Studies Of Phylogeography And Domestication Origin Of Chinese Bayberry(Morella Rubra)

Chinese bayberry(Morella rubra Lour., Myricaceae) is an economically important subtropical evergreen fruit crop native to China and other adjacent countries in East Asia. There are four Morella species (M. rubra, M. esculenta, M. adenophora and M. nand) distributed in China, among which, M. rubra is the only one to be domesticated as an economic fruit. Wild populations of M. rubra are distributed in eleven provinces in eastern China, southern China and southwest China, and are important germplasm resource for Chinese bayberry breeding. Due to its long cultivating history of more than 2000 years in China, Zhejiang, Jiangsu, Fujian and Guangdong have been the major planting areas. However, problems concerning M. rubra remain unsolved with respect to its phylogeny, polulation genetics, domestication origins and relationships among cultivars. Our study aims to elucidate the phylogenetic status of M. rubra based on Restriction-site Associated DNA sequencing (RAD-seq), one of the innovative Next-Generation Sequencing (NGS) techniques, followed by chloroplast genomes assembling and polymorphic screening of cpDNA fragments within the species. Combined with different genetic markers including cpDNA, nuclear SSR and RAD-seq, we studied its genetic diversity, phylogeography, domestication origin and relationships among different cultivars in M. rubra. Main results of the research are as follows:(1) Phylogeny, inter- and intra-specific divergence in M. rubraWe used restriction site-associated DNA sequencing (RAD-seq) to identify candidate loci that will help in determining phylogenetic relationships among M. rubra, M. adenophora, M. nana and M. esculenta. Comptonia peregrina was set as outgroup, and three methods for inferring phylogeny, maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI), were applied to data sets including as many as 4253 RAD loci with 8360 parsimony informative variable sites. All three methods significantly favored the topology of (((M. rubra, M. adenophora), M. nand), M. esculenta), and two species from North America (M. cerifera and M. pensylvanica) were placed as sister to the four Chinese species.According to BEAST analysis, we deduced speciation of M. rubra to be at about the Miocene-Pliocene boundary (5.28 Ma). Intra-specific divergence in M. rubra occurred in the late Pliocene (3.39 Ma), which was likely driven by the uplift of the QTP and the foration of the Hengduan Mountains. Additionally, we identified 3808 unlinked SNP sites across the four populations of M. rubra and discovered genes associated with fruit ripening and senescence, fruit quality and disease/defense metabolism based on KEGG database.(2) Sequencing and analysis of complete chloroplast genomes of M. rubraBased on the statistical parsimony networks estimated from psbA-trnH, three individuals of M. rubra, representing the haplotype H1, H3 and H4 respectively, were selected for complete chloroplast genome sequencing. The genome sequencing was performed on an Illumina Hiseq2000. We recovered more than 4.76G raw data from each sample with read length of 125 bp, and the Q20 of each sample was from 93.14% to 93.45%. After cleaning, quality filtering and de novo assembling, we obtained the chloroplast genome length were 159,440 bp,159,612 bp and 159,567 bp respectively for the three samples.The chloroplast genome of M. rubra was determined to be a circular DNA molecule with a quadripartite structure typical of the majority of angiosperm chloroplast genomes. It includes two inverted repeat regions (IRs) separated by a large single copy region (LSC) and a small single copy region (SSC), respectively. A total of 117 unique genes were identified in the M. rubra cpDNA genome. Seventy four of the 117 genes are polypeptide genes,32 are distinct tRNA genes, four are rRNA genes, four are conserved reading frames and three are pseudogenes. We detected 322 variant sites, including 272 single nucleotide polymorphisms (SNPs) and 49 insertion and deletion (indel), through aligning cpDNA genome of the three samples, nine polymorphic chloroplast fragments within species were developed for the study of population genetics in M. rubra.