Persistence of fertilization effects on soil organic carbon in degraded alpine wetlands in the Yellow River source region

In the restoration of degraded wetlands,fertilization can improve the vegetation-soil-microorganisms complex,thereby affecting the organic carbon content.However,it is currently unclear whether these effects are sustainable.This study employed Biolog-Eco surveys to investigate the changes in vegetation characteristics,soil physicochemical properties,and soil microbial functional diversity in degraded alpine wetlands of the source region of the Yellow River at 3 and 15 months after the application of nitrogen,phosphorus,and organic mixed fertilizer.The following results were obtained:The addition of nitrogen fertilizer and organic compost significantly affects the soil organic carbon content in degraded wetlands.Three months after fertilization,nitrogen addition increases soil organic carbon in both lightly and severely degraded wetlands,whereas after 15 months,organic compost enhanced the soil organic carbon level in severely degraded wetlands.Structural equation modeling indicates that fertilization decreases the soil pH and directly or indirectly influences the soil organic carbon levels through variations in the soil water content and the aboveground biomass of vegetation.Three months after fertilization,nitrogen fertilizer showed a direct positive effect on soil organic carbon.However,organic mixed fertilizer indirectly reduced soil organic carbon by increasing biomass and decreasing soil moisture.After 15 months,none of the fertilizers significantly affected the soil organic carbon level.In summary,it can be inferred that the addition of nitrogen fertilizer lacks sustainability in positively influencing the organic carbon content.

Effects of long-term grazing exclusion on vegetation structure,soil water holding capacity,carbon and nitrogen sequestration capacity in an alpine meadow on the Tibetan Plateau

Grazing exclusion is one of the primary management practices used to restore degraded grasslands on the Tibetan Plateau.However,to date,the effects of long-term grazing exclusion measures on the process of restoring degraded alpine meadows have not been evaluated.In this study,moderately degraded plots,in which the vegetation coverage was approximately 65%and the dominant plant species was Potentilla anserina L,with grazing exclusion for 2 to 23 years,were selected in alpine meadows of Haibei in Qinghai-Tibet Plateau.Plant coverage,plant height,biomass,soil bulk density,saturated water content,soil organic carbon(SOC)and total nitrogen(TN)were evaluated.The results were as follows:(1)With aboveground biomass and total saturated water content at 0-40 cm depth,the average SOC and TN contents in moderately degraded alpine meadows increased as a power function,and the plant height increased as a log function.(2)The average soil bulk density at 0-40 cm depth first decreased and then increased with increasing grazing exclusion duration,and the minimum value of 0.90 g·cm^(-3) was reached at 15.23 years.The plant coverage,total belowground biomass at 0-40 cm depth,total aboveground and belowground biomass first increased and then decreased,their maximum values(80.49%,2452.92g·m^(-2),2891.06 g·m^(-2))were reached at 9.41,9.46 and 10.25 years,respectively.Long-term grazing exclusion is apparently harmful for the sustainable restoration of degraded alpine meadows.The optimal duration of grazing exclusion for the restoration of moderately degraded alpine meadows was 10 years.This research suggests that moderate disturbance should be allowed in moderately degraded alpine meadows after 10years of grazing exclusion.

Cryptic diversity and rampant hybridization in annual gentians on the Qinghai-Tibet Plateau revealed by population genomic analysis

