Zhan-Lin Liu

Intragenomic and interspecific 5S rDNA sequence variation in five Asian pines

Patterns of intragenomic and interspecific variation of 5S rDNA in Pinus (Pinaceae) were studied by cloning and sequencing multiple 5S rDNA repeats from individual trees. Five pines, from both subgenera, Pinus and Strobus, were selected. The 5S rDNA repeat in pines has a conserved 120-base pair (bp) transcribed region and an intergenic spacer region of variable length (382-608 bp). The evolutionary rate in the spacer region is three- to sevenfold higher than in the genic region. We found substantial sequence divergence between the two subgenera. Intragenomic sequence heterogeneity was high for all species, and more than 86% of the clones within each individual were unique. The 5S gene tree revealed that different 5S repeats within individuals are polyphyletic, indicating that their ancestral divergence preceded the speciation events. The degrees of interspecific and intragenomic divergence among diploxylon pines are similar. The observed sequence patterns suggest that concerted evolution has been acting after the diversification of the two subgenera but very weak after the speciation of the four diploxylon pines. Sequence patterns in P. densata are consistent with hybrid origin. It had higher intragenomic diversity and maintained polymorphic copies of the parental types in addition to new and recombinant types unique to the hybrid.

Strong phylogeographic pattern of cpDNA variation reveals multiple glacial refugia for Saruma henryi Oliv. (Aristolochiaceae), an endangered herb endemic to China

Saruma henryi Oliv. (Aristolochiaceae) is an endangered herb endemic to China. In this study, chloroplast microsatellites (cpSSRs) and sequences of the atpB-rbcL intergenic spacers were employed to reveal its genetic diversity and phylogeographic patterns. We detected high within-species genetic diversity (H(T)=0.939 for cpSSR; H(T)=0.862 for atpB-rbcL) and pronounced among-population genetic differentiation (H(S)=0.182, G(ST)=0.811, R(ST)=0.9, F(ST)=0.93 for cpSSR; H(S)=0.238, G(ST)=0.724, N(ST)=0.758, F(ST)=0.79 for atpB-rbcL) with a strong phylogeographic pattern (R(ST)>G(ST), P<0.01 for cpSSR; N(ST)>G(ST), U=0.25 for atpB-rbcL). Eleven haplotypes were distinguished by cpSSRs and atpB-rbcL intergenic spacers, respectively. The molecular phylogenetic data, together with the geographic distribution of the haplotypes, suggested the existence of multiple localized glacial refugia in Mts. Qinling, eastern Mts. Bashan and the southeastern edge of Yunnan-Guizhou Plateau. Nested clade analysis (NCA) and population genetic analyses supported the limited gene flow (caused by low dispersal capacity and complex topography of its habitats) as the major factor responsible for the strong population differentiation and phylogeographic pattern. Past fragmentation and allopatric fragmentation were inferred as important processes responsible for the modern phylogeograhpic pattern. In addition, contiguous range expansions occurred in western Mts. Qinling and eastern Mts. Bashan.

Development of Chloroplast and Nuclear DNA Markers for Chinese Oaks (Quercus Subgenus Quercus) and Assessment of Their Utility as DNA Barcodes

