4CL, CHI, CHS, PP2C, ARF_3, and AHP_4 play pivotal regulatory roles in flavonoid biosynthesis and hormone signalling pathways. Differential expression of the LOX gene emerged as a key factor in the elevated levels of free fatty acids. Upregulation of P5CS_1 and GOT1/2 contributed significantly to the accumulation of Pro and Phe contents. ERF19 emerged as a principal positive regulator governing the synthesis of the aforementioned compounds. Furthermore, observations suggest that Xinjiang-Daye alfalfa may exhibit widespread post-transcriptional regulatory mechanisms in adapting to drought stress. The study findings unveil the critical mechanisms by which Xinjiang-Daye alfalfa adapts to drought stress, offering novel insights for the improvement of alfalfa germplasm resources.

The chromosome-level assembly of the wild diploid alfalfa genome provides insights into the full landscape of genomic variations between cultivated and wild alfalfa.

Alfalfa (Medicago sativa L.) is one of the most important forage legumes in the world, including autotetraploid (M. sativa ssp. sativa) and diploid alfalfa (M. sativa ssp. caerulea, progenitor of autotetraploid alfalfa). Here, we reported a high-quality genome of ZW0012 (diploid alfalfa, 769 Mb, contig N50 = 5.5 Mb), which was grouped into the Northern group in population structure analysis, suggesting that our genome assembly filled a major gap among the members of M. sativa complex. During polyploidization, large phenotypic differences occurred between diploids and tetraploids, and the genetic information underlying its massive phenotypic variations remains largely unexplored. Extensive structural variations (SVs) were identified between ZW0012 and XinJiangDaYe (an autotetraploid alfalfa with released genome). We identified 71 ZW0012-specific PAV genes and 1296 XinJiangDaYe-specific PAV genes, mainly involved in defence response, cell growth, and photosynthesis. We have verified the positive roles of MsNCR1 (a XinJiangDaYe-specific PAV gene) in nodulation using an Agrobacterium rhizobia-mediated transgenic method. We also demonstrated that MsSKIP23_1 and MsFBL23_1 (two XinJiangDaYe-specific PAV genes) regulated leaf size by transient overexpression and virus-induced gene silencing analysis. Our study provides a high-quality reference genome of an important diploid alfalfa germplasm and a valuable resource of variation landscape between diploid and autotetraploid, which will facilitate the functional gene discovery and molecular-based breeding for the cultivars in the future.

Characterization of the seed virome of alfalfa (Medicago sativa L)

BackgroundSeed transmission of plant viruses can be important due to the role it plays in their dissemination to new areas and subsequent epidemics. Seed transmission largely depends on the ability of a virus to replicate in reproductive tissues and survive during the seed maturation process. It occurs through the infected embryo or mechanically through the contaminated seed coat. Alfalfa (Medicago sativa L.) is an important legume forage crop worldwide, and except for a few individual seedborne viruses infecting the crop, its seed virome is poorly known. The goal of this research was to perform initial seed screenings on alfalfa germplasm accessions maintained by the USDA ARS National Plant Germplasm System in order to identify pathogenic viruses and understand their potential for dissemination.MethodsFor the detection of viruses, we used high throughput sequencing combined with bioinformatic tools and reverse transcription-polymerase chain reactions.ResultsOur results suggest that, in addition to common viruses, alfalfa seeds are infected by other potentially pathogenic viral species that could be vertically transmitted to offspring.ConclusionsTo the best of our knowledge, this is the first study of the alfalfa seed virome carried out by HTS technology. This initial screening of alfalfa germplasm accessions maintained by the NPGS showed that the crop’s mature seeds contain a broad range of viruses, some of which were not previously considered to be seed-transmitted. The information gathered will be used to update germplasm distribution policies and to make decisions on the safety of distributing germplasm based on viral presence.

Genetic evaluation and germplasm identification analysis on ITS2, trnL-F, and psbA-trnH of alfalfa varieties germplasm resources.

