Xiaoping Zhou

New primers for sex identification in the Chinese Egret and other ardeid species

Using the universal P2/P8 primers, we were able to obtain the gene segments of chromo‐helicase‐DNA binding protein (CHD)‐Z and CHD‐W from ten species of ardeid birds including Chinese egret (Egretta eulophotes), little egret (E. garzetta), eastern reef egret (E. sacra), great egret (Ardea alba), grey heron (A. cinerea), Chinese pond‐heron (Ardeola bacchus), cattle egret (Bubulcus ibis), black‐crowned night‐heron (Nycticorax nycticorax), cinnamon bittern (Ixobrychus cinnamomeus) and yellow bittern (I. sinensis). Based on conserved regions inside the P2/P8‐derived sequences, we designed new PCR primers for sex identification in these ardeid species. Using agarose gel electrophoresis, the PCR products showed two bands for females (140 bp derived from CHD‐W and the other 250 bp from CHD‐ZW), whereas the males showed only the 250 bp band. The results indicated that our new primers could be used for accurate and convenient sex identification in ardeid species.

The complete mitochondrial genomes of sixteen ardeid birds revealing the evolutionary process of the gene rearrangements

BackgroundThe animal mitochondrial genome is generally considered to be under selection for both compactness and gene order conservation. As more mitochondrial genomes are sequenced, mitochondrial duplications and gene rearrangements have been frequently identified among diverse animal groups. Although several mechanisms of gene rearrangement have been proposed thus far, more observational evidence from major taxa is needed to validate specific mechanisms. In the current study, the complete mitochondrial DNA of sixteen bird species from the family Ardeidae was sequenced and the evolution of mitochondrial gene rearrangements was investigated. The mitochondrial genomes were then used to review the phylogenies of these ardeid birds.ResultsThe complete mitochondrial genome sequences of the sixteen ardeid birds exhibited four distinct mitochondrial gene orders in which two of them, named as “duplicate tRNAGlu–CR” and “duplicate tRNAThr–tRNAPro and CR”, were newly discovered. These gene rearrangements arose from an evolutionary process consistent with the tandem duplication - random loss model (TDRL). Additionally, duplications in these gene orders were near identical in nucleotide sequences within each individual, suggesting that they evolved in concert. Phylogenetic analyses of the sixteen ardeid species supported the idea that Ardea ibis, Ardea modesta and Ardea intermedia should be classified as genus Ardea, and Ixobrychus flavicollis as genus Ixobrychus, and indicated that within the subfamily Ardeinae, Nycticorax nycticorax is closely related to genus Egretta and that Ardeola bacchus and Butorides striatus are closely related to the genus Ardea.ConclusionsThe duplicate tRNAThr–CR gene order is found in most ardeid lineages, suggesting this gene order is the ancestral pattern within these birds and persisted in most lineages via concerted evolution. In two independent lineages, when the concerted evolution stopped in some subsections due to the accumulation of numerous substitutions and deletions, the duplicate tRNAThr–CR gene order was transformed into three other gene orders. The phylogenetic trees produced from concatenated rRNA and protein coding genes have high support values in most nodes, indicating that the mitochondrial genome sequences are promising markers for resolving the phylogenetic issues of ardeid birds when more taxa are added.

PERMANENT GENETIC RESOURCES: A set of primer pairs for amplifying the complete mitochondrial DNA of endangered Chinese egret (Aves, Ardeidae, Egretta eulophotes).

The Chinese egret is a globally endangered species. Here we describe a set of primer pairs to amplify its entire mtDNA. The polymerase chain reaction products (1000–2000 bp) were successfully amplified by using this primer set and were then sequenced and aligned. The contiguous mtDNA sequences of the Chinese egret were assembled to be a circular molecule (17 579 bp). This primer set was also confirmed to be useful for six other species of ardeid birds. The versatility of this primer set will provide a groundwork for further studies on the genetic structure and molecular evolution of the ardeid species.

A set of primer pairs for amplifying the complete mitochondrial DNA of endangered Chinese egret (Aves, Ardeidae, Egretta eulophotes)

The Chinese egret is a globally endangered species. Here we describe a set of primer pairs to amplify its entire mtDNA. The polymerase chain reaction products (1000–2000 bp) were successfully amplified by using this primer set and were then sequenced and aligned. The contiguous mtDNA sequences of the Chinese egret were assembled to be a circular molecule (17 579 bp). This primer set was also confirmed to be useful for six other species of ardeid birds. The versatility of this primer set will provide a groundwork for further studies on the genetic structure and molecular evolution of the ardeid species.