Understanding the evolutionary and ecological processes involved in population differentiation and speciation provides critical insights into biodiversity formation. In this study, we employed 29,865 single nucleotide polymorphisms(SNPs) and complete plastomes to examine genomic divergence and hybridization in Gentiana aristata, which is endemic to the Qinghai-Tibet Plateau(QTP) region. Genetic clustering revealed that G. aristata is characterized by geographic genetic structures with five clusters(West, East, Central, South and North). The West cluster has a specific morphological character(i.e., blue corolla) and higher values of FSTcompared to the remaining clusters, likely the result of the geological barrier formed by the Yangtze River. The West cluster diverged from the other clusters in the Early Pliocene;these remaining clusters diverged from one another in the Early Quaternary. Phylogenetic reconstructions based on SNPs and plastid data revealed substantial cyto-nuclear conflicts. Genetic clustering and D-statistics demonstrated rampant hybridization between the Central and North clusters,along the Bayankala Mountains, which form the geological barrier between the Central and North clusters. Species distribution modeling demonstrated the range of G. aristata expanded since the Last Interglacial period. Our findings provide genetic and morphological evidence of cryptic diversity in G. aristata, and identified rampant hybridization between genetic clusters along a geological barrier.These findings suggest that geological barriers and climatic fluctuations have an important role in triggering diversification as well as hybridization, indicating that cryptic diversity and hybridization are essential factors in biodiversity formation within the QTP region.

Effects of land-use types on soil organic carbon stocks:a case study across an altitudinal gradient within a farmpastoral area on the eastern Qinghai-Tibetan Plateau, China

A crucial region for China's ‘Grain-forGreen Policy' is located within a traditional farmpastoral area, between 2000 to 3000 m above sea level, on the eastern Qinghai-Tibetan Plateau.However, the responses of soil organic carbon(SOC) to different land-use patterns in this region are unclear. Here, we determined the SOC(0–20 cm) content of grasslands and forests that are being converted from farmlands, as well as in abandoned arable land and arable land in this region. The factors influencing the reclaimed lands were analyzed along altitudes from 2030 to 3132 m. Our results showed that SOC content was higher for grassland and abandoned arable land than forest and arable land. The SOC content increased with the increase in altitude for total land-use patterns. Further, the grassland and abandoned arable land had higher SOC content than the forest with almost parallel trends along the increase in altitude. However, the proportion of regulated factors of altitude and species richness varied among forest, grassland, and abandoned arable land. Our results indicated that the land-use pattern of returning farmland to grassland and abandoned arable land was more effective in terms of the SOC storage in the superficial layer in this altitude range in the Qinghai-Tibetan Plateau, thereby being beneficial to optimizing land management in this region.

The Influence of Fence on Body Mass and Population Dynamics of Plateau Pika

To determine the influence of the fence on plateau pika (Ochotona curzoniae), we measured the body mass, breeding status, population densities, using live-trapping and line-transect sampling in April and September 2006. In April, body mass of both male and female plateau pika inside of fence were significant higher than that outside of fence (♀: F = 6.583, df = 1.20, p = 0.019;♂: F = 6.49, df = 1.17, p = 0.021). The ratio of spermary drop of an adult male was also higher (92.31% vs 57.14%). In September, body mass of adult male in both study sites showed no significant difference (F = 0.001, df = 1.23, p = 0.975), but were all significant higher than that of April (F = 121.713, df = 1.22, p < 0.001;F = 105.819, df = 1.20, p < 0.001). In April, the population densities of both study sites showed on significant difference (F = 2.388, df = 1.38, p = 0.131), but the population density inside of fence was much higher than that outside of fence (F = 7.534, df = 1.38, p = 0.009) in September. Our results show that the changes of quality and quantity of food that available to plateau pika in winter, can depress the range of reducing body mass of plateau pika, make breeding season ahead, and then promote the increase of population.