In this study, genetic diversity and germplasm identification of 28 alfalfa germplasm cultivars materials were evaluated by analyzing their internal transcribed spacer 2 (ITS2), trnL-F, and psbA-trnH sequences to provide the innovative reference of alfalfa varieties genetic diversity and identify research. The results showed that the fragment average length of ITS2, trnL-F, and psbA-trnH sorting sequences were 455.7 bp, 230.3 bp, and 345.6 bp, respectively. The ITS2 sequence was too conservative to reflect the individual differences between intercultivars and intracultivars in the preliminary experiment. Furthermore, trnL-F and psbA-trnH sequence differences were relatively small between intercultivars but significant between intracultivars. Alfalfa cultivars were divided into four groups by sequence similarity clustering. Alfalfa cultivars trnL-F and psbA-trnH sequences have apparent differences, showing that chloroplast conservative sequences were independent evolution. Compared with trnL-F and psbA-trnH sequences of alfalfa cultivars, psbA-trnH sequence has abundant variation sites and can better reflect the differences between cultivars than the trnL-F sequence. Therefore, the psbA-trnH sequence can identify different alfalfa cultivars and establish the DNA sequence fingerprint.

ABA-insensitivity of alfalfa (Medicago sativa L.) during seed germination associated with plant drought tolerance

Abscisic acid (ABA) is a vital stress hormone of plants in coping with adverse environmental conditions, such as drought stress. Sensitivity of seed germination to exogenous ABA treatment could be linked to varied drought stress responses in different plant species. Polyploid and outcrossing nature of alfalfa makes it an extensive reservoir of genetic variation, and each single alfalfa seed may have a different genetic background. We speculate the alfalfa seeds resistant to exogenous ABA treatment during germination could lead to a strong plant drought tolerance. Here, we selected the alfalfa seedlings (S0–50) from seeds germinated under 50 μM ABA treatment. The S0–50 alfalfa plants showed more sensitivity in stomatal closure to exogenous ABA and PEG treatments, and also stronger drought tolerance than the plants of ABA-sensitive seeds during germination (S0–0). Leaf ABA content analysis indicated that the S0–50 plants had a higher ABA content in normal and under drought stress growth conditions than the S0–0 plants. The seeds of the S0–50 plants (S1–50) showed significantly higher germination percentage under 50 μM ABA treatment and had longer roots after 15% PEG6000 treatment than the segregated ABA-sensitive seeds (S1–0). We found a cytosol-nucleus dual-localized PPR protein gene MsSOAR1 was significantly highly expressed in the S0–50 than in the S0–0 plants. Overexpression of AtSOAR1 , a negative regulator in ABA-mediated seed germination inhibition in Arabidopsis and a homologous gene of MsSOAR1 , led to significantly improved drought tolerance, increased branch number and plant height, and reduced expression of the ABA receptor genes MsPYL5 and MsPYL6 of AtSOAR1 transgenic alfalfa plants (TGs) . The results suggest that ABA-insensitivity during seed germination is associated with the impaired ABA signaling transduction. Screening alfalfa seedlings during seed germination with exogenous ABA is a way to obtain drought tolerant alfalfa germplasm. • During vegetative stage, the ABA-insensitive ‘Zhongmu No.1’ alfalfa plants, during seeds germination, showed stronger drought tolerance. • Higher ABA content and higher expression of MsSOAR1 gene may contributed to improved drought tolerance of alfalfa plants. • Ectopic overexpression of AtSOAR1 significantly improved alfalfa drought tolerance. • Screening alfalfa seedlings during seed germination with exogenous ABA is a way to obtain drought tolerant alfalfa germplasm.

A novel approach to determine tropical persistence on alfalfa germplasm

AbstractPersistence plays a key role in alfalfa (Medicago sativa L. ssp. sativa) cultivation in tropical areas, but it is still a restriction for breeding programs. The objectives of this study were to identify persistent alfalfa accessions evaluated under tropical conditions, and to propose a method for selecting persistent accessions based on random regression (RR) models using artificial neural networks (ANN). Dry matter yield (DMY) of 77 alfalfa accessions from 24 cuts was measured to evaluate persistence using different RR models. A persistence method was proposed based on the trajectory curves of the accessions. The fitted curves showed a great amplitude regarding DMY over time, which suggest high persistence variability. The three‐step method for accessing persistence presented in this study included RR modeling to obtain trends of persistence, k‐means to define different persistence clusters, and ANN to perform persistence classification in an automated way. When new accessions are evaluated by an alfalfa breeding program, they will be classified according to their genetic values scores using the same ANN previously fitted. The ANN will greatly enhance the decision‐making process.

