Development and validation of sex-linked molecular markers for rapid and accurate identification of male and female Hippophae tibetana plants.

Hippophae tibetana, one of the highest-altitude woody plants endemic to the Qinghai-Tibet Plateau, primarily thrives on riverbanks formed by glacial meltwater. As a dioecious species, it demonstrates significant ecological and economic value in extreme alpine environments. However, the lack of sex identification techniques outside of the flowering period severely limits research on sex ratio, differentiation, and breeding. There is an urgent need to develop effective sex-linked molecular markers that are independent of developmental stages, but current research in this area remains limited. This study developed a set of accurate sex-linked molecular markers for the rapid identification of male and female individuals of H. tibetana. Through whole-genome resequencing of 32 sexually differentiated H. tibetana samples, this study offers strong evidence supporting chromosome 2 as the sex chromosome and successfully identified key loci related to sex determination on this chromosome. Utilizing these loci, we, for the first time, developed three reliable pairs of sex-specific molecular markers, which exhibited high accuracy during validation across various geographic populations, offering an effective tool for the sex identification of H. tibetana. Additionally, this study lays the groundwork for further research into the mechanisms of sex determination and the evolution of sex chromosomes in H. tibetana.

Complete mitochondrial genome of Hippophae tibetana: insights into adaptation to high-altitude environments.

Hippophae tibetana, belonging to the Elaeagnaceae family, is an endemic plant species of the Qinghai-Tibet Plateau, valued for its remarkable ecological restoration capabilities, as well as medicinal and edible properties. Despite being acknowledged as a useful species, its mitochondrial genome data and those of other species of the Elaeagnaceae family are lacking to date. In this study, we, for the first time, successfully assembled the mitochondrial genome of H. tibetana, which is 464,208 bp long and comprises 31 tRNA genes, 3 rRNA genes, 37 protein-coding genes, and 3 pseudogenes. Analysis of the genome revealed a high copy number of the trnM-CAT gene and a high prevalence of repetitive sequences, both of which likely contribute to genome rearrangement and adaptive evolution. Through nucleotide diversity and codon usage bias analyses, we identified specific genes that are crucial for adaptation to high-altitude conditions. Notably, genes such as atp6, ccmB, nad4L, and nad7 exhibited signs of positive selection, indicating the presence of unique adaptive traits for survival in extreme environments. Phylogenetic analysis confirmed the close relationship between the Elaeagnaceae family and other related families, whereas intergenomic sequence transfer analysis revealed a substantial presence of homologous fragments among the mitochondrial, chloroplast, and whole genomes, which may be linked to the high-altitude adaptation mechanisms of H. tibetana. The findings of this study not only enrich our knowledge of H. tibetana molecular biology but also advance our understanding of the adaptive evolution of plants on the Qinghai-Tibet Plateau. This study provides a solid scientific foundation for the molecular breeding, conservation, and utilization of H. tibetana genetic resources.

The complete chloroplast genome of Meconopsis torquata (Papaveraceae), a traditional Tibetan medicine.

Meconopsis torquata Prain 1906, a national second-class rare and endangered plant, is reported here for the first time for its complete chloroplast genome. The genome is 153,290 bp in length, comprising a large single-copy region (LSC, 83,918 bp), a small single-copy region (SSC, 17,740 bp), and two inverted repeat sequences (IRa and IRb, each 25,816 bp). The overall GC content is 38.7%, with the IR region having the highest content (43.1%). The genome is annotated with 112 unique genes, including 4 rRNA genes, 29 tRNA genes, and 79 protein-coding genes. Analysis of codon usage bias reveals that codons ending in A/T account for 96.7% of those with a Relative Synonymous Codon Usage (RSCU) value above 1. This predominance of A/T-ending codons might be indicative of M. torquata adaptation to high-altitude environments. Phylogenetic analysis reveals a close kinship between M. torquata and M. pinnatifolia and M. paniculata, indicating that the ancestral groups of these species might have a complex evolutionary history. This study uncovers the genetic characteristics and adaptive evolution of M. torquata, offering a new perspective in understanding the phylogenetic relationships within the genus. The findings not only provide a solid theoretical foundation for the conservation and sustainable use of this rare and endangered species but also offer significant scientific support for the conservation of biodiversity.

