Title of dissertation Elucidation of Genetic Relationships of Citrus and its Relatives including Bhutanese accessions based on sequence of Nuclear and Chloroplast Genomes RONPAKU Fellow Name Tshering Penjor Position Dupty Chief Research Officer ID No. R11401 Department Department of Plant Protection Institution Renewable Natural Resource Research Nationality Bhutan Development Centre, Wengkar, Mongar Japanese Advisor Name Masashi YAMAMOTO Position Professor Institution Kagoshima University The citrus fruits and their relatives are economically important and belong to subfamily Aurantioideae under family Rutaceae. Among many classification systems proposed under this subfamily, Swingle and Reece (1967) and Tanaka (1977) classification systems were widely accepted ones although there is huge discrepancy and controversy between these two systems in regards to the treatment of genus Citrus. In addition, area for the origin of many Citrus species is considered to be from northeastern India to southwestern China (Gmitter and Hu 1990, Tanaka 1959). Since Bhutan belongs to this region, there may be rich citrus genetic resources in Bhutan which were not properly studied or documented before. Therefore, the current studies were carried out with the following objectives:1) to elucidate the general phylogenetic relationship within subfamily Aurantioideae, using an extensive number of accessions (more than 100 accessions from 22 genera of the Aurantioideae) by Sanger sequencing of chloroplast rbcL and matK; 2) Intraspecies analysis of certain minor accessions using more robust methods such as RAD-Seq/ddRAD-Seq by the high-throughput sequencer; 3) to conduct survey and characterize the Bhutanese citrus accessions morphologically and genetically. Some of the significant outputs from each study were briefly described below: 1. Phylogenetic relationships of Citrus and its relatives based on rbcL gene sequences variations In this study we sequenced the rbcL genes of 64 accessions from 24 genera of Citrus relatives and analyzed them by neighbor-joining and maximum parsimony methods. The study supported Swingle and Reece’s (1967) treatment of the subfamily Aurantioideae as monophyletic. However, it did not support Swingle and Reece’s treatment of tribes and subtribes. The subgenera Citrus and Papeda were not clustered clearly. The analysis associated the Fortunella group with mandarin, Poncirus with Citrus ichangensis, Severinia buxifolia with Atalantia ceylanica, Microcitrus with Eremocitrus and Citrus micrantha, and Hesperethusa crenulata with Citropsis. 2. Phylogenetic relationships of Citrus and its relatives based on matK gene sequences In this study, we analyzed the chloroplast matK genes of 135 accessions from 22 genera of Aurantioideae. The subfamily Aurantioideae has been classified into 2 tribes, Clauseneae and Citreae. The analysis showed that Feroniella oblata is not nested in Citrus species and is closely related with Feronia limonia. Murraya paniculata was found to be similar to Merrillia caloxylon but dissimilar to Murraya koenigii at matK level. The group ‘‘true citrus fruit trees’’ was divided into 2 subclusters. One subcluster included Citrus, Fortunella, and Poncirus, while the other cluster included Microcitrus and Eremocitrus. The members (93 accessions) in the genus Citrus were classified into 3 clusters: citron, pummelo and mandarin clusters. The most mandarin accessions grouped to the mandarin cluster, with few exceptions. The study also revealed genetic information of various species of acid citrus grown in Japan. 3. Exploration of Local Citrus Genetic Resources in Bhutan and their Chloroplast DNA Analysis We characterized and discussed the various Bhutanese indigenous citrus accessions collected during several explorations in Bhutan. Among 14 accessions investigated, all accessions belonged to genus Citrus except one Bael (Aegle marmelos). Ichang papeda relative was found at elevations of around 2,000 m where no other citrus can grow at that altitude due to freezing temperature in winter. Analysis of matK sequences revealed that chloroplast DNA of Bhutanese Ichang papeda relative and a preserved tree of Ichang papeda in Faculty of Agriculture, Saga University was different. However, matK sequence of Bhutanese Ichang papeda was identical with that of a preserved tree of Khasi papeda in Saga University. Diversity also found in matK sequence on group of lime relatives locally known as "Kagati". 4. Characterization of lime (Citrus aurantifolia (Cristm.) Swingle) grown in Bhutan and Indonesia using next generation sequencing In the current study, we characterized limes grown in Bhutan and Indonesia both morphologically and genetically using Restriction site-associated DNA (RAD-seq) employing high output sequencing method. The limes were separated into two groups based on their morphology. The RAD-seq analysis also separated the eight accessions into two clusters. One cluster contained four accessions from Bhutan, whereas the other cluster contained one accession from Bhutan and the three accessions from Indonesia. The genetic classification supported the morphological classification of limes. The study further suggests that the properties associated with asexual reproduction, and somatic homologous recombination, must have contributed to the genetic diversification of limes. 5. RAD-Seq analysis of typical and minor Citrus accessions, including Bhutanese varieties In this study we analyzed the reduced-representation genome sequences of Citrus species by double-digest restriction site-associated DNA sequencing (ddRAD-Seq) using 44 accessions containing typical and minor accessions, including Bhutanese varieties. The results supported the recent hypothesis that citron, papeda, pummelo, and mandarin are the ancestral species, and that most Citrus species are derivatives or hybrids of these four species. The study also showed that ddRAD-Seq analysis could readily detect conservation of heterozygosity, which was useful to discriminate citrus varieties from closely-related species. One Citrus accession from Bhutan was morphologically similar to Mexican lime was now designated a new name as Himalayan lime. The analysis confirmed that Himalayan lime is a probable hybrid between mandarin and citron. In addition to Himalayan lime, the study also suggested that several accessions were formed by previously undescribed combinations. In conclusion the current studies provided new insights and information to solve some of the important controversies involved in the evolution of the tribes, sub-tribes under subfamily Aurantioideae and also in interspecific relationships within the groups. Identification of Himalayan lime from Bhutan was one of key finding from these studies. The genetic background information of various citrus accessions generated from these studies would be also useful for citrus breeders and nursery growers. .