Alfalfa (Medicago sativa L.) pho2 mutant plants hyperaccumulate phosphate.

In this article, we describe a set of novel alfalfa (Medicago sativa L.) plants that hyper-accumulate Phosphate ion (Pi) at levels 3- to 6-fold higher than wild-type. This alfalfa germplasm will have practical applications reclaiming Pi from contaminated or enriched soil or be used in conservation buffer strips to protect waterways from Pi run-off. Hyper-accumulating alfalfa plants were generated by targeted mutagenesis of PHOSPHATE2 (PHO2) using newly created CRISPR/Cas9 reagents and an improved mutant screening strategy. PHO2 encodes a ubiquitin conjugating E2 enzyme (UBC24) previously characterized in Arabidopsis thaliana, Medicago truncatula, and Oryza sativa. Mutations of PHO2 disrupt Pi homeostasis resulting in Pi hyper-accumulation. Successful CRISPR/Cas9 editing of PHO2 demonstrates that this is an efficient mutagenesis tool in alfalfa despite its complex autotetraploid genome structure. Arabidopsis and M. truncatula ortholog genes were used to identify PHO2 haplotypes in outcrossing tetraploid M. sativa with the aim of generating heritable mutations in both PHO2-like genes (PHO2-B and PHO2-C). After delivery of the reagent and regeneration from transformed leaf explants, plants with mutations in all haplotypes of PHO2-B and PHO2-C were identified. These plants were evaluated for morphology, Pi accumulation, heritable transmission of targeted mutations, segregation of mutant haplotypes and removal of T-DNA(s). The Agrobacterium-mediated transformation assay and gene editing reagents reported here were also evaluated for further optimization for future alfalfa functional genomic studies.

Validation of DNA marker-assisted selection for forage biomass productivity under deficit irrigation in alfalfa.

Drought and limited irrigation resources threaten agricultural sustainability in many regions of the world. Application of genomic-based breeding strategies may benefit crop variety development for these environments. Here, we provide a first report on the effect of deploying DNA marker-assisted selection (MAS) for the drought resilience quantitative trait in alfalfa (Medicago sativa L.). The goals of this study were to validate the effect of several quantitative trait loci (QTL) associated with alfalfa forage and crown-root (CR) biomass during drought and to determine their potential to improve forage yield of elite germplasm under water-limited conditions. Marker assisted selection was employed to introgress favorable or unfavorable DNA marker alleles affiliated with 10 biomass QTL into three elite backgrounds. Thirty-two populations were developed and evaluated for forage productivity over 3 yr under continuous deficit irrigation management in New Mexico, USA. Significant yield differences (ranging from -13 to 26%) were detected among some MAS-derived populations in all three elite backgrounds. Application of QTL MAS generally resulted in expected phenotypic responses within an elite genetic background that was similar to that in which the QTL were originally identified. However, relative performance of the populations varied substantially across the three genetic backgrounds. These outcomes indicate that QTL MAS can significantly affect forage productivity of elite alfalfa germplasm in drought-stressed environments. However, if biomass QTL are detected in donor germplasm that is genetically dissimilar to targeted elite populations, characterization of donor alleles may be warranted within elite backgrounds of interest to confirm their phenotypic effects prior to implementing MAS-based breeding.

