Molecular Phylogeny Of The True Citrus Fruit Trees Group (Aurantioideae, Rutaceae) And The Origin Of Cultivated Citrus

Citrus is one of the most important fruit crops in the world. It is widely grown in the tropical and subtropical areas of the world. The genus Citrus established by Carl Linneaus in 1753 belongs to the subfamily Aurantioideae of the family Rutaceae. Since the establishment of the genus, Citrus has attracted a lot of research interest for more than 250 years. However, until now, the phylogenetic relationships between Citrus and its closely related genera have remained unresolved, and the number of "basic species" types in Citrus is still in controversy, and the phylogenetic relationship between the key wild species of Citrus still needs to be clarified. Mainly due to sexual compatibility between Citrus and related genera, high frequency of bud mutations, long history of cultivation, the questions concerning the taxonomy and evolution of the genus Citrus, and the genetic relationship between the cultivated species and their putative wild progenitors have remained unresolved.In the present study, the amplified fragment length polymorphisms (AFLP) fingerprints, nuclear internal transcribed spacer (ITS), and three chloroplast DNA regions (psbH-petB, trnL-trnF and trnS-trnG) of the genus Citrus and its five closely related genera were analyzed in attempt to reconstruct the molecular phylogenetic relationship in the true citrus fruit trees group, establish the phylogenetic framework of the genus Citrus and identify the origin of the cultivated citrus. The main results obtained are as follows:1. Using Murraya paniculata, Atalantia buxifolia, and Severinia buxifolia as outgroups, AFLP fingerprints, ITS sequence, and three chloroplast DNA regions of 59 accessions of the genus Citrus and its five closely related genera, and 25 accessions of the key "non-hybrid" wild species were analyzed respectively in an attempt to reconstruct the molecular phylogenetic relationship of the true citrus fruit trees group (Citrinae, Citreae, Aurantioideae, Rutaceae). Our results showed that the true citrus fruit trees group was monophyletic, and Poncirus was possibly the first genus separated from the other five genera in the group.The genera Microcitrus, Eremocitrus and Clymenia, may be of the same origin, and the genus Microcitrus was more closely related to Eremocitrus, Fortunella might have a close relationship with Citrus. In addition, our cpDNA data indicated that Microcitrus, Eremocitrus and Clymenia together formed a sister relationship to C. medica. Our AFLP dendrogram showed that Microcitrus, Eremocitrus and Clymenia were distant from the genus Citrus. Our ITS data demonstrated that Fortunella had more close relationship with C. reticulata. Our cpDNA and ITS data suggested that some species of M. australasica might have been hybrid origin and papeda might be one of its parents.2. Using Poncirus as outgroup, AFLP fingerprints, ITS sequence, and three chloroplast DNA regions of 47 accessions of genus Citrus, and 17 accessions of "non-hybrid" wild species of the genus Citrus, and 15 accessions of "non-hybrid" wild species of the genus Citrus were analyzed respectively in an attempt to establish the phylogenetic framework of the genus Citrus. All of our molecular data supported that the genus Citrus was monophyletic and the four basic biotypes of Citrus, i.e., Papeda, pummelo, citron and tangerine were of monophyletic origin. Our cpDNA data showed that C. grandis had close relationship with the Papeda species, C. medica, Ichang papeda and C. reticulata were more closely related to each other than they were to other types in the genus. Our AFLP dendrogram showed that Ichang papeda, C. reticulata and C. grandis were more closely related to each other than they were to other types in the genus, and papeda was distant from C. reticulata. Our ITS sequence and combined data analysis demonstrated that C. grandis was most closely related with the Papeda species, C. medica was more closely related to C. grandis and Papeda species than to C. reticulata, and that C. reticulata most likely have a sister relationship to the common ancestor of C. grandis, C. medica, and Papeda species.3. Using Poncirus as outgroup, AFLP fingerprints, ITS sequence, and three chloroplast DNA regions of 27 accessions of the cultivated citrus and their putative wild ancestors were analyzed in an attempt to identify both their paternal and maternal origins. Our cpDNA tree clearly indicated that pummelo (C. grndis) was female parent of sweet orange, sour orange and grapefruit; Papeda was the female parent of mexican lime (C. aurantifolia); The female parent of rough lemon (C. limon), rangpur lime (C. limonia) and guangxi local lemon (C. limonia) was C. reticulata. The chloroplast genome of bergamot (C. aurantifolia) and lemon was derived from sour orange. Our AFLP tree suggested that C. reticulata was probable a parent of, sweet orange and sour orange; Sweet orange was probable a parent of grapefruit; Rough lemon was most likely a parent of rangpur lime and guangxi local lemon; C. medica could be a parent of mexican lime, bergamot and lemon. Our ITS data demonstrated that sweet orange and sour orange were hybrids of mandarin (C. reticulata) and pummelo (C. grandis); Mexican lime was a hybrid of Papeda and C. medica; Sweet orange was a parent of grapefruit; C. medica could be a parent of C. limon'Femminello', bergamot, rough lemon, rangpur lime and guangxi local lemon; Mandarin could take part in the origin of rough lemon, bergamot, lemon and lime.