Genome assembly of Melilotus officinalis provides a new reference genome for functional genomics.

BACKGROUND: Sweet yellow clover (Melilotus officinalis) is a diploid plant (2n = 16) that is native to Europe. It is an excellent legume forage. It can both fix nitrogen and serve as a medicine. A genome assembly of Melilotus officinalis that was collected from Best corporation in Beijing is available based on Nanopore sequencing. The genome of Melilotus officinalis was sequenced, assembled, and annotated. RESULTS: The latest PacBio third generation HiFi assembly and sequencing strategies were used to produce a Melilotus officinalis genome assembly size of 1,066 Mbp, contig N50 = 5 Mbp, scaffold N50 = 130 Mbp, and complete benchmarking universal single-copy orthologs (BUSCOs) = 96.4%. This annotation produced 47,873 high-confidence gene models, which will substantially aid in our research on molecular breeding. A collinear analysis showed that Melilotus officinalis and Medicago truncatula shared conserved synteny. The expansion and contraction of gene families showed that Melilotus officinalis expanded by 565 gene families and shrank by 56 gene families. The contacted gene families were associated with response to stimulus, nucleotide binding, and small molecule binding. Thus, it is related to a family of genes associated with peptidase activity, which could lead to better stress tolerance in plants. CONCLUSIONS: In this study, the latest PacBio technology was used to assemble and sequence the genome of the Melilotus officinalis and annotate its protein-coding genes. These results will expand the genomic resources available for Melilotus officinalis and should assist in subsequent research on sweet yellow clover plants.

A High-Quality Genome Assembly of Sorghum dochna.

Sweet sorghum (Sorghum dochna) is a high-quality bio-energy crop that also serves as food for humans and animals. However, there is little information on the genomic characteristics of S. dochna. In this study, we presented a high-quality assembly of S. dochna with PacBio long reads, Illumina short reads, high-throughput chromosome capture technology (Hi-C) sequencing data, gene annotation, and a comparative genome analysis. The results showed that the genome of S. dochna was assembled to 777 Mb with a contig N50 of 553.47 kb and a scaffold N50 of 727.11 kb. In addition, the gene annotation predicted 37,971 genes and 39,937 transcripts in the genome of S. dochna. A Venn analysis revealed a set of 7,988 common gene annotations by integrating five databases. A Cafe software analysis showed that 191 gene families were significantly expanded, while 3,794 were significantly contracted in S. dochna. A GO enrichment analysis showed that the expanded gene families were primarily clustered in the metabolic process, DNA reconstruction, and DNA binding among others. The high-quality genome map constructed in this study provides a biological basis for the future analysis of the biological characteristics of S. dochna, which is crucial for its breeding.

A de novo assembled high-quality chromosome-scale Trifolium pratense genome and fine-scale phylogenetic analysis.

BACKGROUND: Red clover (Trifolium pratense L.) is a diploid perennial temperate legume with 14 chromosomes (2n = 14) native to Europe and West Asia, with high nutritional and economic value. It is a very important forage grass and is widely grown in marine climates, such as the United States and Sweden. Genetic research and molecular breeding are limited by the lack of high-quality reference genomes. In this study, we used Illumina, PacBio HiFi, and Hi-C to obtain a high-quality chromosome-scale red clover genome and used genome annotation results to analyze evolutionary relationships among related species. RESULTS: The red clover genome obtained by PacBio HiFi assembly sequencing was 423 M. The assembly quality was the highest among legume genome assemblies published to date. The contig N50 was 13 Mb, scaffold N50 was 55 Mb, and BUSCO completeness was 97.9%, accounting for 92.8% of the predicted genome. Genome annotation revealed 44,588 gene models with high confidence and 52.81% repetitive elements in red clover genome. Based on a comparison of genome annotation results, red clover was closely related to Trifolium medium and distantly related to Glycine max, Vigna radiata, Medicago truncatula, and Cicer arietinum among legumes. Analyses of gene family expansions and contractions and forward gene selection revealed gene families and genes related to environmental stress resistance and energy metabolism. CONCLUSIONS: We report a high-quality de novo genome assembly for the red clover at the chromosome level, with a substantial improvement in assembly quality over those of previously published red clover genomes. These annotated gene models can provide an important resource for molecular genetic breeding and legume evolution studies. Furthermore, we analyzed the evolutionary relationships among red clover and closely related species, providing a basis for evolutionary studies of clover leaf and legumes, genomics analyses of forage grass, the improvement of agronomic traits.

