Forage quality determined mainly by protein content and fiber composition has a crucial influence on digestibility and nutrition intake for animal feeding. To explore the genetic basis of quality traits, we conducted QTL mapping based on the phenotypic data of crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and lignin of an F1 alfalfa population generated by crossing of two alfalfa parents with significant difference in quality. In total, 83 QTLs were identified with contribution to the phenotypic variation (PVE) ranging from 1.45 to 14.35%. Among them, 47 QTLs interacted significantly with environment and 12 QTLs were associated with more than one trait. Epistatic effect was also detected for 73 pairs of QTLs with PVE of 1.08–14.06%. The results suggested that the inheritance of quality-related traits was jointly affected by additive, epistasis and environment. In addition, 83.33% of the co-localized QTLs were shared by ADF and NDF with the same genetic direction, while the additive effect of crude protein-associated QTLs was opposite to that fiber composition on the same locus, suggesting that the loci may antagonistically contribute to protein content and fiber composition. Further analysis of a QTL related to all the three traits of fiber composition (qNDF1C, qADF1C-2, and qlignin1C-2) showed that five candidate genes were homologs of cellulose synthase-like protein A1 in Medicago truncatula, indicating the potential role in fiber synthesis. For the protein-associated loci we identified, qCP4C-1 was located in the shortest region (chr 4.3 39.3–39.4 Mb), and two of the seven corresponding genes in this region were predicted to be E3 ubiquitin-protein ligase in protein metabolism. Therefore, our results provide some reliable regions significantly associated with alfalfa quality, and identification of the key genes would facilitate marker-assisted selection for favorable alleles in breeding program of alfalfa quality improvement.

Background Leaf size affects crop canopy morphology and photosynthetic efficiency, which can influence forage yield and quality. It is of great significance to mine the key genes controlling leaf development for breeding new alfalfa varieties. In this study, we mapped leaf length (LL), leaf width (LW), and leaf area (LA) in an F1 mapping population derived from a cultivar named ZhongmuNo.1 with larger leaf area and a landrace named Cangzhou with smaller leaf area. Results This study showed that the larger LW was more conducive to increasing LA. A total of 24 significant quantitative trait loci (QTL) associated with leaf size were identified on both the paternal and maternal linkage maps. Among them, nine QTL explained about 11.50–22.45% phenotypic variation. RNA-seq analysis identified 2,443 leaf-specific genes and 3,770 differentially expressed genes. Combining QTL mapping, RNA-seq alalysis, and qRT-PCR, we identified seven candidate genes associated with leaf development in five major QTL regions. Conclusion Our study will provide a theoretical basis for marker-assisted breeding and lay a foundation for further revealing molecular mechanism of leaf development in alfalfa.

Alfalfa (Medicago sativa L.) is a perennial forage crop known as the “Queen of Forages.” To dissect the genetic mechanism of flowering time (FT) in alfalfa, high−density linkage maps were constructed for both parents of an F1 mapping population derived from a cross between Cangzhou (P1) and ZhongmuNO.1 (P2), consisting of 150 progenies. The FT showed a transgressive segregation pattern in the mapping population. A total of 13,773 single-nucleotide polymorphism markers was obtained by using restriction-site associated DNA sequencing and distributed on 64 linkage groups, with a total length of 3,780.49 and 4,113.45 cM and an average marker interval of 0.58 and 0.59 cM for P1 and P2 parent, respectively. Quantitative trait loci (QTL) analyses were performed using the least square means of each year as well as the best linear unbiased prediction values across 4 years. Sixteen QTLs for FT were detected for P1 and 22 QTLs for P2, accounting for 1.40–16.04% of FT variation. RNA-Seq analysis at three flowering stages identified 5,039, 7,058, and 7,996 genes that were differentially expressed between two parents, respectively. Based on QTL mapping, DEGs analysis, and functional annotation, seven candidate genes associated with flowering time were finally detected. This study discovered QTLs and candidate genes for alfalfa FT, making it a useful resource for breeding studies on this essential crop.

Alfalfa (Medicago sativa) is an important food and feed crop which rich in mineral sources. The WUSCHEL-related homeobox (WOX) gene family plays important roles in plant development and identification of putative gene families, their structure, and potential functions is a primary step for not only understanding the genetic mechanisms behind various biological process but also for genetic improvement. A variety of computational tools, including MAFFT, HMMER, hidden Markov models, Pfam, SMART, MEGA, ProtTest, BLASTn, and BRAD, among others, were used. We identified 34 MsWOX genes based on a systematic analysis of the alfalfa plant genome spread in eight chromosomes. This is an expansion of the gene family which we attribute to observed chromosomal duplications. Sequence alignment analysis revealed 61 conserved proteins containing a homeodomain. Phylogenetic study sung reveal five evolutionary clades with 15 motif distributions. Gene structure analysis reveals various exon, intron, and untranslated structures which are consistent in genes from similar clades. Functional analysis prediction of promoter regions reveals various transcription binding sites containing key growth, development, and stress-responsive transcription factor families such as MYB, ERF, AP2, and NAC which are spread across the genes. Most of the genes are predicted to be in the nucleus. Also, there are duplication events in some genes which explain the expansion of the family. The present research provides a clue on the potential roles of MsWOX family genes that will be useful for further understanding their functional roles in alfalfa plants.

The transition to flowering at the right time is very important for adapting to local conditions and maximizing alfalfa yield. However, the understanding of the genetic basis of the alfalfa flowering time remains limited. There are few reliable genes or markers for selection, which hinders progress in genetic research and molecular breeding of this trait in alfalfa. We sequenced 220 alfalfa cultivars and conducted a genome-wide association study (GWAS) involving 875,023 single-nucleotide polymorphisms (SNPs). The phenotypic analysis showed that the breeding status and geographical origin strongly influenced the alfalfa flowering time. Our GWAS revealed 63 loci significantly related to the flowering time. Ninety-five candidate genes were detected at these SNP loci within 40 kb (20 kb up- and downstream). Thirty-six percent of the candidate genes are involved in development and pollen tube growth, indicating that these genes are key genetic mechanisms of alfalfa growth and development. The transcriptomic analysis showed that 1,924, 2,405, and 3,779 differentially expressed genes (DEGs) were upregulated across the three growth stages, while 1,651, 2,613, and 4,730 DEGs were downregulated across the stages. Combining the results of our GWAS and transcriptome analysis, in total, 38 candidate genes (7 differentially expressed during the bud stage, 13 differentially expressed during the initial flowering stage, and 18 differentially expressed during the full flowering stage) were identified. Two SNPs located in the upstream region of the Msa0888690 gene (which is involved in isop renoids) were significantly related to flowering. The two significant SNPs within the upstream region of Msa0888690 existed as four different haplotypes in this panel. The genes identified in this study represent a series of candidate targets for further research investigating the alfalfa flowering time and could be used for alfalfa molecular breeding.