A Preliminary Study on Conservation Genetics of Three Endangered Orchid Species

采用随机扩增多态DNA(RAPD)分析研究了中国 3种珍稀濒危兰科植物硬叶兜兰 (PaphiopedilummicranthumTangetWang)、麻栗坡兜兰 (P .malipoenseS .C .ChenetTsi)和独花兰 (ChangnieniaamoenaChien)的遗传多样性与群体遗传结构。 12个RAPD引物在 2种兜兰中共扩增出 131条带。对 4个硬叶兜兰群体的检测表明其物种水平的多态条带百分率 (PPB)为 71.6 % ,Nei的基因多样度 (h)为 0 .2 171,Shannon多样性指数 (I)为 0 .330 1;4个群体的平均多样性水平为PPB =45 .2 % ,h =0 .145 7,I =0 .2 2 0 4,低于远交兰花的平均水平。在总遗传变异中 ,群体间遗传变异占 2 0 .31% ,略高于远交物种的平均水平。在物种水平上 ,麻栗坡兜兰的PPB为 49.5 % ,h为 0 .1174,I为0 .176 4,均大大低于硬叶兜兰。对 11个独花兰群体采用 16个RAPD引物共扩增出 119条带。物种水平PPB =76 .5 % ,h =0 .1941,I=0 .30 5 8;在群体水平上 ,上述 3个指标的平均值则分别为 37.2 %、0 .1197和 0 .1810 ,均低于远交兰花的平均水平。群体间的遗传变异占 45 .2 7% ,遗传分化明显高于远交物种的平均水平。导致 3个物种遗传多样性偏低而群体间遗传分化较高的主要原因在于人为的过度采挖和生境的片断化。

Evolution and conservation genetics of pangolins

Pangolins(Pholidota,Manidae)are classified as an evolutionarily distinct and globally endangered mammal due to their unique morphology(nail-like scales and a myrmecophagous diet)and being the victim of heavy poaching and worldwide trafficking.As such,pangolins serve as a textbook example for studying the special phenotypic evolutionary adaptations and conservation genetics of an endangered species.Recent years have demonstrated significant advancements in the fields of molecular genetics and genomics,which have translated to a series of important research achievements and breakthroughs concerning the evolution and conservation genetics of pangolins.This review comprehensively presents the hitherto advances in phylogeny,adaptive evolution,conservation genetics,and conservation genomics that are related to pangolins,which will provide an ample understanding of their diversity,molecular adaptation mechanisms,and evolutionary potentials.In addition,we highlight the priority of investigating species/population diversity among pangolins and suggest several avenues of research that are highly relevant for future pangolin conservation.

Conservation genetics and genomics of threatened vertebrates in China

Conservation genetics and genomics are two independent disciplines that focus on using new techniques in genetics and genomics to solve problems in conservation biology. During the past two decades,conservation genetics and genomics have experienced rapid progress. Here, we summarize the research advances in the conservation genetics and genomics of threatened vertebrates(e.g., carnivorans, primates, ungulates, cetaceans, avians, amphibians and reptiles) in China. First, we introduce the concepts of conservation genetics and genomics and their development. Second, we review the recent advances in conservation genetics research, including noninvasive genetics and landscape genetics. Third, we summarize the progress in conservation genomics research, which mainly focuses on resolving genetic problems relevant to conservation such as genetic diversity, genetic structure, demographic history, and genomic evolution and adaptation. Finally, we discuss the future directions of conservation genetics and genomics.

Population genomic data reveal low genetic diversity,divergence and local adaptation among threatened Reeves's Pheasant(Syrmaticus reevesii)

Population genomic data could provide valuable information for conservation efforts;however,limited studies have been conducted to investigate the genetic status of threatened pheasants.Reeves’s Pheasant(Syrmaticus reevesii)is facing population decline,attributed to increases in habitat loss.There is a knowledge gap in understanding the genomic status and genetic basis underlying the local adaptation of this threatened bird.Here,we used population genomic data to assess population structure,genetic diversity,inbreeding patterns,and genetic divergence.Furthermore,we identified candidate genes linked with adaptation across the current distribution of Reeves’s Pheasant.The present study assembled the first de novo genome sequence of Reeves’s Pheasant and annotated 19,458 genes.We also sequenced 30 individuals from three populations(Dabie Mountain,Shennongjia,Qinling Mountain)and found that there was clear population structure among those populations.By comparing with other threatened species,we found that Reeves’s Pheasants have low genetic diversity.Runs of homozygosity suggest that the Shennongjia population has experienced serious inbreeding.The demographic history results indicated that three populations experienced several declines during the glacial period.Local adaptative analysis among the populations identified 241 candidate genes under directional selection.They are involved in a large variety of processes,including the immune response and pigmentation.Our results suggest that the three populations should be considered as three different conservation units.The current study provides genetic evidence for conserving the threatened Reeves’s Pheasant and provides genomic resources for global biodiversity management.

