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.

The PLATZ family is a novel class of plant-specific zinc finger transcription factors with important roles in plant growth and development and abiotic stress responses. PLATZ members have been identified in many plants, including Oryza sativa, Zea mays, Triticum aestivum, Fagopyrum tataricum, and Arabidopsis thaliana; however, due to the complexity of the alfalfa reference genome, the members of the PLATZ gene family in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed. In this study, 55 Medicago sativa PLATZ genes (MsPLATZs) were identified in the alfalfa “Xinjiangdaye” reference genome. Basic bioinformatic analysis was performed, including the characterization of sequence lengths, protein molecular weights, genomic positions, and conserved motifs. Expression analysis reveals that 7 MsPLATZs are tissue-specifically expressed, and 10 MsPLATZs are expressed in all examined tissues. The transcriptomic expression of these genes is obvious, indicating that these MsPLATZs have different functions in the growth and development of alfalfa. Based on transcriptome data analysis and real-time quantitative PCR (RT-qPCR), we identified 22, 22, and 21 MsPLATZ genes that responded to salt, cold, and drought stress, respectively, with 20 MsPLATZs responding to all three stresses. This study lays a foundation for further exploring the functions of MsPLATZs, and provides ideas for the improvement of alfalfa varieties and germplasm innovation.

NAC (NAM, ATAF1/2, and CUC2) transcription factors compose one of the largest families of plant-specific transcription factors; they are widely involved in plant growth and development and have especially important roles in improving stress resistance in plants. However, NAC gene family members in alfalfa (Medicago sativa L.) have not been systematically identified and analyzed genome-wide due to the complexity of the alfalfa reference genome. In this study, a total of 421 M. sativa NAC genes (MsNACs) were identified from the alfalfa “Xinjiangdaye” reference genome. Basic bioinformatics analysis, including characterization of sequence length, protein molecular weight and genome position and conserved motif analysis, was conducted. Expression analysis showed that 47 MsNACs had tissue-specific expression, and 64 MsNACs were expressed in all tissues. The transcriptomic profiles of the genes were very different, indicating that these MsNACs have various functions in alfalfa growth and development. We identified 25, 42 and 47 MsNACs that respond to cold, drought and salt stress based on transcriptome data analysis and real-time quantitative PCR (RT–qPCR). Furthermore, 22 MsNACs were found to respond to both salt and drought stress, and 15 MsNACs were found to respond to cold, salt and drought stress. The results of this study could provide valuable information for further functional analysis of MsNACs and for the improvement of stress resistance in alfalfa.

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.

Polycyclic aromatic hydrocarbons (PAHs) are among the most toxic air pollutants in China. However, because there are unsubstantial data on indoor and outdoor particulate PAHs, efforts in assessing inhalation exposure and cancer risk to PAHs are limited in China. This study measured 12 individual PAHs in indoor and outdoor environments at 36 homes during the non‐heating period and heating period in 2009. Indoor PAH concentrations were comparable with outdoor environments in the non‐heating period, but were lower in the heating period. The average indoor/outdoor ratios in both sampling periods were lower than 1, while the ratios in the non‐heating period were higher than those in the heating period. Correlation analysis and coefficient of divergence also verified the difference between indoor and outdoor PAHs, which could be caused by high ventilation in the non‐heating period. To support this conclusion, linear and robust regressions were used to estimate the infiltration factor to compare outdoor PAHs to indoor PAHs. The calculated infiltration factors obtained by the two models were similar in the non‐heating period but varied greatly in the heating period, which may have been caused by the influence of ventilation. Potential sources were distinguished using a diagnostic ratio and a mixture of coal combustion and traffic emission, which are major sources of PAHs.

Appropriate root system architecture (RSA) can improve alfalfa yield, yet its genetic basis remains largely unexplored. Here, we evaluated six RSA traits in 171 alfalfa genotypes under controlled greenhouse conditions. We also analyzed five yield-related traits in normal and drought stress environments, finding a significant correlation (0.50) between root dry weight (RDW) and alfalfa dry weight under normal conditions (N_DW). A genome-wide association study (GWAS) was performed using 1,303,374 single nucleotide polymorphisms (SNPs) to explore the relationships between RSA traits. Sixty significant SNPs (-log₁₀(P) ≥ 5) were identified, with genes within 50 kb upstream and downstream ranges primarily enriched in GO terms related to root development, hormone synthesis and signaling, as well as morphological development. Further analysis identified 19 high-confidence candidate genes, including AUXIN RESPONSE FACTORs (ARFs), LATERAL ORGAN BOUNDARIES-DOMAIN (LBD), and WUSCHEL-RELATED HOMEOBOX (WOX). We verified that the forage dry weight under both normal and drought conditions exhibited significant differences among materials with different numbers of favourable haplotypes. Alfalfa containing more favourable haplotypes exhibited higher forage yields, whereas favourable haplotypes were not subjected to human selection during alfalfa breeding. Genomic prediction (GP) utilized SNPs from GWAS and machine learning for each RSA trait, achieving prediction accuracies ranging from 0.70 for secondary root position (SRP) to 0.80 for root length (RL), indicating robust predictive capability across the assessed traits. These findings provide new insights into the genetic underpinnings of root development in alfalfa, potentially informing future breeding strategies aimed at improving yield.