Chloroplast DNA (cpDNA) is frequently used for species demography, evolution, and species discrimination of plants. However, the lack of efficient and universal markers often brings particular challenges for genetic studies across different plant groups. In this study, chloroplast genomes from two closely related species (Quercus rubra and Castanea mollissima) in Fagaceae were compared to explore universal cpDNA markers for the Chinese oak species in Quercus subgenus Quercus, a diverse species group without sufficient molecular differentiation. With the comparison, nine and 14 plastid markers were selected as barcoding and phylogeographic candidates for the Chinese oaks. Five (psbA-trnH, matK-trnK, ycf3-trnS, matK, and ycf1) of the nine plastid candidate barcodes, with the addition of newly designed ITS and a single-copy nuclear gene (SAP), were then tested on 35 Chinese oak species employing four different barcoding approaches (genetic distance-, BLAST-, character-, and tree-based methods). The four methods showed different species identification powers with character-based method performing the best. Of the seven barcodes tested, a barcoding gap was absent in all of them across the Chinese oaks, while ITS and psbA-trnH provided the highest species resolution (30.30%) with the character- and BLAST-based methods, respectively. The six-marker combination (psbA-trnH + matK-trnK + matK + ycf1 + ITS + SAP) showed the best species resolution (84.85%) using the character-based method for barcoding the Chinese oaks. The barcoding results provided additional implications for taxonomy of the Chinese oaks in subg. Quercus, basically identifying three major infrageneric clades of the Chinese oaks (corresponding to Groups Quercus, Cerris, and Ilex) referenced to previous phylogenetic classification of Quercus. While the morphology-based allocations proposed for the Chinese oaks in subg. Quercus were challenged. A low variation rate of the chloroplast genome, and complex speciation patterns involving incomplete lineage sorting, interspecific hybridization and introgression, possibly have negative impacts on the species assignment and phylogeny of oak species.

The Complete Chloroplast Genome Sequences of Six Rehmannia Species

Rehmannia is a non-parasitic genus in Orobanchaceae including six species mainly distributed in central and north China. Its phylogenetic position and infrageneric relationships remain uncertain due to potential hybridization and polyploidization. In this study, we sequenced and compared the complete chloroplast genomes of six Rehmannia species using Illumina sequencing technology to elucidate the interspecific variations. Rehmannia plastomes exhibited typical quadripartite and circular structures with good synteny of gene order. The complete genomes ranged from 153,622 bp to 154,055 bp in length, including 133 genes encoding 88 proteins, 37 tRNAs, and 8 rRNAs. Three genes (rpoA, rpoC2, accD) have potentially experienced positive selection. Plastome size variation of Rehmannia was mainly ascribed to the expansion and contraction of the border regions between the inverted repeat (IR) region and the single-copy (SC) regions. Despite of the conserved structure in Rehmannia plastomes, sequence variations provide useful phylogenetic information. Phylogenetic trees of 23 Lamiales species reconstructed with the complete plastomes suggested that Rehmannia was monophyletic and sister to the clade of Lindenbergia and the parasitic taxa in Orobanchaceae. The interspecific relationships within Rehmannia were completely different with the previous studies. In future, population phylogenomic works based on plastomes are urgently needed to clarify the evolutionary history of Rehmannia.

Efficient Identification of the Forest Tree Species in Aceraceae Using DNA Barcodes

Aceraceae is a large forest tree family that comprises many economically and ecologically important species. However, because interspecific and/or intraspecific morphological variations result from frequent interspecific hybridization and introgression, it is challenging for non-taxonomists to accurately recognize and identify the tree species in Aceraceae based on a traditional approach. DNA barcoding is a powerful tool that has been proposed to accurately distinguish between species. In this study, we assessed the effectiveness of three core standard markers (matK, rbcL and ITS) plus the chloroplast locus trnS-trnG as Aceraceae barcodes. A total of 231 sequences representing 85 species in this forest family were collected. Of these four barcode markers, the discrimination power was highest for the ITS (I) region (50%) and was progressively reduced in the other three chloroplast barcodes matK (M), trnS-trnG (T) and rbcL (R); the discrimination efficiency of the ITS marker was also greater than any two-locus combination of chloroplast barcodes. However, the combinations of ITS plus single or combined chloroplast barcodes could improve species resolution significantly; T+I (90.5% resolution) and R+M+T+I (90.5% resolution) differentiated the highest portion of species in Aceraceae. Our current results show that the nuclear ITS fragment represents a more promising DNA barcode marker than the maternally inherited chloroplast barcodes. The most efficient and economical method to identify tree species in Aceraceae among single or combined DNA barcodes is the combination of T+I (90.5% resolution).

