Genetic analyses of ancient tea trees provide insights into the breeding history and dissemination of Chinese Assam tea (Camellia sinensis var. assamica).

Chinese Assam tea (Camellia sinensis var. assamica) is an important tea crop with a long history of cultivation in Yunnan, China. Despite its potential value as a genetic resource, its genetic diversity and domestication/breeding history remain unclear. To address this issue, we genotyped 469 ancient tea plant trees representing 26 C. sinensis var. assamica populations, plus two of its wild relatives (six and three populations of C. taliensis and C. crassicolumna, respectively) using 16 nuclear microsatellite loci. Results showed that Chinese Assam tea has a relatively high, but comparatively lower gene diversity (HS = 0.638) than the wild relative C. crassicolumna (HS = 0.658). Clustering in STRUCTURE indicated that Chinese Assam tea and its two wild relatives formed distinct genetic groups, with considerable interspecific introgression. The Chinese Assam tea accessions clustered into three gene pools, corresponding well with their geographic distribution. However, NewHybrids analysis indicated that 68.48% of ancient Chinese Assam tea plants from Xishuangbanna were genetic intermediates between the Puer and Lincang gene pools. In addition, 10% of the ancient Chinese Assam tea individuals were found to be hybrids between Chinese Assam tea and C. taliensis. Our results suggest that Chinese Assam tea was domesticated separately in three gene pools (Puer, Lincang and Xishuangbanna) in the Mekong River valley and that the hybrids were subsequently selected during the domestication process. Although the domestication history of Chinese Assam tea in southwestern Yunnan remains complex, our results will help to identify valuable genetic resources that may be useful in future tea breeding programs.

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

Genetic imprints of grafting in wild iron walnut populations in southwestern China.

BACKGROUND: Anthropogenic activities are causing unprecedented loss of genetic diversity in many species. However, the effects on genetic diversity from large-scale grafting onto wild plants of crop species are largely undetermined. Iron walnut (Juglans sigillata Dode) is a deciduous nut tree crop endemic to southwestern China with a long history of cultivation. Due to the rapid expansion of the walnut industry, many natural populations are now being replaced by cultivars grafted onto wild rootstocks. However, little is known about the potential genetic consequences of such action on natural populations. RESULTS: We sampled the scion and the rootstock from each of 149 grafted individuals within nine wild populations of J. sigillata from Yunnan Province which is the center of walnut diversity and cultivation in China, and examined their genetic diversity and population structure using 31 microsatellite loci. Scions had lower genetic diversity than rootstocks, and this pattern was repeated in seven of the nine examined populations. Among those seven populations, AMOVA and clustering analyses showed a clear genetic separation between all rootstocks and all scions. However, the two remaining populations, both from northern Yunnan, showed genetic similarity between scions and rootstocks, possibly indicating that wild populations here are derived from feralized local cultivars. Moreover, our data indicated probable crop-to-wild gene flow between scions and rootstocks, across all populations. CONCLUSIONS: Our results indicate that large-scale grafting has been causing genetic diversity erosion and genetic structure breakdown in the wild material of J. sigillata within Yunnan. To mitigate these effects, we caution against the overuse of grafting in wild populations of iron walnut and other crop species and recommend the preservation of natural genotypes through in situ  and ex situ conservation.

Genetic analysis of walnut cultivars from southwest China: Implications for germplasm improvement.

Walnuts are highly valued for their rich nutritional profile and wide medicinal applications. This demand has led to the intensification of breeding activities in major walnut production areas such as southwest China, in order to develop more superior cultivars. With the increasing number of cultivars, accurate identification becomes fundamental to selecting the right cultivar for grafting, industrial processing or development of new cultivars. To ensure proper identification of cultivars and understand the genetic structure of wild and cultivated material, we genotyped 362 cultivated and wild individuals of walnut trees from southwest China (with two additional populations from Xinjiang, plus three cultivars from Canada, France and Belgium) using 36 polymorphic microsatellite loci. We found relatively low indices of genetic diversity (H O = 0.570, H E = 0.404, N A = 2.345) as well as a high level of clonality (>85% of cultivars), indicating reliance on genetically narrow sources of parental material for breeding. Our STRUCTURE and PCoA analyses generally delineated the two species, though considerable levels of introgression were also evident. More significantly, we detected a distinct genetic group of cultivated Juglans sigillata, which mainly comprised individuals of the popular 'Yangbidapao' landrace. Finally, a core set of 18 SSR loci was selected, which was capable of identifying 32 cultivars. In a nutshell, our results call for more utilization of genetically disparate material, including wild walnut trees, as parental sources to breed for more cultivars. The data reported herein will significantly contribute towards the genetic improvement and conservation of the walnut germplasm in southwest China.