Mother-reliant or self-reliant: the germination strategy of seeds in a species-rich alpine meadow is associated with the existence of pericarps.

BACKGROUND AND AIMS: Some plants germinate their seeds enclosed by a pericarp, whereas others lack the outer packaging. As a maternal tissue, the pericarp might impart seeds with different germination strategies. Plants in a community with different flowering times might separately disperse and germinate their seeds; therefore, flowering time can be considered as one manifestation of maternal effects on the offspring. The mass of the seed is another important factor influencing germination and represents the intrinsic resource of the seed that supports germination. Using seeds from a species-rich alpine meadow located in the Hengduan Mountains of China, a global biodiversity hotspot, we aimed to illustrate whether and how the type of seed (with or without a pericarp) modulates the interaction of flowering time and seed mass with germination. METHODS: Seeds were germinated in generally favourable conditions, and the speed of germination [estimated by mean germination time (MGT)] was calculated. We quantified the maternal conditions by separation of flowering time for 67 species in the meadow, of which 31 produced seeds with pericarps and 36 yielded seeds without pericarps. We also weighed 100 seeds of each species to assess their mass. KEY RESULTS: The MGT varied between the two types of seeds. For seeds with pericarps, MGT was associated with flowering time but not with seed mass. Plants with earlier flowering times in the meadow exhibited more rapid seed germination. For seeds without a pericarp, the MGT depended on seed mass, with smaller seeds germinating more rapidly than larger seeds. CONCLUSIONS: The distinct responses of germination to flowering time and seed mass observed in seeds with and without a pericarp suggest that germination strategies might be mother-reliant for seeds protected by pericarps but self-reliant for those without such protection. This new finding improves our understanding of seed germination by integrating ecologically mediated maternal conditions and inherent genetic properties.

How to survive in the world's third poplar: Insights from the genome of the highest altitude woody plant, Hippophae tibetana (Elaeagnaceae).

Hippophae tibetana (Tibetan sea-buckthorn) is one of the highest distributed woody plants in the world (3,000-5,200 meters a.s.l.). It is characterized by adaptation to extreme environment and important economic values. Here, we combined PacBio Hifi platform and Hi-C technology to assemble a 1,452.75 Mb genome encoding 33,367 genes with a Contig N50 of 74.31 Mb, and inferred its sexual chromosome. Two Hippophae-specific whole-genome duplication events (18.7-21.2 million years ago, Ma; 28.6-32.4 Ma) and long terminal repeats retroelements (LTR-RTs) amplifications were detected. Comparing with related species at lower altitude, Ziziphus jujuba (<1, 700 meters a.s.l.), H. tibetana had some significantly rapid evolving genes involved in adaptation to high altitude habitats. However, comparing with Hippophae rhamnoides (<3, 700 meters a.s.l.), no rapid evolving genes were found except microtubule and microtubule-based process genes, H. tibetana has a larger genome, with extra 2, 503 genes (7.5%) and extra 680.46 Mb transposable elements (TEs) (46.84%). These results suggest that the changes in the copy number and regulatory pattern of genes play a more important role for H. tibetana adapting to more extreme and variable environments at higher altitude by more TEs and more genes increasing genome variability and expression plasticity. This suggestion was supported by two findings: nitrogen-fixing genes of H. tibetana having more copies, and intact TEs being significantly closer genes than fragmentary TEs. This study provided new insights into the evolution of alpine plants.

Comparative phylogeography study reveals introgression and incomplete lineage sorting during rapid diversification of Rhodiola.

