Molecular Biological Study On Phylogeny Of Subgenus Amygdalus And Genetic Diversity Of Prunus Persica

Plants in subgenus Amygdalus are an important resource of stone fruits, of which P. persica and P. communis are major fruit species in the world for fruit production. The study aims to disclose phylogeny among subgenera in stone fruit plants and species in P. persica, taxonomic rank, and genetic diversity of P. persica by molecular biological technology and methods. It will provide molecular evidences for solving problems in taxonomy of subgenus Amygdalus, conservation and utility of germplasm in P. persica.The internal transcribed spacer (ITS) regions of nuclear ribosomal DNA from 8 species of subgenus Amygdalus were sequenced, and analyzed together with other ITS data (from GenBank) of 18 species representing subgenus Cerasus, subgenus Armeniaca and subgenus Primus by using of Padus racemosa (Lam.) Gilib. and Padus buergeriana (Miq.) Yüet Ku as outgroup for studying phylogeny of subgenera in stone fruit plants. 223 accessions from subgenus Amygdalus, subgenus Prunus, subgenus Armeniaca, subgenus Cerasus were studied by RAPD technology and clustered according to mentioned phylogenic contents. Polymorphic information contents (PIC) were calculated among species in subgenera and demes in P. persica. Meanwhile, genetic diversity, genetic structure, genetic differences of demes were analyzed with RAPD markers. The main results as follow:1. A database of germplasm in subgenus Amygdalus (including 20 relative species in other subgenera) was established, which included (1) origin sites, collection places, common names, scientific names and fruit characters of 223 accessions; (2) 40140 data obtained from 180 loci and 22 primers; (3) 9 gene sequences of rDNA ITS in subgenus Amygdalus.2. Phylogeny of subgenus Amygdalus in stone fruit plants was analyzed by RAPD markers, indicating that apricot and mume in subgenus Armeniaca clustered firstly, and the two together with subgenus Prunus, further the three with subgenus Amygdalus, finally the four with subgenus Cerasus. If the cluster was made a joint line at the level of 0.533, there were four groups as above motioned subgenera. The consensus tree by ITS gene sequences indicated that subgenus Cerasus was basal to the other subgenera with a bootstrap value of 68% and occupied an isolated position (cladeâ… ). Subgenus Prunus, subgenus Armeniaca and subgenus Amygdalus formed a monophyletic group (cladeâ…¡) with a bootstrap value of 100%, which implied they had close relationships with each other and probably a common origin. Cladeâ…¡was divided into two subclades: One was subgenus Amygdalus with bootstrap values of 100%, the other was subgunus Prunus and subgenus Armeniaca with bootstrap values of 54%, but species in subgenus Armeniaca with bootstrap values of 85%.3. Phylogeny of species in subgenus Amygdalus was analyzed through markers in 180 loci and made a cluster, which inferred 8 groups: Neimengguchangbingbiantao(P. pedunculata); Guanghetao(P. mira); Shantao(P davidiana), Ganshutao(P. kansuensis); Badan(P. communis); Xinjiangtao, Maoto (P. persica ) from joint line of 0.87. Treating gaps as missing, the parsimony analyses generated five equally shortest trees with a length of 80 steps, a consistency index (CI) of 0.925, a retention index (RI) of 0.941, a rescaled consistency index (RC) of 0.871. The topology of each tree was identical except for placement of Shantao (P. davidiana) and Shanganshantao(P. potanini). In every case, P. ledebouriana and P. tenella were basal to the other species in subgenus Amygdalus, followed by P. communis which occupied an isolated position. Above these species, a tetrachotomy separated four monophyletic groups, the first one consisting of P. mira and P. kansuensis, the second one consisting of P. persica and P. persica var.ferganensis, and the rest two including P. potanini and P. davidiana, respectively. A strict consensus tree depicted sister groups of (P. mira, P. kansuensis), (P. persica, P. persica var. ferganensis), and (P. ledebouriana, P. tenella).4. In allusion to dispute on taxonomic ranks about Xingjiangtao, Shantao, Shanganshantao, they were analyzed with ITS gene sequences and RAPD markers. A bootstrap value of 90% supported extreme relative relationship between Xingjiangtao and common peach. 