Phylogeny Of Tribe Anthemideae (Asteraceae) From East Asia And Intergeneric Cross Between Dendranthema×grandiflorum (Ramat.) Kitam. And Ajania Pacifica (Nakai) K.Bremer & Humphries

Anthemideae Cass. (Asteraceae, subfamily Asteroideae) taxa from east Asia werecollected widely. The phylogeny of this tribe from east Asia was illustrated based on thepollen morphology, ITS sequences, chloroplast sequences and AFLP marker. Closerelationship between Dendranthema and Ajania was observed, therefore, in order toinnovate the chrysanthemum germplasm, intergeneric cross between D.×grandiflorum(Ramat.) Kitam. and A.pacifica (Nakai) K.Bremer & Humphries was attemptedSubsequently, their integeneric hybrid was selfed or backcrossed with the cultivar which isone of the parents. The morphological genetics and the chromosome behavior in F₁ F₂ andBC₁ progenies were investigated. Novel and charming morphological characteristics werealso observed in the descendents of the two genera. The promising role of A. pacifica in theenriching the chrysanthemum germplasm was also discussed.Partâ… : The phylogeny of Anthemideae from east AsiaSixty eight taxa including 17 genera (54 species or variety) were collected from 16provinces or states of our country, Japan and Korea. The chromosome numbers of sometaxa were detected. The result showed that they were mostly diploid or tetraploid. However,many monotypic genera, confined to east Asia such as Opisthopappus, Crossostephium etc.,were diploid, it can be inferred that the diploidy was the plesiomorph characteristics of thistribe. The ploidy diversity in Ajania was observed not only in different species but also indifferent populations in the same specie. The chromosome number of the following taxa,Ajania fastigiata (2n=36), A.variifolia (2n=18), A.potaninii (2n=18), Hippolytiaalashanensis (2n=18), Opisthopappus taihangensis (2n=18), Achillea acuminata (2n=18)and A.wilsoniana (2n=36) was reported for the first time, and the chromosome number ofAjania khartensis (2n=36) was the new count.Pollen morphology of 15 genera (35 species) were investigated using scanning electron microscope (SEM). According to the our SEM results in this research and the previousdiscription of pollen morphology, the taxa of Anthemideae from east Asia can be devidedinto three groups: the first group including Chrysanthemum and Argyranthemum of subtribeChrysantheminae, Hippolytia, Opisthopappus, Pyrethrum and Tanacetum of subtribeTanacetinae, Leucanthemum and Nipponanthemum of subtribe Leucantheminae, Matricariaof subtribe Matricariinae, Achillea of subtribe Achilleinae, Dendranthema, Arctanthemum,Ajania and Brachanthemum of subtribe Artemisiinae,whose pollen were with obviousspines in largest size (＞1.5μm) and foveolae or perforations scatterred on the base of spine;the second group including Artemisia, Seriphidium, Elachanthemum, Crossostephium,Neopallasia, Ajaniopsis, Filifolium and Kaschgaria of subtribe Artemisiinae, whose pollenwere with tiny spinules (＜0.5μm); the third group including two species of Ajania (Asalicifolia and A.variifolia) and Stilpnolepis, whose pollen were with short and obviousspines in middle size (1.0-1.5μm) as compared to those of the first and second groups.According to the morphology of pollen exine, A salicifolia and A.variifolia should betransferred from Ajania to Phaeostigma. In addition, A.latifolia Shih and A.ramosa (Chang)Shih should be a member of Phaeostigma too.Sequences of the nrDNA internal transcribed spacer (ITS) region and the cpDNAtrnL/trnF intergenic spacer (IGS) region were analysed for 67 representatives ofAnthemideae from east Asia. The constructed phylogenic tree was comprised of two majorclades (A and B). The clade A including all taxa of subtribe Chrysantheminae which werestrongly supported monophyletic group, the majority taxa of subtribe Leucantheminae,Tanacetinae and Achilleinae was located at the base of the tree,. All taxa of subtribeArtemisiinae, Hippolytia and Opisthopappus of subtribe Tanacetinae and Nipponanthemumof subtribe Leucantheminae formed the clade B. Subtribe Artemisiinae was also stronglysupported monophyletic group if excluding the doubting genera (e.g. Hippolytia,Opisthopappus and Nipponanthemum). The clade B mainly included two subclades, onesubclade included Dendranthema, Arctanthemum, Ajania, Opisthopappus andElachanthemum was well supported, the remaining taxa including Artemisia,Crossostephium and Neopallasia). Stilpnolepis, Phaeostigma, Kaschgaria, Filifolium andBrachanthemum located as the base branches within the clade B, it can be inferred thesetaxa were likely the ancient forms of subtribe Artemisiinae. Opisthopappus was closelyrelated to the Dendranthema group but far from the taxa of subtribe Tanacetinae.AFLP (amplified fragment length polymorphism) was used to assess the phylogeny of subtribe Artemisiinae and its allied taxa including 51 representatives. A total 1705polyporphism bands using 9 primer pairs were obtained. UPGMA cluster analysis showedBrachanthemum was located at the base of the Dendranthema group and the Neopallasiaand Elachanthemum were located at the base of all taxa. The taxa in Dendranthema andAjania were both divided into two