Effect of Light Intensity on Morphology, Photosynthesis and Carbon Metabolism of Alfalfa (Medicago sativa) Seedlings.

To understand how light intensity influences plant morphology and photosynthesis in the forage crop alfalfa (Medicago sativa L. cv. Zhongmu 1), we investigated changes in leaf angle orientation, chlorophyll fluorescence, parameters of photosynthesis and expression of genes related to enzymes involved in photosynthesis, the Calvin cycle and carbon metabolism in alfalfa seedlings exposed to five light intensities (100, 200, 300, 400 and 500 µmol m-2 s-1) under hydroponic conditions. Seedlings grown under low light intensities had significantly increased plant height, leaf hyponasty, specific leaf area, photosynthetic pigments, leaf nitrogen content and maximal PSII quantum yield, but the increased light-capturing capacity generated a carbon resource cost (e.g., decreased carbohydrates and biomass accumulation). Increased light intensity significantly improved leaf orientation toward the sun and upregulated the genes for Calvin cycle enzymes, thereby increasing photosynthetic capacity. Furthermore, high light (400 and 500 µmol m-2 s-1) significantly enhanced carbohydrate accumulation, accompanied by gene upregulation and increased activity of sucrose and starch-synthesis-related enzymes and those involved in carbon metabolism. Together, these results advance our understanding of morphological and physiological regulation in shade avoidance in alfalfa, which would guide the identification of suitable spatial planting patterns in the agricultural system.

Weeds in the Alfalfa Field Decrease Rhizosphere Microbial Diversity and Association Networks in the North China Plain.

The competition between weeds and crops for soil nutrients is affected by soil microorganisms, which drive diverse ecological processes and are critical in maintaining the stability of agroecosystems. However, the effects of plant species identity, particularly between forage and weed, on soil microbial diversity, composition, and association are not well understood. Here, we investigate the soil physicochemical properties and bacterial/fungal communities in an agroecosystem with native alfalfa [Medicago stativa (Ms)] and five common weed species (Digitaria sanguinalis, Echinochloa crusgalli, Acalypha australis, Portulaca oleracea, and Chenopodium album) in the North China Plain. The five weeds had a lower plant carbon content than Ms. while the opposite was true for plant nitrogen and phosphorus concentrations. The Shannon diversity of bacterial and fungal communities of the five weeds were significantly lower than in Ms. Soil pH and PO4 3--P were identified as the most important factors in shaping the relative abundances of bacteria (Sphingomonadaceae) and fungi (Pleosporaceae), respectively. Importantly, the weeds greatly inhibited the growth of pathogenic fungi (Nectriaceae and Pleosporaceae). Bacterial co-occurrence networks depended on specific species, indicating that Ms. harbored co-occurrence networks that were more complex than those in the bacterial communities of other weed groups. Our study examines how soil nutrients and the soil microbial community structure of five weed species changed in an Ms. field. This analysis of the microbial ecological network enhances our understanding of the influence of weeds on the soil microbiome in agroecosystems.

High-quality chromosome-scale de novo assembly of the Paspalum notatum 'Flugge' genome.

BACKGROUND: Paspalum notatum 'Flugge' is a diploid with 20 chromosomes (2n = 20) multi-purpose subtropical herb native to South America and has a high ecological significance. It is currently widely planted in tropical and subtropical regions. Despite the gene pool of P. notatum 'Flugge' being unearthed to a large extent in the past decade, no details about the genomic information of relevant species in Paspalum have been reported. In this study, the complete genome information of P. notatum was established and annotated through sequencing and de novo assembly of its genome. RESULTS: The latest PacBio third-generation HiFi assembly and sequencing revealed that the genome size of P. notatum 'Flugge' is 541 M. The assembly result is the higher index among the genomes of the gramineous family published so far, with a contig N50 = 52Mbp, scaffold N50 = 49Mbp, and BUSCOs = 98.1%, accounting for 98.5% of the estimated genome. Genome annotation revealed 36,511 high-confidence gene models, thus providing an important resource for future molecular breeding and evolutionary research. A comparison of the genome annotation results of P. notatum 'Flugge' with other closely related species revealed that it had a close relationship with Zea mays but not close compared to Brachypodium distachyon, Setaria viridis, Oryza sativa, Puccinellia tenuiflora, Echinochloa crusgalli. An analysis of the expansion and contraction of gene families suggested that P. notatum 'Flugge' contains gene families associated with environmental resistance, increased reproductive ability, and molecular evolution, which explained its excellent agronomic traits. CONCLUSION: This study is the first to report the high-quality chromosome-scale-based genome of P. notatum 'Flugge' assembled using the latest PacBio third-generation HiFi sequencing reads. The study provides an excellent genetic resource bank for gramineous crops and invaluable perspectives regarding the evolution of gramineous plants.