High regional genetic differentiation of an endangered relict plant Craigia yunnanensis and implications for its conservation

Of the genus Craigia,widespread in the Tertiary,only two relict species survived to modern times.One species is now possibly extinct and the other one,Craigia yunnanensis,is severely endangered.Extensive surveys have located six C.yunnanensis populations in Yunnan province,southwest China.Using fluorescent amplified fragment length polymorphism(AFLP),the genetic diversity and population structure of these populations were examined.It was found that genetic diversity of C.yunnanensis was moderate at the species level,but low at regional and population levels.Analysis of population structure showed significant genetic differentiation between Wenshan and Dehong regions,apparently representing two geographically isolated for long time refuges.There are also clear indications of isolation between populations,which,together with anthropogenically caused decline of population size,will lead to general loss of the species genetic variation with subsequent loss of adaptive potential.To conserve the genetic integrity of C.yunnanensis,we recommend that ex-situ conservation should include representative samples from every population of the two differentiated regions(e.g.Wenshan and Dehong).The crosses between individuals originated from different regions should be avoided because of a high risk of outbreeding depression.As all the extant populations of C.yunnanensis are in unprotected areas with strong anthropogenic impact,there is no alternative to reintroduction of C.yunnanensis into suitable protected locations.

Study on genetic diversity and resource conservation of amphioxus (Branchiostoma balcheri Gray) population

Amphioxus is the ancestor of vertebrates 5×108 a ago, it is a typical transitional sample of evolution from invertebrates to vertebrates. Inter simple sequence repeats (ISSRs) and random amplified polymorphic DNAs (RAPDs) technologies were applied to detect the genetic variation of 3 bulking samples and individually sampled populations in nowadays Xiamen sea areas (Xiekou, Nanxian and Huangcuo) where the amphioxuses are alive. For the bulking sampled populations, 5 ISSR and 10 RAPD primers generated 357 bands, of which 181 (50.7%) were polymorphic. Nei index and UPGMA statistical analysis indicated that amphioxuses in these 3 areas could be divided into 2 groups. The genetic distance between animals in Nanxian and Huangcuo areas was 0.07 and classified into 1 group, while the population in Xiekou belonged to another group because its genetic distances in Nanxian and Huangcuo were 0.12 and 0.14, respectively. The result was in accordance with the morphological comparison among animals from those areas. For individually sampled population, Shannon's index of genetic diversity was used to partition the diversity of the animals among these 3 sea areas, and the results showed that the indices in populations of Xiekou, Nanxian and Huangcuo, were 0.583, 0.482 and 0.374, respectively. The linear regression equation analysis for amphioxus' genetic diversity versus the environment factors revealed that granularity/sorting coefficient and water depth were the most important factors that affect amphioxus genetic diversity. On the basis of the results, the suggestions for amphioxus resource conservation in Xiamen sea areas are put forward.

Research advances in genetics and breeding of Populus davidiana Dode in China

In this paper a general introduction is given to research advances in genetics improvement and breeding of Chinese aspen (Populus davidiana Dode) in China. This introduction includes natural distribution and collection, conservation, gene diversity, provenance trial, crossing breeding, vegetative propagation and disease resistant etc. Based on the current situation of forest tree breeding in China, some strategic suggestions concerning the future development of Chinese aspen genetics improvement in China are presented, taking into consideration the existing domestic demands of forestry production and international trends in forest tree breeding.

