Conservation genetics of endangered Trillium govanianum Wall. ex D. Don - A pharmaceutically prized medicinal plant from the Himalaya and implications for species recovery.

Understanding the genetic diversity and population structure of pharmaceutically important endangered plant species is crucial for their conservation and sustainable use. Despite the continuous population decline in Trillium govanianum Wall. ex D. Don, a highly prized medicinal plant endemic to the Himalaya, information regarding its conservation genetics has been lacking. Here, we employed a conservation genetics approach to investigate how drastically declining populations in natural habitats impact population genetic diversity and structure of this endangered species across the Kashmir Himalaya. We used Start codon targeted (SCoT) and Simple sequence repeat (SSR) markers to assess the intra- and inter-population genetic variation in seven sites across the study region. Based on these markers, we found a very low genetic diversity in T. govanianum populations. Very low levels of observed heterozygosity (Ho = 0.000) and that expected (He = 0.064) in the populations indicate high heterozygote deficiency and high levels of inbreeding depression (FIS = 1.000). A high genetic differentiation was observed among the populations for both SCoT (Gst = 0.719) and SSR (Fst = 0.707) markers. Both the markers showed low gene flow, SCoT (Nm = 0.195) and SSR (Nm = 0.119), depicting high among-population variation than within-population variation. Analysis of molecular variance also indicated a higher genetic variation between the populations than within populations. We also observed a significant positive correlation between genetic divergence and geographical distance, indicating that genetic differentiation in T. govanianum follows a pattern of isolation by distance. Bayesian structure and cluster analysis grouped the populations according to their geographical proximity. Further, redundancy analysis (RDA) revealed the presence of one polymorphic locus for each marker with high discriminatory power. Overall, our findings reveal a very low genetic diversity, high levels of inbreeding, and high genetic differentiation among the populations; likely resulting from habitat fragmentation, population isolation, bottleneck effect, low gene flow, and predominantly asexual reproduction currently operative in the species. Finally, based on the insights gained, we discuss the potential implications of our findings in guiding species recovery and habitat rehabilitation of T. govanianum in the Himalaya with conservation lessons for elsewhere in the world.

Spatial transcriptome analysis provides insights of key gene(s) involved in steroidal saponin biosynthesis in medicinally important herb Trillium govanianum.

Trillium govanianum, an endangered medicinal herb native to the Himalaya, is less studied at the molecular level due to the non-availability of genomic resources. To facilitate the basic understanding of the key genes and regulatory mechanism of pharmaceutically important biosynthesis pathways, first spatial transcriptome sequencing of T. govanianum was performed. 151,622,376 (~11.5 Gb) high quality reads obtained using paired-end Illumina sequencing were de novo assembled into 69,174 transcripts. Functional annotation with multiple public databases identified array of genes involved in steroidal saponin biosynthesis and other secondary metabolite pathways including brassinosteroid, carotenoid, diterpenoid, flavonoid, phenylpropanoid, steroid and terpenoid backbone biosynthesis, and important TF families (bHLH, MYB related, NAC, FAR1, bZIP, B3 and WRKY). Differentially expressed large number of transcripts, together with CYPs and UGTs suggests involvement of these candidates in tissue specific expression. Combined transcriptome and expression analysis revealed that leaf and fruit tissues are the main site of steroidal saponin biosynthesis. In conclusion, comprehensive genomic dataset created in the current study will serve as a resource for identification of potential candidates for genetic manipulation of targeted bioactive metabolites and also contribute for development of functionally relevant molecular marker resource to expedite molecular breeding and conservation efforts in T. govanianum.

Pollen-mediated gene dispersal within continuous and fragmented populations of a forest understorey species, Trillium cuneatum.

Pollen movement plays a critical role in the distribution of genetic variation within and among plant populations. Direct measures of pollen movement in the large, continuous populations that characterize many herbaceous plant species are often technically difficult and biologically unreliable. Here, we studied contemporary pollen movement in four large populations of Trillium cuneatum. Three populations, located in the Georgia Piedmont, are exposed to strong anthropogenic disturbances, while the fourth population, located in the Southern Appalachian Mountains, is relatively undisturbed. Using the recently developed TwoGener analysis, we extracted estimates of the effective number of pollen donors (N(ep)), effective mating neighbourhood size (A(ep)) and the average distance of pollen movement (delta) for each population. We extended the TwoGener method by developing inference on the paternal gametic contribution to the embryo in situations where offspring genotypes are inferred from seeds and elaiosomes of species with bisporic megagametogenesis. Our estimates indicate that maternal plants do not sample pollen randomly from a global pool; rather, pollen movement in all four populations is highly restricted. Although the effective number of pollen donors per maternal plant is low (1.22-1.66) and pollen movement is highly localized in all populations, N(ep) in the disturbed Piedmont populations is higher and there is more pollen movement than in the mountains. The distance pollen moves is greater in disturbed sites and fragmented populations, possibly due to edge effects in Trillium habitats.

Genetic diversity and genetic structure of an endangered species, Trillium tschonoskii.

The genetic diversity and genetic structure of Trillium tschonoskii (Maxim) were investigated using amplified fragment length polymorphism markers. Eight primer combinations were carried out on 105 different individuals sampled from seven populations. Of the 619 discernible DNA fragments generated, 169 (27.3%) were polymorphic. The percentage of polymorphic bands within populations ranged from 4.52 to 10.50. Genetic diversity (H(E)) within populations ranged from 0.0130 to 0.0379, averaging 0.0536 at the species level. Genetic differentiation among populations was detected based on Nei's genetic diversity analysis (53.03%) and analysis of molecular variance (AMOVA) (52.43%). AMOVA indicated significant genetic differentiation among populations (52.43% of the variance) and within populations (47.57% of the variance) (p < 0.0002). Gene flow was low (0.4429) among populations. Species breeding system and limited gene flow among populations are plausible reasons for the high genetic differentiation observed for this species. We propose an appropriate strategy for conserving the genetic resources of T. tschonoskii in China.