203 species, varieties, cultivars and forms clustered with RAPD markers, inferring that all species except Xingjiangtao in subgenus Amygdalus displayed in outer of the dendrogram, while 5 accessions of Xingjiangtao did not cluster out of common peach and mixed with accessions in common peach. Systematic tree was constructed by ITS sequences, indicating that Clades of Shanganshantao and Shantao existed parallel, turning into own group at the base of the other species in subgenus Amygdalus. The dendrogram from RAPD markers supported that Shantao was in an independent group. 5. Core germplasm in each deme was put forward and listed as Liemage, Haluhong, Bailey, Akelawa in rootstock group; Hongchongban, Shouxintao(Red), etc. in shouxingtao group; Hongbitao, Honghuabitao, Renmiantao, etc. in bitao group; Zhubo 3, Zhubo 6, Luogehongye etc. in red leaf peach group; Dahongpao, Wuyuexian, Qinglingdongtao, Yixianhong, Yangquanroutao, Datiantao, etc. in crisp peach group; Qiumi, Taiyuanshuimi, Shenzhouhongmi, Wenzhoushuimi, etc. in honey peach group; Okayamahaku, Spring time, Rebin, Hakuho, Xiahui No2, Zhaoyian, Fenghuayulu, etc. in juicy peach group; Yulupantao, Wuyuexianbiangan, Sulianpantao, Jiaqing, Yangzhou 124, etc. in flat peach group; Shuguang, Ruiguang No.2, Mayfire, Aimila, Hongliguang, Armking, etc. in nectarine group; Fertinimoroteini, Xizhuang No.1, Nong 1-2-4, Gold queen, Red haven, Jinxiu, Chengxiang, Elberta, Jingcheng, etc. in yellow peach group. Among above mentioned germplasm, Yulupantao, Qiumi, Elberta, Fertinimoroteini appeared very special because of both RAPD markers and clustering place in dendrogram.6. Molecular checking indexes which identified the experimented accessions were edited according to special markers. Typical bands were used to analyze relation with ecotypes and characters of fruits, indicating that there was no relation between typical bands and ecotypes, but some extent relation between bands and characters of fruits existed. If all cultivars were divided into freestone and clingstone, melting and nonmelting, both freestone (ratio shared total survey, 41/56=73.2%) and clingstone (ratio shared total survey, 33/79=41.8%) appeared in the loci of S₁₆₇-1050 bp linked marker raised by Warburton et al. It was apparently not strong linkage between the marker and the character, and exchange rate of genes was high. If combinations of S₁₆₇-850bp and S₁₆₇-1050bp or S₁₆₇-850bp and S₁₆₇-1400bp were used to distinguish characters of fruits, 6 freestone accessions without clingstone ones and 6 melting accessions without nonmelting ones displayed in the first combination of markers, 20 clingstone accessions and 2 semi-freestone ones without freestone ones appeared in the second combination of markers.7. Shouxintao group, weeping peach group, red leaf peach group and bitao group, crisp peach group, mitao group, honey peach group, flat peach group, nectarine group, yellow peach group were studied from aspects of polymorphic markers, frequency of markers, variation degree of amplified loci, genetic diversity, genetic differentiation and genetic structure. There were 11.9% and 88.1% of genetic variation existing among and within demes, respectively. Genetic diversity of the demes was expressed as follow: yellow peach group＞honey peach group＞flat peach group＞red leaf peach group＞crisp peach group＞bitao group and juicy peach group＞nectarine group＞shouxingtao group＞weeping peach group. Demes clustered to be divided into five sections at joint line of 0.9586, the first one was cultivated cultivars including crisp peach, honey peach, juicy peach, flat peach, nectarine, yellow peach, of which crisp peach and juicy peach had 0.9806 of highest similarity; the others were shouxingtao, bitao, red leaf peach, weeping peach, of which red leaf peach and weeping peach had 0.9160 of lowest similarity.According to above results and former research achievements of morphology, cytology, pollen, isozyme, plants of stone fruits should be classified as subgenus Amygdalus, subgenus Prunus, subgenus Armeniaca, subgenus Cerasus; Xingjiangtao is a variant and suggested scientific name as Prunus persica var. ferganensis; Shantao as Prunus davidiana. Although shanganshantao clustered as independent group in the experiment, it needs studying further about taxonomical rank. There were special germplasm by means of genetic diversity. Different demes had various genetic variations, which of yellow peach was highest. Genetic variation in demes appeared 8 times higher than among demes. The paper also gave a few methods such as dendrogram, statistic, frequency of markers, polymorphic bands and special loci, genetic variations within and among demes for conservation and utility, molecular checking indexes for cultivars can provide basic information for protection of intellectual property rights and selection of wanted cultivars.