Soil physicochemical properties and vegetation structure along an elevation gradient and implications for the response of alpine plant development to climate change on the northern slopes of the Qilian Mountains

Elevation is one of key factors to affect changes in the environment, particularly changes in conditions of light, water and heat. Studying the soil physicochemical properties and vegetation structure along an elevation gradient is important for understanding the responses of alpine plants andtheir growing environment to climate change. In this study, we studied plant coverage, plant height, species richness, soil water-holding capacity, soil organic carbon(SOC) and total nitrogen(N) on the northern slopes of the Qilian Mountains at elevations from2124 to 3665 m. The following conclusions were drawn:(1) With the increase of elevation, plant coverage and species richness first increased and then decreased, with the maximum values being at 3177 m.Plant height was significantly and negatively correlated with elevation(r=–0.97, P<0.01), and the ratio of decrease with elevation was 0.82 cm·100 m-1.(2) Both soil water-holding capacity and soil porosity increased on the northern slopes of the Qilian Mountains with the increase of elevation. The soil saturated water content at the 0-40 cm depth first increased and then stabilized with a further increase of elevation, and the average ratio of increase was2.44 mm·100 m-1. With the increase of elevation, the average bulk density at the 0-40 cm depth first decreased and then stabilized at 0.89 g/cm3.(3) With the increase of elevation, the average SOC content at the 0-40 cm depths first increased and then decreased,and the average total N content at the 0-40 cm depth first increased and then stabilized. The correlation between average SOC content and average total N content reached significant level. According to the results of this study, the distribution of plants showed a mono-peak curve with increasing elevation on the northern slopes of the Qilian Mountains. The limiting factor for plant growth at the high elevation areas was not soil physicochemical properties, and therefore,global warming will likely facilitate the development of plant at high elevation areas in the Qilian Mountains.

Offering the win-win solutions between ecological conservation and livelihood development:National parks in Qinghai, China

National parks as protected areas have been on the spotlight because they are essential in achieving ecological civilization in China.Despite the profound achievements of national parks to conserve nature,the benefits of national parks for local residents remain lacking.Under the theory of harmonious coexistence between human and nature,this study focused on the development of local people’s livelihood in Qinghai’s national parks.We systematically explored the establishment of the national parks in Qinghai Province,analyzed the current prob-lems,and provided the main measures for developing people’s livelihood in protected areas in Qinghai.This study put forward the viewpoint that national parks should set a long-term goal,and gradually advance based on the environmental conditions and appropriately prioritize the improvement of local people’s wellbeing in the future.

The weak effects of fencing on ecosystem respiration,CH4,and N2O fluxes in a Tibetan alpine meadow during the growing season

Fencing is the most common land-management practice to protect grassland degradation from livestock overgrazing on the Tibetan Plateau. However, it is unclear whether fencing reduces CO_2, CH_4, and N_2O emission. Here, we selected four vegetation types of alpine meadow(graminoid, shrub, forb, and sparse vegetation) to determine fencing effects on ecosystem respiration(Re), CH_4, and N_2O fluxes during the growing season. Despite increased average monthly ecosystem respiration(Re) for fenced graminoid vegetation at the end of the growing season, there was no significant difference between grazing and fencing across all vegetation types. Fencing significantly reduced average CH_4 uptake by about 50% in 2008 only for forb vegetation and increased average N_2O release for graminoid vegetation by 38% and 48% in 2008 and 2009,respectively. Temperature, moisture, total organic carbon, C/N, nitrate, ammonia, and/or bulk density of soil, as well as above-and belowground biomass, explained 19%~71% and 6%~33% of variation in daily and average Re and CH_4 fluxes across all vegetation types, while soil-bulk density explained 27% of variation in average N_2O fluxes. Stepwise regression showed that soil temperature and soil moisture controlled average Re, while soil moisture and bulk density controlled average CH_4 fluxes. These results indicate that abiotic factors control Re, CH_4, and N_2O fluxes; and grazing exclusion has little effect on reducing their emission—implying that climatic change rather than grazing may have a more important influence on the budgets of Re and CH_4 for the Tibetan alpine meadow during the growing season.