Genetic Diversity and Structure of Persian Walnut (Juglans regia L.) in Pakistan: Implications for Conservation.

Persian (Common) walnut (Juglans regia L.) is a famous fruit tree species valued for its nutritious nuts and high-quality wood. Although walnut is widely distributed and plays an important role in the economy and culture of Pakistan, the genetic diversity and structure of its populations in the country remains poorly understood. Therefore, using 31 nuclear microsatellites, we assessed the genetic diversity and population structure of 12 walnut populations sampled across Pakistan. We also implemented the geostatistical IDW technique in ArcGIS to reveal "hotspots" of genetic diversity. Generally, the studied populations registered relatively low indices of genetic diversity (NA = 3.839, HO = 0.558, UHE = 0.580), and eight populations had positive inbreeding coefficient (FIS) values. Low among-population differentiation was indicated by AMOVA, pairwise FST and DC. STRUCTURE, PCoA and neighbor joining (NJ) analysis revealed a general lack of clear clustering in the populations except that one population in Upper Dir was clearly genetically distinct from the rest. Furthermore, the Mantel test showed no correlation between the geographic and genetic distance (r = 0.14, p = 0.22), while barrier analysis suggested three statistically significant genetic barriers. Finally, the spatial interpolation results indicated that populations in Ziarat, Kashmir, Dir, Swat, Chitral, and upper Dir had high intrapopulation genetic diversity, suggesting the need to conserve populations in those areas. The results from this study will be important for future breeding improvement and conservation of walnuts in Pakistan.

Deep Insights Into the Plastome Evolution and Phylogenetic Relationships of the Tribe Urticeae (Family Urticaceae).

Urticeae s.l., a tribe of Urticaceae well-known for their stinging trichomes, consists of more than 10 genera and approximately 220 species. Relationships within this tribe remain poorly known due to the limited molecular and taxonomic sampling in previous studies, and chloroplast genome (CP genome/plastome) evolution is still largely unaddressed. To address these concerns, we used genome skimming data-CP genome and nuclear ribosomal DNA (18S-ITS1-5.8S-ITS2-26S); 106 accessions-for the very first time to attempt resolving the recalcitrant relationships and to explore chloroplast structural evolution across the group. Furthermore, we assembled a taxon rich two-locus dataset of trnL-F spacer and ITS sequences across 291 accessions to complement our genome skimming dataset. We found that Urticeae plastomes exhibit the tetrad structure typical of angiosperms, with sizes ranging from 145 to 161 kb and encoding a set of 110-112 unique genes. The studied plastomes have also undergone several structural variations, including inverted repeat (IR) expansions and contractions, inversion of the trnN-GUU gene, losses of the rps19 gene, and the rpl2 intron, and the proliferation of multiple repeat types; 11 hypervariable regions were also identified. Our phylogenomic analyses largely resolved major relationships across tribe Urticeae, supporting the monophyly of the tribe and most of its genera except for Laportea, Urera, and Urtica, which were recovered as polyphyletic with strong support. Our analyses also resolved with strong support several previously contentious branches: (1) Girardinia as a sister to the Dendrocnide-Discocnide-Laportea-Nanocnide-Zhengyia-Urtica-Hesperocnide clade and (2) Poikilospermum as sister to the recently transcribed Urera sensu stricto. Analyses of the taxon-rich, two-locus dataset showed lower support but was largely congruent with results from the CP genome and nuclear ribosomal DNA dataset. Collectively, our study highlights the power of genome skimming data to ameliorate phylogenetic resolution and provides new insights into phylogenetic relationships and chloroplast structural evolution in Urticeae.

Determinants of Genetic Structure in a Highly Heterogeneous Landscape in Southwest China.

Intra-specific genetic diversity is a fundamental component of biodiversity, and is key to species adaptation and persistence. However, significant knowledge gaps still exist in our understanding of the patterns of genetic diversity and their key determinants. Most previous investigations mainly utilized single-species and/or a limited number of explanatory variables; so here we mapped the patterns of plastid genetic diversity within 15 plant species, and explored the key determinants shaping these patterns using a wide range of variables. Population-level cpDNA sequence data for 15 plant species from the Longitudinal Range Gorge Region (LRGR), southwest China, were retrieved from literature and used to estimate haplotype diversity (H D) and population pairwise genetic differentiation (F ST) indices. Genetic diversity and divergence landscape surfaces were then generated based on the H D and F ST, respectively, to clarify the patterns of genetic structure in the region. Subsequently, we analyzed the relationships between plastid genetic diversity and 16 explanatory variables (classified as anthropogenic, climatic, and topographic). We found that the highest genetic diversity occurred in the Yulong Mountain region, with a significant proportion (~74.81%) of the high diversity land area being located outside of protected areas. The highest genetic divergence was observed approximately along the 25°N latitudinal line, with notable peaks in the western and eastern edges of the LRGR. Genetic diversity (H D) was weakly but significantly positively correlated with both Latitude (lat) and Annual Mean Wet Day Frequency (wet), yet significantly negatively correlated with all of Longitude (long), Annual Mean Cloud Cover Percent (cld), Annual Mean Anthropogenic Flux (ahf), and Human Footprint Index (hfp). A combination of climatic, topographic, and anthropogenic factors explained a significant proportion (78%) of genetic variation, with topographic factors (lat and long) being the best predictors. Our analysis identified areas of high genetic diversity (genetic diversity "hotspots") and divergence in the region, and these should be prioritized for conservation. This study contributes to a better understanding of the features that shape the distribution of plastid genetic diversity in the LRGR and thus would inform conservation management efforts in this species-rich, but vulnerable region.

