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.

Salinity is one of major abiotic stresses limiting alfalfa (Medicago sativa L.) production in the arid and semi-arid regions in US and other counties. In this study, we used a diverse panel of alfalfa accessions previously described by Zhang et al. (2015) to identify molecular markers associated with salt tolerance during germination using genome-wide association study (GWAS) and genotyping-by-sequencing (GBS). Genotyping was done by germinating alfalfa seeds under different levels of salt stress. Phenotypic data of adjusted germination rates and SNP markers generated by GBS were used for marker-trait association. Thirty six markers were significantly associated with salt tolerance in at least one level of salt treatments. Alignment of sequence tags to the Medicago truncatula genome revealed genetic locations of the markers on all chromosomes except chromosome 3. Most significant markers were found on chromosomes 1, 2 and 4. BLAST search using the flanking sequences of significant markers identified 14 putative candidate genes linked to 22 significant markers. Most of them were repeatedly identified in two or three salt treatments. Several loci identified in the present study had similar genetic locations to the reported QTL associated with salt tolerance in M. truncatula. A locus identified on chromosome 6 by this study overlapped with that by drought in our previous study. To our knowledge, this is the first report on mapping loci associated with salt tolerance during germination in autotetraploid alfalfa. Further investigation on these loci and their linked genes would provide insight into understanding molecular mechanisms by which salt and drought stresses affect alfalfa growth. Functional markers closely linked to the resistance loci would be useful for MAS to improve alfalfa cultivars with enhanced resistance to drought and salt stresses.

Drought resistance is an important breeding target for enhancing alfalfa productivity in arid and semi-arid regions. Identification of genes involved in drought tolerance will facilitate breeding for improving drought resistance and water use efficiency in alfalfa. Our objective was to use a diversity panel of alfalfa accessions comprised of 198 cultivars and landraces to identify genes involved in drought tolerance. The panel was selected from the USDA-ARS National Plant Germplasm System alfalfa collection and genotyped using genotyping by sequencing. A greenhouse procedure was used for phenotyping two important traits associated with drought tolerance: drought resistance index (DRI) and relative leaf water content (RWC). Marker-trait association identified nineteen and fifteen loci associated with DRI and RWC, respectively. Alignments of target sequences flanking to the resistance loci against the reference genome of M. truncatula revealed multiple chromosomal locations. Markers associated with DRI are located on all chromosomes while markers associated with RWC are located on chromosomes 1, 2, 3, 4, 5, 6 and 7. Co-localizations of significant markers between DRI and RWC were found on chromosomes 3, 5 and 7. Most loci associated with DRI in this work overlap with the reported QTLs associated with biomass under drought in alfalfa. Additional significant markers were targeted to several contigs with unknown chromosomal locations. BLAST search using their flanking sequences revealed homology to several annotated genes with functions in stress tolerance. With further validation, these markers may be used for marker-assisted breeding new alfalfa varieties with drought resistance and enhanced water use efficiency.

Soil salinity is a growing problem in world production agriculture. Continued improvement in crop salt tolerance will require the implementation of innovative breeding strategies such as marker-assisted selection (MAS) and genomic selection (GS). Genetic analyses for yield and vigor traits under salt stress in alfalfa breeding populations with three different phenotypic datasets was assessed. Genotype-by-sequencing (GBS) developed markers with allele dosage and phenotypic data were analyzed by genome-wide association studies (GWAS) and GS using different models. GWAS identified 27 single nucleotide polymorphism (SNP) markers associated with salt tolerance. Mapping SNPs markers against the Medicago truncatula reference genome revealed several putative candidate genes based on their roles in response to salt stress. Additionally, eight GS models were used to estimate breeding values of the training population under salt stress. Highest prediction accuracies and root mean square errors were used to determine the best prediction model. The machine learning methods (support vector machine and random forest) performance best with the prediction accuracy of 0.793 for yield. The marker loci and candidate genes identified, along with optimized GS prediction models, were shown to be useful in improvement of alfalfa with enhanced salt tolerance. DNA markers and the outcome of the GS will be made available to the alfalfa breeding community in efforts to accelerate genetic gains, in the development of biotic stress tolerant and more productive modern-day alfalfa cultivars.

Salinity tolerance is highly desirable to sustain alfalfa production in marginal lands that have been rendered saline. In this study, we used a diverse panel of 198 alfalfa accessions for mapping loci associated with plant growth and forage production under salt stress using genome-wide association studies (GWAS). The plants were genotyped using genotyping-by-sequencing (GBS). A greenhouse procedure was used for phenotyping four agronomic and physiological traits affected by salt stress, including dry weight (DW), plant height (PH), leaf chlorophyll content (LCC) and stomatal conductance (SC). For each trait, a stress susceptibility index (SSI) was used to evaluate plant performance under stressed and non-stressed conditions. Marker-trait association identified a total of 42 markers significantly associated with salt tolerance. They were located on all chromosomes except chromosome 2 based on the alignment of their flanking sequences to the reference genome (Medicago truncatula). Of those identified, 13 were associated with multiple traits. Several loci identified in the present study were also identified in previous reports. BLAST search revealed that 19 putative candidate genes linked to 24 significant markers. Among them, B3 DNA-binding protein, Thiaminepyrophosphokinase and IQ calmodulin-binding motif protein were identified among multiple traits in the present and previous studies. With further investigation, these markers and candidates would be useful for developing markers for marker-assisted selection in breeding programs to improve alfalfa cultivars with enhanced tolerance to salt stress.

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.