Integrative analysis of the metabolome and transcriptome reveal the phosphate deficiency response pathways of alfalfa.

Understanding the mechanisms underlying the responses to inorganic phosphate (Pi) deficiency in alfalfa will help enhance Pi acquisition efficiency and the sustainable use of phosphorous resources. Integrated global metabolomic and transcriptomic analyses of mid-vegetative alfalfa seedlings under 12-day Pi deficiency were conducted. Limited seedling growth were found, including 13.24%, 16.85% and 33.36% decreases in height, root length and photosynthesis, and a 24.10% increase in root-to-shoot ratio on day 12. A total of 322 and 448 differentially abundant metabolites and 1199 and 1061 differentially expressed genes were identified in roots and shoots. Increased (>3.68-fold) inorganic phosphate transporter 1;4 and SPX proteins levels in the roots (>2.15-fold) and shoots (>2.50-fold) were related to Pi absorption and translocation. The levels of phospholipids and Pi-binding carbohydrates and nucleosides were decreased, while those of phosphatases and pyrophosphatases in whole seedlings were induced under reduced Pi. In addition, nitrogen assimilation was affected by inhibiting high-affinity nitrate transporters (NRT2.1 and NRT3.1), and nitrate reductase. Increased delphinidin-3-glucoside might contribute to the gray-green leaves induced by Pi limitation. Stress-induced MYB, WRKY and ERF transcription factors were identified. The responses of alfalfa to Pi deficiency were summarized as local systemic signaling pathways, including root growth, stress-related responses consisting of enzymatic and nonenzymatic systems, and hormone signaling and systemic signaling pathways including Pi recycling and Pi sensing in the whole plant, as well as Pi recovery, and nitrate and metal absorption in the roots. This study provides important information on the molecular mechanism of the response to Pi deficiency in alfalfa.

Evolutionary balance between LRR domain loss and young NBS-LRR genes production governs disease resistance in Arachis hypogaea cv. Tifrunner.

BACKGROUND: Cultivated peanut (Arachis hypogaea L.) is an important oil and protein crop, but it has low disease resistance; therefore, it is important to reveal the number, sequence features, function, and evolution of genes that confer resistance. Nucleotide-binding site-leucine-rich repeats (NBS-LRRs) are resistance genes that are involved in response to various pathogens. RESULTS: We identified 713 full-length NBS-LRRs in A. hypogaea cv. Tifrunner. Genetic exchange events occurred on NBS-LRRs in A. hypogaea cv. Tifrunner, which were detected in the same subgenomes and also found in different subgenomes. Relaxed selection acted on NBS-LRR proteins and LRR domains in A. hypogaea cv. Tifrunner. Using quantitative trait loci (QTL), we found that NBS-LRRs were involved in response to late leaf spot, tomato spotted wilt virus, and bacterial wilt in A. duranensis (2 NBS-LRRs), A. ipaensis (39 NBS-LRRs), and A. hypogaea cv. Tifrunner (113 NBS-LRRs). In A. hypogaea cv. Tifrunner, 113 NBS-LRRs were classified as 75 young and 38 old NBS-LRRs, indicating that young NBS-LRRs were involved in response to disease after tetraploidization. However, compared to A. duranensis and A. ipaensis, fewer LRR domains were found in A. hypogaea cv. Tifrunner NBS-LRR proteins, partly explaining the lower disease resistance of the cultivated peanut. CONCLUSIONS: Although relaxed selection acted on NBS-LRR proteins and LRR domains, LRR domains were preferentially lost in A. hypogaea cv. Tifrunner compared to A. duranensis and A. ipaensis. The QTL results suggested that young NBS-LRRs were important for resistance against diseases in A. hypogaea cv. Tifrunner. Our results provid insight into the greater susceptibility of A. hypogaea cv. Tifrunner to disease compared to A. duranensis and A. ipaensis.

