A genome assembly for Orinus kokonorica provides insights into the origin, adaptive evolution and further diversification of two closely related grass genera.

Ancient whole-genome duplication (WGD) or polyploidization is prevalent in plants and has played a crucial role in plant adaptation. However, the underlying genomic basis of ecological adaptation and subsequent diversification after WGD are still poorly understood in most plants. Here, we report a chromosome-scale genome assembly for the genus Orinus (Orinus kokonorica as representative) and preform comparative genomics with its closely related genus Cleistogenes (Cleistogenes songorica as representative), both belonging to a newly named subtribe Orininae of the grass subfamily Chloridoideae. The two genera may share one paleo-allotetraploidy event before 10 million years ago, and the two subgenomes of O. kokonorica display neither fractionation bias nor global homoeolog expression dominance. We find substantial genome rearrangements and extensive structural variations (SVs) between the two species. With comparative transcriptomics, we demonstrate that functional innovations of orthologous genes may have played an important role in promoting adaptive evolution and diversification of the two genera after polyploidization. In addition, copy number variations and extensive SVs between orthologs of flower and rhizome related genes may contribute to the morphological differences between the two genera. Our results provide new insights into the adaptive evolution and subsequent diversification of the two genera after polyploidization.

The genome sequence provides insights into salt tolerance of Achnatherum splendens (Gramineae), a constructive species of alkaline grassland.

Achnatherum splendens Trin. (Gramineae) is a constructive species of the arid grassland ecosystem in Northwest China and is a major forage grass. It has good tolerance of salt and drought stress in alkaline habitats. Here, we report its chromosome-level genome, determined through a combination of Illumina HiSeq sequencing, PacBio sequencing and Hi-C technology. The final assembly of the ~1.17 Gb genome sequence had a super-scaffold N50 of 40.3 Mb. A total of 57 374 protein-coding genes were annotated, of which 54 426 (94.5%) genes have functional protein annotations. Approximately 735 Mb (62.37%) of the assembly were identified as repetitive elements, and among these, LTRs (40.53%) constitute the highest proportion, having made a major contribution to the expansion of genome size in A. splendens. Phylogenetic analysis revealed that A. splendens diverged from the Brachypodium distachyon-Hordeum vulgare-Aegilops tauschii subclade around 37 million years ago (Ma) and that a clade comprising these four species diverged from the Phyllostachys edulis clade ~47 Ma. Genomic synteny indicates that A. splendens underwent an additional species-specific whole-genome duplication (WGD) 18-20 Ma, which further promoted an increase in copies of numerous saline-alkali-related gene families in the A. splendens genome. By transcriptomic analysis, we further found that many of these duplicated genes from this extra WGD exhibited distinct functional divergence in response to salt stress. This WGD, therefore, contributed to the strong resistance to salt stress and widespread arid adaptation of A. splendens.

Extensive genomic rearrangements mediated by repetitive sequences in plastomes of Medicago and its relatives.

BACKGROUND: Although plastomes are highly conserved with respect to gene content and order in most photosynthetic angiosperms, extensive genomic rearrangements have been reported in Fabaceae, particularly within the inverted repeat lacking clade (IRLC) of Papilionoideae. Two hypotheses, i.e., the absence of the IR and the increased repeat content, have been proposed to affect the stability of plastomes. However, this is still unclear for the IRLC species. Here, we aimed to investigate the relationships between repeat content and the degree of genomic rearrangements in plastomes of Medicago and its relatives Trigonella and Melilotus, which are nested firmly within the IRLC. RESULTS: We detected abundant repetitive elements and extensive genomic rearrangements in the 75 newly assembled plastomes of 20 species, including gene loss, intron loss and gain, pseudogenization, tRNA duplication, inversion, and a second independent IR gain (IR ~ 15 kb in Melilotus dentata) in addition to the previous first reported cases in Medicago minima. We also conducted comparative genomic analysis to evaluate plastome evolution. Our results indicated that the overall repeat content is positively correlated with the degree of genomic rearrangements. Some of the genomic rearrangements were found to be directly linked with repetitive sequences. Tandem repeated sequences have been detected in the three genes with accelerated substitution rates (i.e., accD, clpP, and ycf1) and their length variation could be explained by the insertions of tandem repeats. The repeat contents of the three localized hypermutation regions around these three genes with accelerated substitution rates are also significantly higher than that of the remaining plastome sequences. CONCLUSIONS: Our results suggest that IR reemergence in the IRLC species does not ensure their plastome stability. Instead, repeat-mediated illegitimate recombination is the major mechanism leading to genome instability, a pattern in agreement with recent findings in other angiosperm lineages. The plastome data generated herein provide valuable genomic resources for further investigating the plastome evolution in legumes.