BACKGROUND AND AIMS: Many plant taxa in the Qinghai-Tibetan Plateau (QTP) and the Hengduan Mountains (HM) radiated rapidly during the Quaternary but with frequent secondary contact between diverging populations. Incomplete lineage sorting and introgressive hybridization might be involved during the rapid radiation, but their effects on phylogeography have not been fully determined. METHODS: We investigated the chloroplast DNA (cpDNA)/internal transcribed spacer (ITS) sequence variations of 611 samples of Rhodiola bupleuroides, R. discolor, R. fastigiata and R. chrysanthemifolia from the QTP and HM to compare the phylogeographic patterns between the four species with different evolutionary histories, geographic ranges and reproductive modes. KEY RESULTS: The divergence times of these species were consistent with the last peak of in situ speciation in the HM. While closely related species exhibited different phylogeographic patterns, they shared several ribotypes and haplotypes in sympatric populations, suggesting introgressive hybridization. A significant phylogenetic discordance between ribotypes and haplotypes was detected in three species, implying incomplete lineage sorting. Rhodiola discolor houses an extraordinary richness of cpDNA haplotypes, and this finding may be attributed to adaptive radiation. CONCLUSION: In addition to geographic isolation and climate oscillations during the Quaternary, both introgressive hybridization and incomplete lineage sorting play important roles in species that experienced rapid diversification in the QTP and HM.

Evolutionary history and pan-genome dynamics of strawberry (Fragaria spp.).

Strawberry (Fragaria spp.) has emerged as a model system for various fundamental and applied research in recent years. In total, the genomes of five different species have been sequenced over the past 10 y. Here, we report chromosome-scale reference genomes for five strawberry species, including three newly sequenced species' genomes, and genome resequencing data for 128 additional accessions to estimate the genetic diversity, structure, and demographic history of key Fragaria species. Our analyses obtained fully resolved and strongly supported phylogenies and divergence times for most diploid strawberry species. These analyses also uncovered a new diploid species (Fragaria emeiensis Jia J. Lei). Finally, we constructed a pan-genome for Fragaria and examined the evolutionary dynamics of gene families. Notably, we identified multiple independent single base mutations of the MYB10 gene associated with white pigmented fruit shared by different strawberry species. These reference genomes and datasets, combined with our phylogenetic estimates, should serve as a powerful comparative genomic platform and resource for future studies in strawberry.

Genomic analysis of field pennycress (Thlaspi arvense) provides insights into mechanisms of adaptation to high elevation.

BACKGROUND: Understanding how organisms evolve and adapt to extreme habitats is of crucial importance in evolutionary ecology. Altitude gradients are an important determinant of the distribution pattern and range of organisms due to distinct climate conditions at different altitudes. High-altitude regions often provide extreme environments including low temperature and oxygen concentration, poor soil, and strong levels of ultraviolet radiation, leading to very few plant species being able to populate elevation ranges greater than 4000 m. Field pennycress (Thlaspi arvense) is a valuable oilseed crop and emerging model plant distributed across an elevation range of nearly 4500 m. Here, we generate an improved genome assembly to understand how this species adapts to such different environments. RESULTS: We sequenced and assembled de novo the chromosome-level pennycress genome of 527.3 Mb encoding 31,596 genes. Phylogenomic analyses based on 2495 single-copy genes revealed that pennycress is closely related to Eutrema salsugineum (estimated divergence 14.32-18.58 Mya), and both species form a sister clade to Schrenkiella parvula and genus Brassica. Field pennycress contains the highest percentage (70.19%) of transposable elements in all reported genomes of Brassicaceae, with the retrotransposon proliferation in the Middle Pleistocene being likely responsible for the expansion of genome size. Moreover, our analysis of 40 field pennycress samples in two high- and two low-elevation populations detected 1,256,971 high-quality single nucleotide polymorphisms. Using three complementary selection tests, we detected 130 candidate naturally selected genes in the Qinghai-Tibet Plateau (QTP) populations, some of which are involved in DNA repair and the ubiquitin system and potential candidates involved in high-altitude adaptation. Notably, we detected a single base mutation causing loss-of-function of the FLOWERING LOCUS C protein, responsible for the transition to early flowering in high-elevation populations. CONCLUSIONS: Our results provide a genome-wide perspective of how plants adapt to distinct environmental conditions across extreme elevation differences and the potential for further follow-up research with extensive data from additional populations and species.

Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude.

Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the Qinghai-Tibet Plateau (QTP) about 4,000 m above sea level and represents an attractive model system for studying speciation and ecological adaptation in extreme environments. We assembled a draft genome sequence of 234.72 Mb encoding 27,019 genes and investigated its origin and adaptive evolutionary mechanisms. Phylogenomic analyses based on 4,586 single-copy genes revealed that C. himalaica is most closely related to Capsella (estimated divergence 8.8 to 12.2 Mya), whereas both species form a sister clade to Arabidopsis thaliana and Arabidopsis lyrata, from which they diverged between 12.7 and 17.2 Mya. LTR retrotransposons in C. himalaica proliferated shortly after the dramatic uplift and climatic change of the Himalayas from the Late Pliocene to Pleistocene. Compared with closely related species, C. himalaica showed significant contraction and pseudogenization in gene families associated with disease resistance and also significant expansion in gene families associated with ubiquitin-mediated proteolysis and DNA repair. We identified hundreds of genes involved in DNA repair, ubiquitin-mediated proteolysis, and reproductive processes with signs of positive selection. Gene families showing dramatic changes in size and genes showing signs of positive selection are likely candidates for C. himalaica's adaptation to intense radiation, low temperature, and pathogen-depauperate environments in the QTP. Loss of function at the S-locus, the reason for the transition to self-fertilization of C. himalaica, might have enabled its QTP occupation. Overall, the genome sequence of C. himalaica provides insights into the mechanisms of plant adaptation to extreme environments.

Testing the effect of the Himalayan mountains as a physical barrier to gene flow in Hippophae tibetana Schlect. (Elaeagnaceae).

Hippophae tibetana is a small, dioecious wind-pollinated shrub endemic to the Tibetan-Qinghai Plateau. It is one of the shrubs that occur at very high elevations (5250 m a.s.l.). The Himalayan mountains provides a significant geographical barrier to the Qinghai-Tibetan Plateau, dividing the Himalayan area into two regions with Nepal to the south and Tibet to the north. There is no information on how the Himalayan mountains influence gene flow and population differentiation of alpine plants. In this study, we analyzed eight nuclear microsatellite markers and cpDNA trnT-trnF regions to test the role of the Himalayan mountains as a barrier to gene flow between populations of H. tibetana. We also examined the fine-scale genetic structure within a population of H. tibetana on the north slope of Mount (Mt.) Everest. For microsatellite analyses, a total of 241 individuals were sampled from seven populations in our study area (4 from Nepal, 3 from Tibet), including 121 individuals that were spatially mapped within a 100 m × 100 m plot. To test for seed flow, the cpDNA trnT-trnF regions of 100 individuals from 6 populations (4 from Nepal, 2 from Tibet) were also sequenced. Significant genetic differentiation was detected between the two regions by both microsatellite and cpDNA data analyses. These two datasets agree about southern and northern population differentiation, indicating that the Himalayan mountains represent a barrier to H. tibetana limiting gene flow between these two areas. At a fine scale, spatial autocorrelation analysis suggests significant genetic structure within a distance of less than 45 m, which may be attributed mainly to vegetative reproduction and habitat fragmentation, as well as limited gene flow.

Phylogeography of Sophora moorcroftiana Supports Wu's Hypothesis on the Origin of Tibetan Alpine Flora.

Wu hypothesized that the Tibetan flora originated mostly from the paleotropical Tertiary flora in the Hengduan Mountains by adapting to the cold and arid environments associated with the strong uplift of the Qinghai-Tibet Plateau (QTP). Here, we combine the phylogeographic history of Sophora moorcroftiana with that of Sophora davidii to explore the speciation of S. moorcroftiana to test this hypothesis. We collected 151 individuals from 17 populations and sequenced 2 chloroplast fragments and the internal transcribed spacer of rDNA. Five chlorotypes and 9 ribotypes were detected but no significant phylogeographic structure was revealed. The integrated results of phylogeographic studies of these 2 species clearly support the progenitor-derivative relationship between them. We infer that the western peripheral population of S. davidii migrated westwards from the Hengduan Mountains to the middle reaches of the Yarlung Zangbo River and differentiated from its ancestor in the process of adaptation to increasingly cold and arid environments with the uplift of the QTP and finally evolved into S. moorcroftiana during the Late Pliocene. In addition, our findings shed light on the idea that natural selection, as imposed by climate differentiation (especially mean diurnal range and precipitation seasonality), directly drove this peripatric speciation event after geographic isolation. The speciation of S. moorcroftiana is a strong case supporting Wu's hypothesis about the origin of Tibet's flora.