Conservation and variability of hepatitis B core at different chronic hepatitis stages

BACKGROUND Since it is currently not possible to eradicate hepatitis B virus(HBV)infection with existing treatments,research continues to uncover new therapeutic strategies.HBV core protein,encoded by the HBV core gene(HBC),intervenes in both structural and functional processes,and is a key protein in the HBV life cycle.For this reason,both the protein and the gene could be valuable targets for new therapeutic and diagnostic strategies.Moreover,alterations in the protein sequence could serve as potential markers of disease progression.AIM To detect,by next-generation sequencing,HBC hyper-conserved regions that could potentially be prognostic factors and targets for new therapies.METHODS Thirty-eight of 45 patients with chronic HBV initially selected were included and grouped according to liver disease stage[chronic hepatitis B infection without liver damage(CHB,n=16),liver cirrhosis(LC,n=5),and hepatocellular carcinoma(HCC,n=17)].HBV DNA was extracted from patients’plasma.A region between nucleotide(nt)1863 and 2483,which includes HBC,was amplified and analyzed by next-generation sequencing(Illumina Mi Seq platform).Sequences were genotyped by distance-based discriminant analysis.General and intergroup nt and amino acid(aa)conservation was determined by sliding window analysis.The presence of nt insertion and deletions and/or aa substitutions in the different groups was determined by aligning the sequences with genotype-specific consensus sequences.RESULTS Three nt(nt 1900-1929,2249-2284,2364-2398)and 2 aa(aa 117-120,159-167)hyper-conserved regions were shared by all the clinical groups.All groups showed a similar pattern of conservation,except for five nt regions(nt 1946-1992,2060-2095,2145-2175,2230-2250,2270-2293)and one aa region(aa 140-160),where CHB and LC,respectively,were less conserved(P<0.05).Some group-specific conserved regions were also observed at both nt(2306-2334 in CHB and 1935-1976 and 2402-2435 in LC)and aa(between aa 98-103 in CHB and 28-30 and 51-54 in LC)levels.No differences in insertion and deletions frequencies were observed.An aa substitution(P79 Q)was observed in the HCC group with a median(interquartile range)frequency of 15.82(0-78.88)vs 0(0-0)in the other groups(P<0.05 vs CHB group).CONCLUSION The differentially conserved HBC and HBV core protein regions and the P79 Q substitution could be involved in disease progression.The hyper-conserved regions detected could be targets for future therapeutic and diagnostic strategies.

The whole-genome survey of Acer griseum,its polymorphic simple sequence repeats development and application

Background:Acer griseum Pax is an endangered species endemic to China with both ornamental and economic value.However,the lack of information on its genome size and characteristics hinders further work at the genome level.Methods:This paper applied bioinformatics methods to predict the characteristics and patterns of the A.griseum genome,providing an important basis for formulating its whole-genome sequencing scheme.This study also characterized the simple sequence repeats(SSRs)of A.griseum,laying the foundation for the development and application of genome-wide SSR markers.In this study,PE150 sequencing was performed by the BGI MGISEQ platform,and the sequence files were analyzed by the K-mer method to estimate the characteristic information by GCE software.Results:The genome size was finally determined to be 739.63 Mb,its heterozygosity ratio was 1.33%,and the repetition ratio was 65.68%.A total of 825,960 SSR loci were identified in the assembled genome sequence,and primers were successfully designed for 526,020 loci.To verify the effectiveness of these primers,100 pairs of primers were randomly selected and synthesized,and 81 pairs successfully amplified the target fragments.Fourteen pairs of primers with good polymorphism were selected for principal component analysis of 31 A.griseum individuals from two populations,showing favorable heterozygosity and PIC values.According to the findings,these SSRs might identify genetic variations based on geographic areas.Conclusion:It is suggested that Illumina+PacBio assembly strategy should be used for whole-genome sequencing due to the high heterozygosity rate and high repetition rate of the genome.In addition,the SSR primers designed in batches in this study laid a foundation for the in-depth study of population structure and population maintenance mechanism of A.griseum,which is helpful for the effective conservation and sustainable utilization of this germplasm resource.

Augmenting the conservation value of rehabilitated wildlife by integrating genetics and population modeling in the post-rehabilitation decision process

Insular populations are particularly vulnerable to the effects of stochastic events, epidemics, and loss of genetic diversity due to inbreeding and genetic drift. The development of successful man- agement options will require accurate baseline data, establishment of clear objectives, and finally monitoring and implementation of corrective measures, if and when required. This study assessed management options for the genetic rehabilitation of highly inbred woylies obtained from wildlife rehabilitation centers. The study generated genetic data for the woylie Bettongia penicillata from a conservation reserve and calculated measures of genetic diversity and individual relatedness. These data were fed into a population viability analysis （PVA） to test genetic outcomes in relation to different management actions. We demonstrated that a careful selection of the founder cohort produced a population with an expected heterozygosity of -70% for a window of approximately 10 years. A proposal to increase the size of the reserve available to the colony was shown to almost double the time at which the colony would retain heterozygosity levels of≥70%. Additionally, developing a regular program of supplementation of unrelated woylies would result in a further improvement in their genetic value. This study demonstrated how the application of molecular techniques in combination with PVA can be beneficial for the management of rehabilitated wildlife otherwise considered of little conservation value. This approach can be applied to the management of breeding programs, but also to small, closed populations such as those found on islands, fenced enclosures, insurance populations, and in zoological collections.