Isolation and characterization of microsatellite loci in vulnerable Chinese egret (Egretta eulophotes: Aves)

Eighteen polymorphic microsatellite loci were isolated from the vulnerable Chinese egret Egretta eulophotes using the magnetic bead-based enrichment method, and were tested in a sample of 20 individuals from a Chinese egret population distributed in Fujian province of China. The number of alleles range from 2 to 9 per locus with the observed and expected heterozygosity ranging from 0.20 to 0.85 and 0.18 to 0.82, respectively. One locus deviated significantly from Hardy–Weinberg equilibrium and no pairs showed evidence of linkage disequilibrium after sequential bonferroni correction. These loci were also confirmed useful for the cross-amplifications in other five ardeid species. These new microsatellite markers will be useful for the further studies on the genetic structure, molecular evolution and conservation management of this vulnerable species and the other ardeid species.

Characterization, Polymorphism and Selection of Major Histocompatibility Complex (MHC) DAB Genes in Vulnerable Chinese Egret (Egretta eulophotes)

The major histocompatibility complex (MHC) is an excellent molecular marker for the studies of evolutionary ecology and conservation genetics because it is a family of highly polymorphic genes that play a key role in vertebrate immune response. In this study, the functional genes of MHC Class II B (DAB) were isolated for the first time in a vulnerable species, the Chinese egret ( Egretta eulophotes ). Using a full length DNA and cDNA produced by PCR and RACE methods, four potential MHC DAB loci were characterized in the genome of this egret and all four were expressed in liver and blood. At least four copies of the MHC gene complex were similar to two copies of the minimal essential MHC complex of chicken, but are less complex than the multiple copies expressed in passerine species. In MHC polymorphism, 19 alleles of exon 2 were isolated from 48 individuals using PCR. No stop codons or frameshift mutations were found in any of the coding regions. The signatures of positive selection detected in potential peptide-binding regions by Bayesian analysis, suggesting that all of these genes were functional. These data will provide the fundamental basis for further studies to elucidate the mechanisms and significance of MHC molecular adaptation in vulnerable Chinese egret and other ardeids.

Major histocompatibility complex class II DAB alleles associated with intestinal parasite load in the vulnerable Chinese egret (Egretta eulophotes)

Abstract The maintenance of major histocompatibility complex (MHC) polymorphism has been hypothesized to result from many mechanisms such as rare‐allele advantage, heterozygote advantage, and allele counting. In the study reported herein, 224 vulnerable Chinese egrets (Egretta eulophotes) were used to examine these hypotheses as empirical results derived from bird studies are rare. Parasite survey showed that 147 (65.63%) individuals were infected with 1–3 helminths, and 82.31% of these infected individuals carried Ascaridia sp. Using asymmetric polymerase chain reaction technique, 10 DAB1, twelve DAB2, and three DAB3 exon 2 alleles were identified at each single locus. A significant association of the rare allele Egeu‐DAB2*05 (allele frequency: 0.022) with helminth resistance was found for all helminths, as well as for the most abundant morphotype Ascaridia sp. in the separate analyses. Egeu‐DAB2*05 occurred frequently in uninfected individuals, and individuals carrying Egeu‐DAB2*05 had significantly lower helminth morphotypes per individual (HMI) (the number of HMI) and the fecal egg count values. Further, the parasite infection measurements were consistently lower in individuals with an intermediate number of different alleles in the duplicated DAB loci. Significantly, heterozygosity within each DAB locus was not correlated with any parasite infection measurements. These results indicate that the diversity in MHC Egeu‐DAB gene is associated with intestinal parasite load and maintained by pathogen‐driven selection that probably operate through both the rare‐allele advantage and the allele counting strategy, and suggest that Egeu‐DAB2*05 might be a valuable indicator of better resistance to helminth diseases in the vulnerable Chinese egret.