Molecular cytogenetic study on the plants of Elymus nutans with varying fertility on the Qinghai-Tibet Plateau

A molecular cytogenetic investigation was conducted on plants of the allohexaploid species Elymus nutans with varying fertility on the Qinghai-Tibet Plateau.Molecular karyotyping revealed that chromosome variants were distributed unevenly among genomes and among different homologue chromosomes in each genome.The plants with varying fertility exhibited significantly higher numbers of chromosome variants than did the normal fertility samples,although both kinds of plants showed the same pattern of high-to-low polymorphism from the Y to St and H genomes.Heterozygosis and karyotype heterozygosity in the plants with varying fertility were 3-and 13-fold higher than those in normal samples,respectively.Significant negative correlations were found not only between seed setting rates and total genome heterozygosity but also between seed setting rates and heterozygosity of each genome in the plants of varying fertility.Chromosome pairing analysis was performed using genomic in situ hybridization in selected plants of different fertility levels.The pairing of chromosomes at meiotic metaphase I was mostly bivalent,although univalent,trivalent,quadrivalent,and other polyvalents also occurred;in addition,chromosome configuration forms and frequencies varied among the studied samples.ANOVA results showed that the average number of ring bivalents in the Y genome was significantly higher than those in the St and H genomes.Significant positive correlations between pollen grain fertility and ring bivalent number were found in the St and H genomes but not in the Y genome.Furthermore,chromosome configuration parameters(total bivalents,numbers of ring and rod bivalents)were found to be significantly correlated with heterozygosity and seed setting rates in the St and H genomes,respectively,but not in the Y genome.It was inferred that the seed setting rate and pollen grain fertility in E.nutans are strongly influenced by the heterozygosity of each genome,but the Y genome differs from the St and H genomes due to chromosome pair alterations.The St and H genomes may contain more chromosome structural variations than the Y genome in E.nutans.

Molecular identification of Taenia mustelae cysts in subterranean rodent plateau zokors(Eospalax baileyi)

Cestode larvae spend one phase of their two-phase life cycle in the viscera of rodents, but cases of cestodes infecting subterranean rodents have only been rarely observed. To experimentally gain some insight into this phenomenon, we captured approximately 300 plateau zokors(Eospalax baileyi), a typical subterranean rodent inhabiting the Qinghai-Tibet Plateau, and examined their livers for the presence of cysts. Totally, we collected five cysts, and using a mitochondrial gene(cox1) and two nuclear genes(pepck and pold) as genetic markers, we were able to analyze the taxonomy of the cysts. Both the maximum likelihood and Bayesian methods showed that the cysts share a monophyly with Taenia mustelae, while Kimura 2-parameter distances and number of different sites between our sequences and T. mustelae were far less than those found between the examined sequences and other Taeniidae species. These results, alongside supporting paraffin section histology, imply that the cysts found in plateau zokors can be regarded as larvae of T. mustelae, illustrating that zokors are a newly discovered intermediate host record of this parasite.

How precipitation and grazing influence the ecological functions of drought-prone grasslands on the northern slopes of the Tianshan Mountains, China?

Drought-prone grasslands provide a critical resource for the millions of people who are dependent on livestock for food security.However,this ecosystem is potentially vulnerable to climate change(e.g.,precipitation)and human activity(e.g.,grazing).Despite this,the influences of precipitation and grazing on ecological functions of drought-prone grasslands in the Tianshan Mountains remain relatively unexplored.Therefore,we conducted a systematic field investigation and a clipping experiment(simulating different intensities of grazing)in a drought-prone grassland on the northern slopes of the Tianshan Mountains in China to examine the influences of precipitation and grazing on aboveground biomass(AGB),soil volumetric water content(SVWC),and precipitation use efficiency(PUE)during the period of 2014–2017.We obtained the meteorological and SVWC data using an HL20 Bowen ratio system and a PR2 soil profile hydrometer,respectively.We found that AGB was clearly affected by both the amount and seasonal pattern of precipitation,and that PUE may be relatively low in years with either low or excessive precipitation.The PUE values were generally higher in the rapid growing season(April–July)than in the entire growing season(April–October).Overall,moderate grazing can promote plant growth under water stress conditions.The SVWC value was higher in the clipped plots than in the unclipped plots in the rapid growing season(April–July),but it was lower in the clipped plots than in the unclipped plots in the slow growing season(August–October).Our findings can enhance the understanding of the ecological effects of precipitation and grazing in drought-prone grasslands and provide data that will support the effective local grassland management.