(3) Genetic diversity, structure and evolutionary history of wild M. rubraBased on nine SSR markers and four cpDNA markers selected from the above nine cpDNA fragments, we analyzed the genetic diversity and structure of the wild M. rubra populations. Results from nuclear SSR analyses revealed an intermediate level of the genetic diversity within M. rubra. Based on cpDNA sequence variation,53 variable sites and 17 haplotypes were detected in all the wild populations. These sites include 51 single-site mutations and two insertions. Compared with nucleotide SSR, the wild populations showed relatively lower genetic diversity (h=0.195, π=0.0000325) for cpDNA sequences. Linear regression analysis indicated a significant negative relationship between nucleotide diversity (π) and latitude. When latitude increases, the nucleotide diversity decreased (r=0.170, p=0.036). No other genetic diversity parameter was found to be significantly related with longitude and latitude.Results from AMOVA analyses showed that the genetic divergence of M. rubra was larger between populations (88.17%) than within populations (11.83%) on cpDNA level, but was smaller between populations (18.89%) than within populations (81.11%) on SSR level. The Nm values inferred from the genetic divergence index based on SSR (FST=0.189) and cpDNA (FST=0.882) were 1.07 and 0.07, respectively. The ratio of pollen flow and seed flow was 30, indicating pollen flow as the primary gene flow between populations of M. rubra.We firstly used RAD-seq to analyze the genetic relationship between wild populations of M. rubra, and 83,225 unlinked SNP sites were screen from all the samples. Combined results from cpDNA and nucleotide SSR showed that Southwest China is an ancestral distribution region of M. rubra in the mainland of China. Populations in Yunnan province displayed characters of ancestral populations. Mismatch distribution analysis (MDA) did not detect population expansion of wild populations. Analysis of isolation by distance (IBD) did not show significant relationship between the genetic pattern and geographical distribution of M. rubra populations. This is consistent with the anemophilous nature of M. rubra. However, phylogeography of cpDNA haplotype revealed that the polymorphism center of M. rubra populations is located in the subtropical area south of Nanling Mountains. The cpDNA haplotypes in central and east China north of Nanling Mountains are unique, which worth further study and discussion.(4) Genetic diversity, domestication origin and relationships among different cultivars in M. rubraWe analyzed the genetic diversity and structure of wild and cultivated M. rubra populations based on nine SSR markers. As indicated by the gene diversity (Hs) allelic richness (AR), expected heterozygosity (HE), observed heterozygosity (Ho) and specific allele richness (PR), the genetic diversity of M. rubra was higher in wild populations than cultivated populations. This suggests that M. rubra has undergone certain artificial selection in domestication process. Results from whole genome sequencing based on RAD-seq indicated obvious population divergence between wild and cultivated M. rubra populations. Therefore, domestication from nearby wild populations and long term vegetative reproduction might be the main reasons leading to this situation.Based on nucleotide SSR and RAD-seq markers, we analyzed the domestication origin of cultivated Chinese bayberry. Results from two genetic markers revealed multiple independent domestication events of M. rubra cultivated in China. The cultivated populations in Zhejiang Province have undergone two domestication events, cultivated populations in Fujian Province have undergone independent domestication event. Partial of the cultivars from Jiangsu Province was introduced from other areas, while the cultivar "Dongkui" from different planting areas was obtained from single domestication event. Combined with the principals of "nearby domestication", we proposed that the currently presented main cultivars of Chinese bayberry were domesticated from wild populations from Zhejiang, Jiangxi, and Fujian Provinces with multiple domestication events.Similarly, we analyzed the relationships between different cultivars of Chinese bayberry using nucleotide SSR and RAD-seq markers combined with fruit morphological characters. Since RAD-seq can provide more informative loci, results from RAD-seq are usually more clear and reliable than results from nucleotide SSR, ISSR, and RAPD. The genetic relationships of different cultivars were found to be highly related with the produced area of the corresponding cultivars, and most cultivars from adjacent produced area displayed similar germplasm basis. Combined with analyses of morphological character, our results showed that Dongkui is a relatively stable cultivar in terms of genetic and morphological characters. The cultivars named as ’Dayexidi’(SZDY),’Xiaoyexidi’(SZXY) from Suzhou,’Tongzi’ (TZTZ), ’Zaoda’(LHZD),’Fugong No.1’(FJFG),’Tezaomei’(FJTZ),’Zaose’ (HZZS),’Shenhong’(SYSH), and’Fenhong’ (YYFH) that have relatively close genetic characteristics also showed stable morphological features.Our study fully revealed the germplasm structure and genealogical relationships of the currently distributed wild and cultivated populations of M. rubra and laid a foundation of the sustainable utilization and breeding of Chinese bayberry.