Biogeography and Evolutionary Factors Determine Genetic Differentiation of Pinus mugo(Turra) in the Tatra Mountains(Central Europe)

Inter Simple Sequence Repeats （ISSR） markers were used to assess genetic diversity within and among populations of dwarf mountain pine （Pinus rnugo Turra） growing in the Tatra National Park （UNESCO Biosphere Reserve） in Southern Poland （Central Europe）. The analyzed population belongs to two different geobotanical sub-districts： the Western and High Tatras. The level of genetic diversity assessed in this study for P. mugo is generally comparable to that reported for the other pine species in the Pinaceae family assessed by ISSR markers, especially with respect to Nei＇s genetic diversity and the percentage of polymorphic bands. Bayesian analysis clustered the analyzed populations into two groups, corresponding to their geobotanical locations in the Tatras. Significant divergence between the two genetical clusters was supported by the results of Analysis of Molecular Variance （AMOVA）. According to the Mantel test, there was no correlation between the genetic distance and the geographical distance. The present study confirms the existence of two genetically distinct dusters of P. mugo populations in the Tatra Mountains. The observed high population-genetic differentiation of P.mugo in the Tatras could be attributed to several genetic, environmental and historical factors occurring in this mountain area.

Applying molecular genetic tools to tiger conservation

The utility of molecular genetic approaches in conservation of endangered taxa is now commonly recognized.Over the past decade,conservation genetic analyses based on mitochondrial DNA sequencing and microsatellite genotyping have provided powerful tools to resolve taxonomy uncertainty of tiger subspecies,to define conservation units,to reconstruct phylogeography and demographic history,to examine the genetic ancestry of extinct subspecies,to assess population genetic status non-invasively,and to verify genetic background of captive tigers worldwide.The genetic status of tiger subspecies and populations and implications for developing strategies for the survival of this charismatic species both in situ and ex situ are discussed.

Strategic Conservation of Orchard Germplasm Based on Indigenous Knowledge and Genetic Diversity:a Case Study of Sour Orange Populations in China

To effectively conserve sour orange （Citrus aurantium L.） germplasm on two islands at the estuary of the Yangtze River in China, we estimated genetic variation and relationships of the known parental trees and their proposed descendents （young trees） using the fingerprints of random amplified polymorphic DNA （RAPD）. Results based on RAPD analyses showed considerable genetic diversity in the parental populations （He = 0.202）. The overall populations including the parental and young trees showed slightly higher genetic diversity （He = 0.298） than the parents, with about 10% variation between populations. An unweighted pair group method with arithmetic mean analysis dendrogram based on cluster analysis of the Jaccard similarity among individuals demonstrated a more complicated relationship of the parental and young trees from the two islands, although the young trees showed a clear association with parental trees. This indicates a significant contribution of parental trees in establishing the sour orange populations on the two islands. According to farmers＇ knowledge, conservation of only one or two parental trees would be sufficient because they believed that the whole populations were generated from a single mother tree. However, this study suggests that preserving most parental trees and some selected young trees with distant genetic relationships should be an effective conservation strategy for sour orange germplasm on the two islands.

Loss of Genetic Diversity of Domesticated Panax notoginseng F H Chen as Evidenced by ITS Sequence and AFLP Polymorphism： A Comparative Study with P. stipuleanatus H T Tsai et K M Feng

