| There are more than 170 species belonging to 78 genera of wild orchids in Fujian Province,most of which have scarce wild resources,narrow distribution area and special habitat.Oberonia seidenfadenii and Conchidium sinicum are newly recorded species discovered in Fujian in recent years.The former has very few known distribution points,and the latter has a short plant type and habitat.They are epiphytic wild orchids and are able to bloom normallybut have low fruit.The plants are short and the flowers are smaller,causing disputes over their classification.Nowadays,the wild resources,habits and populations of wild orchids are being damaged with the development of tourist industry and the excavation of plant's medicinal value.Oberonia seidenfadenii and Conchidium sinicum are endanger species and treated as national key protected plants.Therefore,it is urgent to take measures to protect the wild germplasm resources.Aim to clarify the taxonomic status of the two species and put forward an effective protection plan,this study focus on the classification,population genetic diversity,aseptic germination,and multiplication culture of Oberonia seidenfadenii and Conchidium sinicum,through the analyze of ITS and ISSR and tissue culture methods.The main results are as follows:(1)Based on the phylogenetic analyze of ITS,the genus Hippeophyllu Schltr.could be combined into another genus Oberonia Lindl.The ITS sequence of Oberonia seidenfadenii from Yongtai county are consistent with others localities(from the NCBI databases),which supports the treatment of this species in Flora of China(FOC).Eria sinica and Eria pusilla original belong to the genus Eria Lindl,are placed in the genus Conchidium Griff.,and suggested to be treatedas as two taxons,Conchidium sinicum and Conchidium pusilla,in line with Flora Reipublicae Popularis Sinicae(FRPS)instead of the treatment of “combine Eria sinica with Conchidium pusilla” in FOC,or the treatment of “place Eria sinica in the genus Porpax Lindl.” by Yan Peng Ng(Ng et al.2018).(2)The ISSR-PCR reaction systemand amplified procedures for Oberonia seidenfadenii are established.In this study,12 primers of 21 samples are selected to amplification,get153 bands,of which 139 are polymorphic bandsand the percentage of polymorphic bands(PPF)is 90.85%.Alignments analyzed by Popgene 1.31 and NTsys2.10 e show that the genetic distance in 21 populations are in the range of 0.0000?3.1781.There are significant differences between Oberonia seidenfadenii populations from Fujian and Zhejiang on the genetic diversity,among which the population genetic diversity of Yongtai is as follows: NPF=130,PPF=85.53%,Na=0.3530,Ne=0.3477,Nei=0.1750,I=0.2366;as for the populations genetic diversity of Ninghai and Fenghua in Zhejiang Province is as follows: NPF=79,PPF=51.97%,Na=0.5013,Ne=0.3943,Nei=0.2100,I=0.2997.Unweighted mean distance map(UPGMA)of 21 samples of Oberonia seidenfadenii are divided into two branches when the genetic distance was 0.560,and the interspecies populations in similar geographical locations were almost clustered together,which illustrates the close relationship between these species.(3)The ISSR-PCR reaction system and amplified procedures for Conchidium sinicum are established.In this study,13 primers of 23 samples are selected to amplification,144 bands are obtained,of which 133 are polymorphic bands,every primer gets 11.08 bands,10.23 polymorphic bandsand in average and PPF is 92.33%.There are differences of Conchidium sinicum populations in Fujian ongenetic diversity,NPF of seven Conchidium sinicum populations are between 34?81,PPF are between 23.61%?56.25%,Na are between 0.4262?0.5005,Ne are between 0.3289?0.4104,the diversity of Nei gene are between 0.1807?0.2131,Shannon's information diversity index is between0.2591?0.3023.The genetic diversity parameter index of genetic diversity of Conchidium sinicum populations are the maximumfrom Nanjing(NJ)(PPF = 53.47%,I = 0.3023),and are minimum from Yunxiao(YX)(PPF = 23.61%,I = 0.2591),indicating that the genetic diversity in populations from NJ are the richest and from YX are the lowest.The genetic distance between two pairs of 23 populations are in the range of 0.1188 ? 0.3051,and the genetic distance of 7 populations are between 0.7370 ? 0.8880.UPGMA of 23 samples of Conchidium sinicum are divided into two branches when the genetic consistency is 0.650,and they are divided into three branches,when the genetic consistency is 0.658,among which thepopulations from Changle(CL)is a single branch.The results of cluster analysis show that the closely related Conchidium sinicum groups are also close to each other geographically.(4)In the orthogonal experiment of aseptic germination in Oberonia seidenfadenii,the influences on seed germination rate from high to low are NAA > organic additive >basic medium type.The optimal formulation of aseptic germination medium: 1/2MS +30.0 g/L sucrose+8.0 g/L agar+ 100 ml/L banana juice+0.1 mg/L NAA,p H=5.8±0.1,the germination rate is up to 90%.The most suitable culture formula for the proliferation and development of protocorm: 1/2MS + 30.0 g/L sucrose+7.0 g/L agar+ 100 ml/L potato juice+1.0 mg/L NAA,p H=5.8±0.1.In the orthogonal experiment of aseptic germination in Conchidium sinicum,the influences on seed germination rate from high to low are activated carbon >6-BA >NAA.The optimal formulation of aseptic germination medium: 1/2MS + 20.0 g/L sucrose+ 7.0g/L agar+ 100 ml/L coconut milk+1.0 mg/L 6-BA+1.0 mg/L NAA+1.0 g/L activated carbon,p H=5.3±0.1,and the germination rate is up to 93%.The horizontal genetic diversity is high in the population of Oberonia seidenfadenii,which shows a strong environmental adaptability.It is suggested that measures,such as local protection,artificial propagation,seedlings production,and bionic cultivation in similar habitat to expand its distribution range and promote gene exchange,should be carried out for Oberonia seidenfadenii population.By contrast,the horizontal genetic diversity is lower in the population of Conchidium sinicum,and which means its weak environmental adaptability.Although Conchidium sinicum is not in danger,it is easily damaged by human beings and should be paid more attention.As it is difficult to do artificial rapid reproduction,local protection is the best strategy for Conchidium sinicum.Additionally,the known distribution points of Oberonia seidenfadenii include Taiwan,Guangdong,Guangxi and other provinces;the known distribution points of Conchidium sinicum are also in Zhejiang,Guangdong,Guangxi,Yunnan,Hunan,Guizhou and other provinces.It is necessary to expand the distribution area,increase the number of samples,and conducta more convincing analysis of the genetic diversity;as well as the most suitable cultivation formula for the proliferation and development of protocorm of Conchidium sinicum,etc.in the future general survey of these two other distribution points in Fujian Province. |
PDF Full Text Request