The complete chloroplast genome sequences of an endemic species of Urticaceae (Debregeasia hekouensis).

Debregeasia hekouensis, which belongs to the nettle family (Urticaceae), is a local endemic species in Hekou County, Yunnan Province, China. To provide a basis for the development of effective molecular markers for its conservation, we sequenced the chloroplast (cp) genome of D. hekouensis in the present study. The total length of the chloroplast(cp) genome was 155,941 bp, and exhibited a typical quadripartite structure, with a pair of IRs (inverted repeats; 25,664 bp in length) being separated by a small single copy (SSC) region of 19,085 bp and a large single copy (LSC) region of 85,528 bp. The cp genome contained a total of 112 genes, including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The GC content of the entire cp genome, LSC region, SSC region, and IR region was 36.3%, 34.0%, 29.4%, and 42.7%, respectively. Phylogenetic analysis indicated that D. hekouensis is evolutionarily closer to Debregeasia orientalis and Debregeasia squamata.

Spatiotemporal maintenance of flora in the Himalaya biodiversity hotspot: Current knowledge and future perspectives.

Mountain ecosystems support a significant one-third of all terrestrial biodiversity, but our understanding of the spatiotemporal maintenance of this high biodiversity remains poor, or at best controversial. The Himalaya hosts a complex mountain ecosystem with high topographic and climatic heterogeneity and harbors one of the world's richest floras. The high species endemism, together with increasing anthropogenic threats, has qualified the Himalaya as one of the most significant global biodiversity hotspots. The topographic and climatic complexity of the Himalaya makes it an ideal natural laboratory for studying the mechanisms of floral exchange, diversification, and spatiotemporal distributions. Here, we review literature pertaining to the Himalaya in order to generate a concise synthesis of the origin, distribution, and climate change responses of the Himalayan flora. We found that the Himalaya supports a rich biodiversity and that the Hengduan Mountains supplied the majority of the Himalayan floral elements, which subsequently diversified from the late Miocene onward, to create today's relatively high endemicity in the Himalaya. Further, we uncover links between this Miocene diversification and the joint effect of geological and climatic upheavals in the Himalaya. There is marked variance regarding species dispersal, elevational gradients, and impact of climate change among plant species in the Himalaya, and our review highlights some of the general trends and recent advances on these aspects. Finally, we provide some recommendations for conservation planning and future research. Our work could be useful in guiding future research in this important ecosystem and will also provide new insights into the maintenance mechanisms underpinning other mountain systems.

From the Wild to the Cup: Tracking Footprints of the Tea Species in Time and Space.

Tea is one of the world's most popular beverages, known for its cultural significance and numerous health benefits. A clear understanding of the origin and history of domestication of the tea species is a fundamental pre-requisite for effective germplasm conservation and improvement. Though there is a general consensus about the center of origin of the tea plant, the evolutionary origin and expansion history of the species remain shrouded in controversy, with studies often reporting conflicting findings. This mini review provides a concise summary of the current state of knowledge regarding the origin, domestication, and dissemination of the species around the world. We note that tea was domesticated around 3000 B.C. either from non-tea wild relatives (probably Camellia grandibracteata and/or C. leptophylla) or intra-specifically from the wild Camellia sinensis var. assamica trees, and that the genetic origins of the various tea varieties may need further inquiry. Moreover, we found that lineage divergence within the tea family was apparently largely driven by a combination of orogenic, climatic, and human-related forces, a fact that could have important implications for conservation of the contemporary tea germplasm. Finally, we demonstrate the robustness of an integrative approach involving linguistics, historical records, and genetics to identify the center of origin of the tea species, and to infer its history of expansion. Throughout the review, we identify areas of debate, and highlight potential research gaps, which lay a foundation for future explorations of the topic.

Integrating a comprehensive DNA barcode reference library with a global map of yews (Taxus L.) for forensic identification.