Complete mitochondrial genomes render the Night Heron genus Gorsachius non-monophyletic

In the present study, the complete mitochondrial genomes of three Night Herons from the genus Gorsachius were sequenced. All the complete mitochondrial genomes in this genus exhibit duplications in the region between cytochrome b and 12S ribosomal RNA. In Gorsachius magnificus, the duplicated regions span from the last 108 base pairs of cytochrome b to the control region, which are nearly identical to each other in nucleotide sequences, suggesting they are evolving in concert. In G. goisagi and G. melanolophus, the duplicated control regions were nearly identical in majority portions within each individual, while the first tRNAPro-ND6-tRNAGlu and the second Cytb-tRNAThr regions have degenerated into non-coding regions. Phylogenetic analyses with Bayesian inference and maximum likelihood based on the nucleotide sequences of two ribosomal RNA genes and 12 protein coding genes indicate that G. magnificus is not monophyletic with the other two Gorsachius species. These new results provide the fundamental basis for further studies to elucidate their phylogenetic positions and relationships with other genera within the subfamily Ardeinae.ZusammenfassungVollständiges mitochondriales Genom ergibt Nicht-Monophylie der Nachtreiher-GattungGorsachius In der vorliegenden Studie wurde das vollständige mitochondriale Genom von drei Nachtreiherarten der Gattung Gorsachius sequenziert. Alle mitochondrialen Genome dieser Gattung wiesen Duplikationen in der Region zwischen Cytochrome b und 12S der ribosomalen RNA auf. Bei G. magnificus umfassten die duplizierten Regionen die letzten 108 Basenpaare von Cytochrome b bis zur Kontrollregion, die nahezu identisch sind in ihren Nukleotidsequenzen. Dies deutet auf eine gemeinsame Entwicklung hin. Bei G. goisagi und G. melanolophus waren die mehrheitlichen Anteile der duplizierten Kontrollregionen nahezu bei jedem Individuum identisch, während die ersten tRNAPro-ND6-tRNAGlu und die zweiten CytbtRNAThr Regionen zu nicht-kodierenden Regionen degeneriert waren. Phylogenetische Analysen mittels Bayes-Inferenz und Maximum Likelihood Schätzung, die auf den Nukleotidsequenzen zweier ribosomaler RNA Gene und 12 Protein kodierender Gene basierten, zeigten, dass G. magnificus nicht der gleichen Stammform entstammt wie die beiden anderen Gorsachius-Arten. Diese neuen Erkenntnisse liefern die fundamentale Basis für weitere Studien zur Aufklärung phylogenetischer Positionen und Verwandtschaftsverhältnisse mit anderen Gattungen innerhalb der Unterfamilie der Ardeinae.

PCR–RFLP technique for species identification of molted feathers in six species of co-occurring Ardeids

Naturally molted feathers provide a valuable tissue resource for avian research. Here we present a convenient and accurate species identification method for the naturally molted white feathers of six co-occurring Ardeid species, Egretta eulophotes, E. garzetta, Ardea alba, Bubulcus ibis, Mesophoyx intermedia and Ardeola bacchus, based on the polymerase chain reaction–restriction fragment length polymorphism of the partial mitochondrial cytochrome c oxidase subunit I gene. The resulting species-specific restriction patterns are easily distinguished by agarose gel electrophoresis. In this study, 81 of 88 naturally molted feathers noninvasively collected from heronries and 139 plucked feathers were all successfully distinguished, while the 7 feathers that could not be successfully distinguished by this method did not provide enough amplified products. This newly developed method for species identification of molted feathers will be useful for further studies on the vulnerable Chinese Egret and the other five co-occurring Ardeid species in East Asia.

Development and cross-species transferability of 23 microsatellite markers from the vulnerable Chinese Egret (Egretta eulophotes) using 454 sequencing

AbstractThe Chinese Egret (Egretta eulophotes) is a vulnerable waterbird breeding on offshore islands. Here, 23 new microsatellite loci were isolated using 454 pyrosequencing, and were tested in 32 individuals from a single population in Fujian province of China. The number of alleles per locus ranged from 2 to 9. Observed heterozygosity and expected heterozygosity ranged from 0.094 to 0.844 and 0.091 to 0.840, respectively. All loci showed Hardy–Weinberg equilibrium with the exception of three loci (Ee10, Ee17 and Ee23), possibly because of null alleles. The new loci also were confirmed a high cross-amplification success rate in the other seven Ardeid species. These new microsatellite data will provide a major resource for future conservation genetics studies on this vulnerable egret and the other Ardeids.