Activation of β_2-Adrenergic Receptor Induced by Three Catecholamine Agonists:a Docking and Molecular Dynamics Study

We studied the activation of β2-adrenergic receptor（β2AR） by norepinephrine, epinephrine and isoprote- renol using docking and molecular dynamics（MD） simulation. The simulation was done on the assumption that β2AR was surrounded with explicit water and infinite lipid bilayer membrane at body temperature. So the result should be close to that under the physiological conditions. We calculated the structure of binding sites in β2AR for the three ac- tivators. We also simulated the change of the conformation ofβ2AR in the transmembrane regions（TMs）, in the mo- lecular switches, and in the conserved DRY（Aspartic acid, Arginine and Tyrosine） motif. This study provides detailed information concerning the structure ofβ2AR during activation process.

Maternal lysozyme concentrations in the eggs of the Great Cormorant (Phalacrocorax carbo) in relation to breeding density and laying order

Background: Females can differentially deposit the immune factor lysozyme into eggs based on conditions of local breeding density and laying order.Materials: We collected 80 eggs from Great Cormorants(Phalacrocorax carbo) and then analyzed whether the level of lysozymes in the eggs is related to breeding density and laying order.Results: Between clutches,the level of lysozyme in eggs is positively related to breeding density; while within a clutch,the level of lysozyme is positively related to the laying order.Conclusion: When parents breed under conditions of high density,they allocate more lysozymes to their offspring,a trait adaptive to the local environment.That the increase in the level of lysozymes is a function of the laying order seems a necessary condition to mitigate the hierarchy among siblings for improving the survival of the entire clutch.

Characterization of a Novel 1Ay Gene and Its Expression Protein in Triticum urartu

High molecular weight glutenin subunit （HMW-GS） plays an important role in determining dough property and breadmaking quality, and the exploration of novel genes for HMW-GS will be crucial for quality improvement program. A gene coding the y type HMW-GS at Glu-A1 locus in Triticum urartu （AA, 2n=2×= 14） with an electrophoretic mobility similar to that of 1Dyl2, was cloned, sequenced, and heterologously expressed. This novel active lay gene FJ404595 was confirmed by structure analyses of nucleotide and deduced amino acid sequences combining with phylogenetic analysis. The open reading frame （ORF） of this gene was 1 830 bp, encoded a protein of 608 amino acid residues containing 46 hexapeptides and 14 nonapeptides, which was mostly similar to the lAy gene AM183223 at a high identity of 99.62% with the two substitutions of both leucine/proline and valine/glutamate, obviously different from the lAy gene EU984504 with 587 residues containing 44 hexapeptides and 13 nonapeptides in T. urartu. The amino acid （leucine） at 446 differed from that （proline） of all the eight compared active lAy subunits. The predicted secondary protein structure implied that this lAy subunit might also have positive impact on flour processing quality.

Comparison of Grain Zinc and Iron Concentration between Synthetic Hexaploid Wheats and Their Parents