Naphthylacetic Acid and Tea Polyphenol Application Promote Biomass and Lipid Production of Nervonic Acid-Producing Microalgae.

Mychonastes afer HSO-3-1 is a potential producer of nervonic acid, which could be accumulated to 2-3% of dry cell weight. Improving the productivity of nervonic acid is critical to promote the commercialization of this product. In this study, 1-naphthylacetic acid (NAA) and tea polyphenol (TP) were selected as bioactive additives to stimulate the growth of M. afer. Supplementing NAA in the early growth stage and TP in the middle and late growth stage led to improved lipid accumulation in M. afer. The cultures supplemented with TP at the late growth stage maintained higher photosynthetic efficiency than the control groups without TP. Furthermore, the intracellular reactive oxygen species (ROS) accumulations in M. afer supplemented with 500 mg/L of TP was 63% lower than the control group. A linear relationship (R2= 0.899) between the values of Fv/Fm and ROS accumulation was established. We hypothesize supplement of bioactive additives at different growth stage could promote the cell growth rate and nervonic acid productivity of M. afer by retrieving intracellular ROS level. Further analysis of photosynthetic system II (PSII) protein in M. afer cultured in presence of NAA and TP indicated the levels of D1 and D2 proteins, the core skeleton proteins of PSII, showed 33.3 and 25.6% higher than the control group. CP43 protein, a critical module in PSII repair cycle, decreased significantly. These implied that TP possesses the function of slowing down the damage of PSII by scavenging excess intracellular ROS.

Ecological diversity and co-occurrence patterns of bacterial community through soil profile in response to long-term switchgrass cultivation.

Switchgrass (Panicum virgatum L.) is a cellulosic biofuel feedstock and their effects on bacterial communities in deep soils remain poorly understood. To reveal the responses of bacterial communities to long-term switchgrass cultivation through the soil profile, we examined the shift of soil microbial communities with depth profiles of 0-60 cm in five-year switchgrass cultivation and fallow plots. The Illumina sequencing of the 16S rRNA gene showed that switchgrass cultivation significantly increased microbial OTU richness, rather than microbial Shannon diversity; however, there was no significant difference in the structure of microbial communities between switchgrass cultivation and fallow soils. Both switchgrass cultivation and fallow soils exhibited significant negative vertical spatial decay of microbial similarity, indicating that more vertical depth distant soils had more dissimilar communities. Specifically, switchgrass cultivation soils showed more beta-diversity variations across soil depth profile. Through network analysis, more connections and closer relationships of microbial taxa were observed in soils under switchgrass cultivation, suggesting that microbial co-occurrence patterns were substantially influenced by switchgrass cultivation. Overall, our study suggested that five-year switchgrass cultivation could generated more beta-diversity variations across soil depth and more complex inter-relationships of microbial taxa, although did not significantly shape the structure of soil microbial community.

Effects of grazing and precipitation on herbage biomass, herbage nutritive value, and yak performance in an alpine meadow on the Qinghai-Tibetan Plateau.

The Qinghai-Tibetan Plateau is a very large land unit and an important terrestrial ecosystem within the Eurasian continent. Because of the harsh climate associated with the high altitude, alpine meadows on the plateau are susceptible to degradation from overgrazing. For this region, and for other alpine meadow pastures internationally, there is a need to define the sustainable stocking rate, to develop sound policy pertaining to future land use. Here we report biomass and liveweight gain per animal and per ha for pastures grazed by yaks at high, medium, or low stocking rates over 4 growing seasons from 2010 to 2013. Measures of herbage nutritive value are reported. The influence of inter-year variation in precipitation on standing herbage biomass was also evaluated. Higher precipitation increased standing herbage biomass and herbage nutritive value, indicating that vegetation suffered summer water deficit even in this environment. The sustainable stocking rate in this environment was determined to be approximately 1 yak ha-1 (grown from 80 kg to 120 kg liveweight in 90 d). At this stocking rate, yak weight gain per ha was 88% of that achieved at higher stocking rates typically used by farmers, but with little or no evidence of land degradation.