sub-groups, respectively. These four sub-groupsintercrossed each other and formed two small clades, moreover the specie-groups of Ajaniawere always located at the base of the small clades. It showed Dendranthema maybederived from the different species of Ajania at the same time in the different regions.Opisthopappus was closely related to the Dendranthema and Ajania clade, it is morereasonable to treat it as a member of subtribe Artemisiinae based on the ITS-IGS sequenceand AFLP polymorphism.Taken together, the results of cytogenetics, pollen morphology, sequence analysis andAFLP polymorphism suggested that the Anthemideae from east Asia originated in Eurasia.The Eurasian ancestors dispersed gradually from Eurasia to east Asia through middle Asia.So the Central Asian taxa such as Kaschgaria etc. should be the initial form or relic speciesof Anthemideae from east Asia. So subtribe Artemisiinae should be divided into threegroups: the Dendranthema group, the Artemisia group and the ancestrol group. TheDendranthema group that included Dendranthema, Ajania, Arctanthemum,Brachanthemum and Opisthopappus and the Artemisia group that included Artemisia,Crossostephium and Neopallasia of subtfibe Artemisiinae were originated separately fromthe ancestral group. Kaschgaria was the member of ancestral group. And Phaeostigma,Filifolium,Stilpnolepis and Elachanthemum should be the members of this group, while itshould be verified by the further research. Dendranthema was closely related to Ajania andit maybe originated from the different species of Ajania at the same time in the differentregion. The position of Hippolytia was unresolved. It should be the close ancestor of theDendranthema group, but more evidience should be provided by further investigation.Partâ…¡: Intergeneric cross between Dendranthema×grandiflorum andAjania pacificaThe intergeneric crosses were made between D.×grandiflorum 'Aoyunhuoju' or'Yidalihong' and A.pacifica. The F₁ progenies were identified as an intergeneric hybridaccording to the morphologies, cytogenetical characteristics and random amplification of polymorphic DNA (RAPD) molecular marker. Specific characteristics of A.pacifica, suchas leaf color, was successfully integrated into the chrysanthemum cultivar, as well as someless appealing morphological characteristics. Characteristics of the intergeneric hybrid,including leaf color, shape of leaf, color of flower, branching and blooming date, wereintermediate between parents. However, the size, number, and shape of ray florets variedfrom individual to individual. Additionally, the chromosome number of the intergenerichybrid was normal with an expected number of 2n=72; meiosis behavior was overallnormal too. The intergeneric hybrid of D.×grandiflorum 'Aoyunhuoju' and A.pacifica andits normal chromosome behavior, as a result of the relatively high cross compatibility,suggested a close relationship between the parents. We supposed that a close relationshipbetween Dendrathema and Ajania.Morphological characteristics such as plant height, canopy and the number of rayflorets and tube florets in F₂ progenies were degressively related to F₁. It showed that therewere inbred depression in F₂ progenies. The heredity of some characteristics such asphylliform, the length and color of ray petal and the bloming date etc. were different amongF₂ progenies, e.g. the ray florets of 21F5 and 21F6 were longer than those of their parentYZ21. The chromosome number of F₂ progenies varied from 51 to 90, mostly from 70 to72 with an exception of 21 F6 with a mainly number of 63.The chromosome number of BC₁ progenies between F₁ progenies and 'Aoyunhuoju' or'Yidalihong' varied from 52 to 63, mostly varied from 60 to 63. Morphologicalcharacteristics such as plant height, canopy, phylliform, epidermal hairs of the leaf,branching and ramification in progenies of backcross were overall heredity universal,which exhibited intermediate characteristics between parents, but more resembled those of'Aoyunhuoju' or 'Yidalihong' than those of F₁ progeny to some extend. It implied that thesecharacteristics were quantitatively heredity traits. However, individuals of progenies ofbackcross differed in the morphology of ray florets. The anterior extremity of ray florets inprogenies of backcross between F₁ progeny ('Yidalihong' and A. pacifica) and'Yidalihong'was not cloven and rich in flower color. However, progenies of backcross between F₁progeny (A. pacifica and 'Yidalihong') and'Yidalihong' showed 1 to 4 lobe-ray-florets(mainly 2 or 3 lobes) and were poor in fower color. It suggested that phenotype of rayflorets in progenies of backcross was likely regulated by cytoplasmic genes. Intergenerichybridization was successfully attempted between A. pacifica and commercial cultivars.When the F₁ progeny of intergeneric hybridization was subsequently backcrossed with commercial cultivar, novel germplasm that both leaves and flowers are of ornamentalvalues was created. Take together, the characteristics genetics and the chromosomebehavior of F₁, F₂ and BC₁ suggested the characteristics of epidermal hair of the leaf andthe ray florets were quantitative traits controlled by the multigene in special materials andhad additive effects.