Rapid and accurate identification of endangered species is a critical component of biosurveillance and conservation management, and potentially policing illegal trades. However, this is often not possible using traditional taxonomy, especially where only small or preprocessed parts of plants are available. Reliable identification can be achieved via a comprehensive DNA barcode reference library, accompanied by precise distribution data. However, these require extensive sampling at spatial and taxonomic scales, which has rarely been achieved for cosmopolitan taxa. Here, we construct a comprehensive DNA barcode reference library and generate distribution maps using species distribution modelling (SDM), for all 15 Taxus species worldwide. We find that trnL-trnF is the ideal barcode for Taxus: It can distinguish all Taxus species and in combination with ITS identify hybrids. Among five analysis methods tested, NJ was the most effective. Among 4,151 individuals screened for trnL-trnF, 73 haplotypes were detected, all species-specific and some population private. Taxonomical, geographical and genetic dimensions of sampling strategy were all found to affect the comprehensiveness of the resulting DNA barcode library. Maps from SDM showed that most species had allopatric distributions, except T. mairei in the Sino-Himalayan region. Using the barcode library and distribution map data, two unknown forensic samples were identified to species (and in one case, population) level and another was determined as a putative interspecific hybrid. This integrated species identification system for Taxus can be used for biosurveillance, conservation management and to monitor and prosecute illegal trade. Similar identification systems are recommended for other IUCN- and CITES-listed taxa.

Domestication Origin and Breeding History of the Tea Plant (Camellia sinensis) in China and India Based on Nuclear Microsatellites and cpDNA Sequence Data.

Although China and India are the two largest tea-producing countries, the domestication origin and breeding history of the tea plant in these two countries remain unclear. Our previous study suggested that the tea plant includes three distinct lineages (China type tea, Chinese Assam type tea and Indian Assam type tea), which were independently domesticated in China and India, respectively. To determine the origin and historical timeline of tea domestication in these two countries we used a combination of 23 nSSRs (402 samples) and three cpDNA regions (101 samples) to genotype domesticated tea plants and its wild relative. Based on a combination of demographic modeling, NewHybrids and Neighbour joining tree analyses, three independent domestication centers were found. In addition, two origins of Chinese Assam type tea were detected: Southern and Western Yunnan of China. Results from demographic modeling suggested that China type tea and Assam type tea first diverged 22,000 year ago during the last glacial maximum and subsequently split into the Chinese Assam type tea and Indian Assam type tea lineages 2770 year ago, corresponding well with the early record of tea usage in Yunnan, China. Furthermore, we found that the three tea types underwent different breeding histories where hybridization appears to have been the most important approach for tea cultivar breeding and improvements: a high proportion of the hybrid lineages were found to be F2 and BCs. Collectively, our results underscore the necessity for the conservation of Chinese Assam type tea germplasm and landraces as a valuable resource for future tea breeding.

Insights into the Genetic Relationships and Breeding Patterns of the African Tea Germplasm Based on nSSR Markers and cpDNA Sequences.

Africa is one of the key centers of global tea production. Understanding the genetic diversity and relationships of cultivars of African tea is important for future targeted breeding efforts for new crop cultivars, specialty tea processing, and to guide germplasm conservation efforts. Despite the economic importance of tea in Africa, no research work has been done so far on its genetic diversity at a continental scale. Twenty-three nSSRs and three plastid DNA regions were used to investigate the genetic diversity, relationships, and breeding patterns of tea accessions collected from eight countries of Africa. A total of 280 African tea accessions generated 297 alleles with a mean of 12.91 alleles per locus and a genetic diversity (H S) estimate of 0.652. A STRUCTURE analysis suggested two main genetic groups of African tea accessions which corresponded well with the two tea types Camellia sinensis var. sinensis and C. sinensis var. assamica, respectively, as well as an admixed "mosaic" group whose individuals were defined as hybrids of F2 and BC generation with a high proportion of C. sinensis var. assamica being maternal parents. Accessions known to be C. sinensis var. assamica further separated into two groups representing the two major tea breeding centers corresponding to southern Africa (Tea Research Foundation of Central Africa, TRFCA), and East Africa (Tea Research Foundation of Kenya, TRFK). Tea accessions were shared among countries. African tea has relatively lower genetic diversity. C. sinensis var. assamica is the main tea type under cultivation and contributes more in tea breeding improvements in Africa. International germplasm exchange and movement among countries within Africa was confirmed. The clustering into two main breeding centers, TRFCA, and TRFK, suggested that some traits of C. sinensis var. assamica and their associated genes possibly underwent selection during geographic differentiation or local breeding preferences. This study represents the first step toward effective utilization of differently inherited molecular markers for exploring the breeding status of African tea. The findings here will be important for planning the exploration, utilization, and conservation of tea germplasm for future breeding efforts in Africa.