Deficiencies of iron (Fe) and zinc (Zn) in human food afflict a large proportion of the world’s population. Wheat is a major food source of minerals. One way to enhance bread wheat’s ability to enrich these minerals would be to take advantage of diversity of wild species by creating synthetic hexaploid wheat (SW). In this study, two minerals (Fe and Zn) concentrated in the grain of Aegilops tauschii Coss. (2n = 2x = 14, DD), Triticum turgidum L. (2n = 4x = 28, AABB), and 33 lines of their corresponding SW (2n = 2x = 42, AABBDD) were evaluated. The results showed that Fe concentration was decreased in most of SW lines compared with their parental Aegilops tauschii accessions, while Zn concentration was greatly increased in most of SW lines compared with their parental Aegilops tauschii accessions. Aegilops tauschii had stronger Fe enrichment than Triticum turgidum while they expressed the same ability for Zn enrichment. The genotypic variance based on their physiological performance was analyzed. SW lines showed less genotypic variance of Fe and Zn concentration than Aegilops tauschii. SW lines showed less genotypic variance of Fe concentration than Triticum turgidum L. lines while they had more genotypic variance of Zn concentration than Triticum turgidum L. lines. Regardless of the fact that the traits expressed in wild relatives of wheat may not predict the traits that will be expressed in SW lines derived from them, production of SW could be a powerful method creating genotypes with enhanced trait expression.

QTL Positioning of Thousand Wheat Grain Weight in Qaidam Basin

TGW is an important agronomic trait that influences the yield and milling quality of wheat. In this paper, after having investigated the phenotype of TGW from 114 ITMI recombinant inbred genealogies in 4 years in Qaidam Basin, a typical plateau oasis agricultural area, and having combined 1410 molecular markers, 7 major QTL loci of the thousand gain weight were screened out with the aid of QTL network software. These loci included qTgw1B (42.6 cm), qTgw2A(77.9 cm), qTgw2D1 (25.4 cm), qTgw2D2 (51.8 cm), qTgw6A1 (56.1 cm), qTgw6A2 (62.2 cm) and qTgw7A(75.7 cm) with their genetic contribution rates between 3.29% - 19.36%. There were two epistatic effect loci2A-2D and2A-6B with their genetic contribution rates as 2.3% and 5.3% respectively. The quantitative genetic locus positioning of thousand hexaploid wheat grain weight in Qaidam Basin can assist us in better understanding the genetic regulatory network formed by TGW, and can also provide a theoretical basis for improving thousand wheat grain weight in this ecological area.

Alpine grassland degradation intensifies the burrowing behavior of small mammals:evidence for a negative feedback loop

Globally,grassland degradation is an acute ecological problem.In alpine grassland on the Tibetan Plateau,increased densities of various small mammals in degraded grassland are assumed to intensify the degradation process and these mammals are subject to lethal control.However,whether the negative impact of small mammals is solely a result of population size or also a result of activity and behavior has not been tested.In this study,we use plateau pika as a model to compare population size,core area of colony,and the number of burrow entrances and latrines between lightly and severely degraded grassland.We test whether the alleged contribution of pika to grassland degradation is a result of increased population size or increased burrowing activities of individuals in response to lower food abundance.We found that grassland degradation resulted in lower plant species richness,plant height,and biomass.Furthermore,the overall population size of pika was not significantly affected by location in lightly and severely degraded grassland.However,pika core areas in severely grassland degradation were significantly larger and had significantly higher densities of burrows and latrines.Our study provides convincing evidence that habitat-induced changes in the behavior of small,burrowing mammals,such as pika,can exacerbate grassland degradation.This finding has significant implications for managing small mammals and restoring degraded grassland ecosystems.

Alterations to biological soil crusts with alpine meadow retrogressive succession affect seeds germination of three plant species