Genome Assembly of Alfalfa Cultivar Zhongmu-4 and Identification of SNPs Associated with Agronomic Traits

Alfalfa (Medicago sativa L.) is the most important legume forage crop worldwide with high nutritional value and yield. For a long time, the breeding of alfalfa was hampered by lacking reliable information on the autotetraploid genome and molecular markers linked to important agronomic traits. We herein reported the de novo assembly of the allele-aware chromosome-level genome of Zhongmu-4, a cultivar widely cultivated in China, and a comprehensive database of genomic variations based on resequencing of 220 germplasms. Approximate 2.74 Gb contigs (N50 of 2.06 Mb), accounting for 88.39% of the estimated genome, were assembled, and 2.56 Gb contigs were anchored to 32 pseudo-chromosomes. A total of 34,922 allelic genes were identified from the allele-aware genome. We observed the expansion of gene families, especially those related to the nitrogen metabolism, and the increase of repetitive elements including transposable elements, which probably resulted in the increase of Zhongmu-4 genome compared with Medicago truncatula. Population structure analysis revealed that the accessions from Asia and South America had relatively lower genetic diversity than those from Europe, suggesting that geography may influence alfalfa genetic divergence during local adaption. Genome-wide association studies identified 101 single nucleotide polymorphisms (SNPs) associated with 27 agronomic traits. Two candidate genes were predicted to be correlated with fall dormancy and salt response. We believe that the allele-aware chromosome-level genome sequence of Zhongmu-4 combined with the resequencing data of the diverse alfalfa germplasms will facilitate genetic research and genomics-assisted breeding in variety improvement of alfalfa.

Phenotyping seedlings for selection of root system architecture in alfalfa (Medicago sativa L.)

BackgroundThe root system architecture (RSA) of alfalfa (Medicago sativa L.) affects biomass production by influencing water and nutrient uptake, including nitrogen fixation. Further, roots are important for storing carbohydrates that are needed for regrowth in spring and after each harvest. Previous selection for a greater number of branched and fibrous roots significantly increased alfalfa biomass yield. However, phenotyping root systems of mature alfalfa plant is labor-intensive, time-consuming, and subject to environmental variability and human error. High-throughput and detailed phenotyping methods are needed to accelerate the development of alfalfa germplasm with distinct RSAs adapted to specific environmental conditions and for enhancing productivity in elite germplasm. In this study methods were developed for phenotyping 14-day-old alfalfa seedlings to identify measurable root traits that are highly heritable and can differentiate plants with either a branched or a tap rooted phenotype. Plants were grown in a soil-free mixture under controlled conditions, then the root systems were imaged with a flatbed scanner and measured using WinRhizo software.ResultsThe branched root plants had a significantly greater number of tertiary roots and significantly longer tertiary roots relative to the tap rooted plants. Additionally, the branch rooted population had significantly more secondary roots > 2.5 cm relative to the tap rooted population. These two parameters distinguishing phenotypes were confirmed using two machine learning algorithms, Random Forest and Gradient Boosting Machines. Plants selected as seedlings for the branch rooted or tap rooted phenotypes were used in crossing blocks that resulted in a genetic gain of 10%, consistent with the previous selection strategy that utilized manual root scoring to phenotype 22-week-old-plants. Heritability analysis of various root architecture parameters from selected seedlings showed tertiary root length and number are highly heritable with values of 0.74 and 0.79, respectively.ConclusionsThe results show that seedling root phenotyping is a reliable tool that can be used for alfalfa germplasm selection and breeding. Phenotypic selection of RSA in seedlings reduced time for selection by 20 weeks, significantly accelerating the breeding cycle.