In the present study, we evaluated the genetic diversity of Panax notoginseng F H Chen, a domesticated species, and P. stipuleanatus H T Tsai et K M Feng, an endangered wild species in southeastern Yunnan and adjacent areas in Vietnam, using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA and amplified fragment length polymorphism (AFLP) markers. Twenty-four accessions from three plantations of P. notoginseng and 51 samples from eight populations of P. stipuleanatus were assayed. A total of 694 bp of partial sequences of 18S, ITS 1, 5.8S, ITS2, and partial sequences of 26S were obtained. No sequence variation was detected within P. notoginseng and nine sites (1.30%) were variable in P. stipuleanatus. Two-thirds of the variable sites were found between Langqiao and other populations. In P. notoginseng, four pairs of AFLP primer combinations generated 312 bands, of which 240 (76.9%) were polymorphic and 60.15% of the polymorphisms were harbored within plantations. Approximately 41.0% and 66.9% of bands were polymorphic in population D7 and 5589, respectively. In P.stipuleanatus, the same four primer combinations produced 346 bands, of which 334 (96.5%) were polymorphic and approximately 62.14% of polymorphisms were maintained within populations. Considerable variations were observed. The percentage of polymorphic bands ranged from 50.2% to 84.9% and the average over populations was 70.9%. Cluster analysis did not show correlation of genetic differentiation with the distinctive leaf morphology of P. stipuleanatus (i.e. one form with bipinnatifid leaflets and the other with undivided leaflets). Because over 40% of genetic variations were maintained among populations and because of the very restricted distribution of P. stipuleanatus, all natural populations of this species should be conserved in situ. Considering that there are variations in P. notoginseng within and among plantations, we suggest establishing a genetic resource conservation garden or reintroducing P. notoginseng into its native habitats in southwestern China. Such reintroduction should be carefully executed after large-scale screening of genetic variation within the species.

Genomics and introgression： Discovery and mapping of thousands of species-diagnostic SNPs using RAD sequencing

Invasive hybridization and introgression pose a serious threat to the persistence of many native species. Understand- ing the effects of hybridization on native populations （e.g., fitness consequences） requires numerous species-diagnostic loci dis- tributed genome-wide. Here we used RAD sequencing to discover thousands of single-nucleotide polymorphisms （SNPs） that are diagnostic between rainbow trout （RBT, Oncorhynchus mykiss）, the world＇s most widely introduced fish, and native westslope cutthroat trout （WCT, （9. clarkii lewisi） in the northern Rocky Mountains, USA. We advanced previous work that identified 4,914 species-diagnostic loci by using longer sequence reads （100 bp vs. 60 bp） and a larger set of individuals （n = 84）. We sequenced RAD libraries for individuals from diverse sampling sources, including native populations of WCT and hatchery broodstocks of WCT and RBT. We also took advantage of a newly released reference genome assembly for RBT to align our RAD loci. In total, we discovered 16,788 putatively diagnostic SNPs, 10,267 of which we mapped to anchored chromosome locations on the RBT genome. A small portion of previously discovered putative diagnostic loci （325 of 4,914） were no longer diagnostic （i.e., fixed between species） based on our wider survey of non-hybridized RBT and WCT individuals. Our study suggests that RAD loci mapped to a draft genome assembly could provide the marker density required to identify genes and chromosomal regions in- fluencing selection in admixed populations of conservation concern and evolutionary interest [Current Zoology 61 （1）： 146-154, 2015].

Isolation of microsatellite loci and reliable genotyping using noninvasive samples of a critically endangered primate, Trachypithecus leucocephalus

Genetic information can be critical in identifying conservation priorities and developing conservation strategies.There is an urgent need for noninvasive genetic tools to study the wild populations of Asian colobine monkeys.The majority of these species are threatened with habitat destruction,population reduction and even extinction,but generally lack information on their genetic diversity and population structure.Genetic sampling and tissue collection have been scarce in these species owing to strict regulations on manipulation of endangered species,and the difficulties and risks associated with capturing these arboreal and fast-moving monkeys in the challeng­ing environments that they inhabit.These difficulties have hindered the development of molecular genetic mark­ers,which are usually derived from tissues or blood.In this study,we present a method for de novo microsatel­lite isolation and genotyping using DNA from noninvasive origins of a critically endangered Asian colobine,the white-headed langur(Trachypithecus leucocephalus).Genomic DNA isolated from hair was shown to be suf­ficient for microsatellite enrichment and isolation,with similar isolation efficiencies as from tissue DNA.We identified and characterized 20 polymorphic microsatellite loci,and evaluated their amplification success and genotyping reliability with 86 field-collected fecal samples.These results show that this panel of loci can pro­duce reliable genotypes from fecal samples,and represent a useful tool for noninvasive investigation of genetic structure,individual identification and kinship assessment in this highly endangered species.Our approach can be applied to conservation genetic studies of other wild species that lack sequence information and tissue sam­ples.