Biological soil crusts(BSCs) are the important components of alpine meadow ecosystems.The extent and morphology of BSCs vary greatly with alpine meadow retrogressive succession due to grazing pressure.There is significant interest in impacts of crust composition on plant seed germination,especial l y in(semi-) arid environments.However,little is known about the influences of BSCs,and their associations with alpine meadow succession,on germination of typical alpine meadow vascular plant species.In a full factorial common-gardenexperiment,we studied effects of:(1) crust type,(2) seed position,and(3) surface texture on seed germination.We chose three typical alpine meadow plant species(i.e.Poa pratensis,Tibetia himalaica and Potentillen nivea),which belonged to different functional groups(graminoids,legumes,and forbs) and play important roles in all alpine meadow succession stages.Crust type and seed position influenced seed germination,and the inhibitory effects of BSCs depended on the crust type and seed species tested.The major factors influencing seed germination were BSC type,seed position,soil texture,and the interactions between BSC type and seed position; species and seed position; species andsurface texture; and species,crust type,and surface texture.Cyanobacteria crust significantly inhibited germination of all seeds.Seed position also had a significant effect on seed germination(p < 0.001).Fewer seedlings germinated on the surface than below the surface,this was especially true for P.nivea.seeds within cyanobacteria and lichen crusts.Only germination rates of T.himalaica on the soil surface were significantly correlated with plant occurrence frequency within the alpine meadow community.The poor correlation for the other two species is possibly that they are perennials.Our results clearly demonstrated that BSCs can be biological filters during seed germination,depending on the BSC succession stage.Through their influences on seed germination,BSCs can strongly influence community assemblages throughout alpine meadow retrogressive succession.

Investigating the Effectiveness of Road-related Mitigation Measures under Semi-controlled Conditions:A Case Study on Asian Amphibians

Road traffic is the main factor causing the decline in amphibian populations worldwide. The proper design of an amphibian tunnel is one of the most efficient measures to mitigate the negative impacts of road traffic on amphibians. However, no study has investigated the effectiveness of amphibian tunnels under semi-controlled conditions in Asian amphibians. Here, we selected two representative amphibian species, the Chinese brown frog, Rana chensinensis, and the Asiatic toad, Bufo gargarizans, which suffer the most severe road mortality along the roads in Northeast China. We placed experimental arrays of culverts of various sizes(diameters of 1.5, 1, and 0.5 m for circular culverts; side lengths of 1.5, 1, and 0.5 m for box culverts), and substrate type(soil, concrete, and metal) to examine the preferences of both species during the migratory season between May and September in 2016 and 2017. The results revealed that the Chinese brown frog preferred mid-and large-sized culverts as well as soil culverts. We concluded that culverts with a side length ≥ 1 m, lined with soil, and accompanied by a ≥ 0.4 m high guide drift fence and ≤ 45° gradient on the roadside ditch wall would best facilitate road crossings for both species and likely for other amphibian species in Northeast China.

A chromosome-scale genome assembly of a diploid alfalfa,the progenitor of autotetraploid alfalfa

Alfalfa(Medicago sativa L.)is one of the most important and widely cultivated forage crops.It is commonly used as a vegetable and medicinal herb because of its excellent nutritional quality and significant economic value.Based on Illumina,Nanopore and Hi-C data,we assembled a chromosome-scale assembly of Medicago sativa spp.caerulea(voucher PI464715),the direct diploid progenitor of autotetraploid alfalfa.The assembled genome comprises 793.2 Mb of genomic sequence and 47,202 annotated protein-coding genes.The contig N50 length is 3.86 Mb.This genome is almost twofold larger and contains more annotated protein-coding genes than that of its close relative,Medicago truncatula(420 Mb and 44,623 genes).The more expanded gene families compared with those in M.truncatula and the expansion of repetitive elements rather than whole-genome duplication(i.e.,the two species share the ancestral Papilionoideae whole-genome duplication event)may have contributed to the large genome size of M.sativa spp.caerulea.Comparative and evolutionary analyses revealed that M.sativa spp.caerulea diverged from M.truncatula~5.2 million years ago,and the chromosomal fissions and fusions detected between the two genomes occurred during the divergence of the two species.In addition,we identified 489 resistance(R)genes and 82 and 85 candidate genes involved in the lignin and cellulose biosynthesis pathways,respectively.The near-complete and accurate diploid alfalfa reference genome obtained herein serves as an important complement to the recently assembled autotetraploid alfalfa genome and will provide valuable genomic resources for investigating the genomic architecture of autotetraploid alfalfa as well as for improving breeding strategies in alfalfa.