Mapping freezing tolerance QTL in alfalfa: based on indoor phenotyping

BackgroundWinter freezing temperature impacts alfalfa (Medicago sativa L.) persistence and seasonal yield and can lead to the death of the plant. Understanding the genetic mechanisms of alfalfa freezing tolerance (FT) using high-throughput phenotyping and genotyping is crucial to select suitable germplasm and develop winter-hardy cultivars. Several clones of an alfalfa F1 mapping population (3010 x CW 1010) were tested for FT using a cold chamber. The population was genotyped with SNP markers identified using genotyping-by-sequencing (GBS) and the quantitative trait loci (QTL) associated with FT were mapped on the parent-specific linkage maps. The ultimate goal is to develop non-dormant and winter-hardy alfalfa cultivars that can produce extended growth in the areas where winters are often mild.ResultsAlfalfa FT screening method optimized in this experiment comprises three major steps: clone preparation, acclimation, and freezing test. Twenty clones of each genotype were tested, where 10 samples were treated with freezing temperature, and 10 were used as controls. A moderate positive correlation (r ~ 0.36, P < 0.01) was observed between indoor FT and field-based winter hardiness (WH), suggesting that the indoor FT test is a useful indirect selection method for winter hardiness of alfalfa germplasm. We detected a total of 20 QTL associated with four traits; nine for visual rating-based FT, five for percentage survival (PS), four for treated to control regrowth ratio (RR), and two for treated to control biomass ratio (BR). Some QTL positions overlapped with WH QTL reported previously, suggesting a genetic relationship between FT and WH. Some favorable QTL from the winter-hardy parent (3010) were from the potential genic region for a cold tolerance gene CBF. The BLAST alignment of a CBF sequence of M. truncatula, a close relative of alfalfa, against the alfalfa reference showed that the gene’s ortholog resides around 75 Mb on chromosome 6.ConclusionsThe indoor freezing tolerance selection method reported is useful for alfalfa breeders to accelerate breeding cycles through indirect selection. The QTL and associated markers add to the genomic resources for the research community and can be used in marker-assisted selection (MAS) for alfalfa cold tolerance improvement.

Pan-transcriptome identifying master genes and regulation network in response to drought and salt stresses in Alfalfa (Medicago sativa L.)

Alfalfa is an important legume forage grown worldwide and its productivity is affected by environmental stresses such as drought and high salinity. In this work, three alfalfa germplasms with contrasting tolerances to drought and high salinity were used for unraveling the transcriptomic responses to drought and salt stresses. Twenty-one different RNA samples from different germplasm, stress conditions or tissue sources (leaf, stem and root) were extracted and sequenced using the PacBio (Iso-Seq) and the Illumina platforms to obtain full-length transcriptomic profiles. A total of 1,124,275 and 91,378 unique isoforms and genes were obtained, respectively. Comparative analysis of transcriptomes identified differentially expressed genes and isoforms as well as transcriptional and post-transcriptional modifications such as alternative splicing events, fusion genes and nonsense-mediated mRNA decay events and non-coding RNA such as circRNA and lncRNA. This is the first time to identify the diversity of circRNA and lncRNA in response to drought and high salinity in alfalfa. The analysis of weighted gene co-expression network allowed to identify master genes and isoforms that may play important roles on drought and salt stress tolerance in alfalfa. This work provides insight for understanding the mechanisms by which drought and salt stresses affect alfalfa growth at the whole genome level.

Genetic variations in Turkey cultivar and ecotype Medicago sativa species: cytological, total protein profile, and molecular characterization

BackgroundAlfalfa(Medicago sativa L.) is a perennial plant, which is high in nutritional value and resistant to environmental conditions, and it is one of most frequently preferred feed crop among the leguminous family. In this study, it was aimed to determine the genetic diversity of some alfalfa ecotypes and their varieties by DNA, protein, nucleus, and chromosome counts. The genetic distance between the populations of control (M. truncatula), five different cultivars (Alsancak, Bilensoy, Iside, Plato, Bilensoy82), and three different ecotypes (Erzurum, Muş, and Konya) was investigated by cytogenetic analysis, flow cytometry, simple sequence repeats (SSR), and SDS PAGE techniques. ResultsCytogenetic analysis of these tested plants has verified the existence of expected levels such as diploid, triploid, and tetraploid as well as aneuploid (2n = 4x = 30) plants. Flow cytometry analysis have displayed that all of tested plants were tetraploid, whereas cytological analysis had either diploid, triploid, or tetraploid. Genetic diversity dendrogram was created using Erzurum, Muş, Konya, Bilensoy82, Alsancak, and Plato varieties. The Iside and Bilensoy were found to be morphogenetic in relationship. Our control plant, M. truncatula, did not have a similarity relationship with other ecotypes and cultivars. The total numbers of protein bands differed among tested plants from 140 kDA to 25 kDa. ConclusionsThis paper first reports on the genetic variation of Turkish alfalfa plants by using detailed analysis techniques. This work provides important findings for the classification, conservation, and innovation of alfalfa germplasm resources.

Potential of dry matter yield from alfalfa germplasm in composing base populations

Potential of dry matter yield from alfalfa germplasm in composing base populations