| Phaius Lour.is a small genus in Orchidaceae,about 40 species distributed in tropical Africa,Madagascar,tropical and subtropical Asia to Oceania.There are 10 species of Phaius in China,mainly distributed in the south of the Yangtze River.Phaius is a genus of large,terrestrial orchid having showy flowers with high ornamental value and medicinal value.Recently,only a few studies on hybridization breeding,propagation technology and embryo development,however the conservation of germplasm resources technology and molecular biology are very scarce.Due to habitat loss and over exploitation,most of Phaius species have been endangered in China.It is urgent to protect the wild plant resources of Phaius,strengthen the collection and conservation of germplasm resources,and carry out basic research on conservation biology.In this study,we selected the widely distributed and common species of Phaius tancarvilleae to study the pollen morphology and preservation of Phaius;meanwhile,we also studied the chloroplast comparative genome of nine Phaius species in China.This study provides a scientific basis for the protection and sustainable utilization of the important ornamental flower resources in China.The main content and results of this study are as follows:(1)The morphology of pollinia of P.tancarvilleaeThe macro-and micro-characters of pollinia of P.tancarvilleae were investigated by stereomicroscope,light microscope and scanning electron microscope.P.tancarvilleae has eight kidneyshaped pollinia,attached by caudicles to a sticky substance.The exine of pollinia are smooth under scanning electron microscope.The pollen tetrads are 2-celled.(2)In vitro germination of pollen of P.tancarvilleaeFresh pollens of P.tancarvilleae were cultured in the in vitro liquid medium contained sucrose,boric acid,calcium chloride and PEG-4000.Pollen germination percentage and tube growth were studied by light microscope.The effects of chemical of medium on pollen germination of P.tancarvilleae were studied.The results of the single factor tests showed that the chemical of medium sucrose,boric acid,calcium chloride and PEG within a certain concentration range were conductive to the pollen germination and pollen tube growth.Sucrose and CaCl2 significantly influence the pollen germination and pollen tube growth of P.tancarvilleae.Pollen germination percentage and tube growth rate showed significant highly with the concentration of 100 mg-L-1 of sucrose,20 mg-L-1 H3 BO3,10 mg-L-1 CaCl2 and 100 mg-L-1 PEG4000.The appropriate amount of sucrose,boric acid,calcium chloride and PEG-4000 were through the orthogonal tests,the optimal culture medium was 100 g·L-1 sucrose+30 mg·L-1 H3 BO3+20 mg·L-1 C aCl2+ 50 mg·L-1 PEG-4000;(3)The storage conditions for pollen of P.tancarvilleaeAfter being dried and stored for 360 days at 4℃,-20℃ and-80℃,the fruit setting rate of 120 days was 20%,60%and 80%,respectively.with the stored pollen for artificial pollination.Research have shown that after one year of pollen storage at-80℃,its viability and fruiting efficiency are very high,it’s the best temperature for pollen storage amony 3 temperatures.(4)Comparative analysis of plastomes of nine Phaius species in ChinaThe plastome of nine Phaius species in China were sequenced,assembled and annotated in this study.Comparison of the nine plastome showed that the nine Phaius species have a typical quadripartite structure with similar structure and gene contents.Genome size ranged from 157,997 to 158,630 bp,average 156,182 bp.The chloroplast genomes included 114 unique genes encoding 80 proteins,30 tRNAs and 4 rRNAs.A total of 2831 mutation events were founded in Chinese Phaius,most events were found in the LSC and the IGS.Among the three types of events,substitution events occur frequently;Dinucleotide inversion is the most common inversion event.A total of 9 intergenic regions,e.g.rps19-psbA,psbC-trnS-UGA,rpl16-rps3,rpoB-trnC-GCA,clpPpsbB,psaC-ndhE,petN-psbM,rps16-trnQ-UUG,rpsl9-trnH-GUG of the most variable hotspot regions were proposed as candidate effective molecular markers for future phylogenetic relationships and species divergence in the genus of Phaius.A total of 33 polymorphic SSR loci detected in this study to Phaius identification.69 CDS phylogenetic analysis based on Maximum Likelihood,Bayesian and Parsimony methods all highly supported the monophyly of the genus of Phaius and two subclade,e.g.P.columnaris subclade including P.columaris,P.flavus and P.tokeoi,and P.tancarvilleae subclade including the remain 6 species.The findings have resolved the phylogenetic relationships of the genus Phaius species,and provide valuable molecular markers to use in accurately identifying species and conservation genetics of endangered Phaius species. |