Whole-Genome Diversification Analysis of the Hornbeam Species Reveals Speciation and Adaptation Among Closely Related Species.

Speciation is the key evolutionary process for generating biological diversity and has a central place in evolutionary and ecological research. How species diverge and adapt to different habitats is one of the most exciting areas in speciation studies. Here, we sequenced 55 individuals from three closely related species in the genus Carpinus: Carpinus tibetana, Carpinus monbeigiana, and Carpinus mollicoma to understand the strength and direction of gene flow and selection during the speciation process. We found low genetic diversity in C. tibetana, which reflects its extremely small effective population size. The speciation analysis between C. monbeigiana and C. mollicoma revealed that both species diverged ∼1.2 Mya with bidirectional gene flow. A total of 291 highly diverged genes, 223 copy number variants genes, and 269 positive selected genes were recovered from the two species. Genes associated with the diverged and positively selected regions were mainly involved in thermoregulation, plant development, and response to stress, which included adaptations to their habitats. We also found a great population decline and a low genetic divergence of C. tibetana, which suggests that this species is extremely vulnerable. We believe that the current diversification and adaption study and the important genomic resource sequenced herein will facilitate the speciation studies and serve as an important methodological reference for future research.

Genomic analysis of Medicago ruthenica provides insights into its tolerance to abiotic stress and demographic history.

Medicago ruthenica has been recently cultivated as a new forage crop and has been recognized as a source of genes to improve abiotic stress tolerance in cultivated alfalfa because of its remarkable tolerance to drought, salinity-alkalinity, and cold and snowy winters. Here, we reveal a chromosome-scale genome sequence of M. ruthenica based on Illumina, PacBio, and Hi-C data. The assembled genome consists of 903.56 Mb with 50,268 annotated protein-coding genes, which is larger and contains relatively more genes than Medicago truncatula (420 Mb and 44,623 genes) and Medicago sativa spp. caerulea (793 Mb and 47,202 genes). All three species shared the ancestral Papilionoideae whole-genome duplication event before their divergence. The more recent expansion of repetitive elements compared to that in the other two species was determined to have contributed greatly to the larger genome size of M. ruthenica. We further found that multiple gene and transcription factor families (e.g., SOS homologous genes, NAC, C2H2, and CAMTA) have expanded in M. ruthenica, which might have led to its enhanced tolerance to abiotic stress. In addition, M. ruthenica harbors more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed two genetic lineages, reflecting the west and east of its geographical distribution, respectively. The two lineages probably diverged during the last glaciation and survived in multiple refugia at the last glacial maximum, followed by recent expansion. Our genomic data provide a genetic basis for further molecular breeding research on M. ruthenica and alfalfa.

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.

Phylogeny and Species Delimitation of Chinese Medicago (Leguminosae) and Its Relatives Based on Molecular and Morphological Evidence.

Medicago and its relatives, Trigonella and Melilotus comprise the most important forage resources globally. The alfalfa selected from the wild relatives has been cultivated worldwide as the forage queen. In the Flora of China, 15 Medicago, eight Trigonella, and four Melilotus species are recorded, of which six Medicago and two Trigonella species are introduced. Although several studies have been conducted to investigate the phylogenetic relationship within the three genera, many Chinese naturally distributed or endemic species are not included in those studies. Therefore, the taxonomic identity and phylogenetic relationship of these species remains unclear. In this study, we collected samples representing 18 out of 19 Chinese naturally distributed species of these three genera and three introduced Medicago species, and applied an integrative approach by combining evidences from population-based morphological clusters and molecular data to investigate species boundaries. A total of 186 individuals selected from 156 populations and 454 individuals from 124 populations were collected for genetic and morphological analyses, respectively. We sequenced three commonly used DNA barcodes (trnH-psbA, trnK-matK, and ITS) and one nuclear marker (GA3ox1) for phylogenetic analyses. We found that 16 out of 21 species could be well delimited based on phylogenetic analyses and morphological clusters. Two Trigonella species may be merged as one species or treated as two subspecies, and Medicago falcata should be treated as a subspecies of the M. sativa complex. We further found that major incongruences between the chloroplast and nuclear trees mainly occurred among the deep diverging lineages, which may be resulted from hybridization, incomplete lineage sorting and/or sampling errors. Further studies involving a finer sampling of species associated with large scale genomic data should be employed to better understand the species delimitation of these three genera.