The complete chloroplast genome of common walnut (Juglans regia)

Abstract Common walnut (Juglans regia L.) is cultivated in temperate regions worldwide for its wood and nuts. The complete chloroplast genome of J. regia was sequenced using the Illumina MiSeq platform. This is the first complete chloroplast sequence for the Juglandaceae, a family that includes numerous species of economic importance. The chloroplast genome of J. regia was 160 367 bp in length, with 36.11% GC content. It contains a pair of inverted repeats (26 035 bp) which were separated by a large single copy (89 872 bp) and a small single copy region (18 425 bp). A total of 137 genes were annotated, which included 86 protein-coding genes, three pseudogenes (two ycf15 and one infA), 40 tRNA genes and eight rRNA genes. The neighbour-joining phylogenetic analysis with the reported chloroplast genomes showed that common walnut chloroplasts are most closely related to those of the Fagaceae family.

The complete chloroplast genome of Armand pine Pinus armandii, an endemic conifer tree species to China

Abstract The complete chloroplast genome (cpDNA) sequence of an endemic conifer species, Armand pine Pinus armandii Franch., is determined in this study. The cpDNA was 117,265 bp in length, containing a pair of 475 bp inverted repeat (IR) regions those distinguished in large and small single copy (LSC and SSC) regions of 64,548 and 51,767 bp in length, respectively. The cpDNA contained 114 genes, including 74 protein-coding genes (74 PCG species), 4 ribosomal RNA genes (four rRNA species) and 36 transfer RNA genes (33 tRNA species). Out of these genes, 12 harbor a single intron and most of the genes occurred in a single copy. The overall AT content of the Armand pine cpDNA was 61.2%, while the corresponding values of the LSC, SSC and IR regions were 62.0%, 60.2% and 62.7%, respectively. A phylogenetic analysis revealed that P. armandii chloroplast genome is closely related to that of the P. koraiensis within the genus Pinus.

Comparative Plastid Genomes of Primula Species: Sequence Divergence and Phylogenetic Relationships

Compared to traditional DNA markers, genome-scale datasets can provide mass information to effectively address historically difficult phylogenies. Primula is the largest genus in the family Primulaceae, with members distributed mainly throughout temperate and arctic areas of the Northern Hemisphere. The phylogenetic relationships among Primula taxa still maintain unresolved, mainly due to intra- and interspecific morphological variation, which was caused by frequent hybridization and introgression. In this study, we sequenced and assembled four complete plastid genomes (Primula handeliana, Primula woodwardii, Primula knuthiana, and Androsace laxa) by Illumina paired-end sequencing. A total of 10 Primula species (including 7 published plastid genomes) were analyzed to investigate the plastid genome sequence divergence and their inferences for the phylogeny of Primula. The 10 Primula plastid genomes were similar in terms of their gene content and order, GC content, and codon usage, but slightly different in the number of the repeat. Moderate sequence divergence was observed among Primula plastid genomes. Phylogenetic analysis strongly supported that Primula was monophyletic and more closely related to Androsace in the Primulaceae family. The phylogenetic relationships among the 10 Primula species showed that the placement of P. knuthiana–P. veris clade was uncertain in the phylogenetic tree. This study indicated that plastid genome data were highly effective to investigate the phylogeny.

Isolation and characterization of polymorphic microsatellites in the perennial herb Euphorbia kansui using paired-end Illumina shotgun sequencing

Euphorbia kansui is a threatened perennial herb endemic to China, and a good knowledge of its population genetics would be essential to the formulation of effective conservation and management strategies. In this study, 12 novel microsatellite markers were developed for E. kansui using Illumina sequencing technology. The number of alleles varied between 2 and 11, with an average of 4.1 alleles per locus. The expected (HE) and observed heterozygosity (HO) ranged from 0.0950 to 0.7875 and from 0.0500 to 0.9500, with an average of 0.4050 and 0.4083, respectively. Following the false discovery rate controlling procedure, significant deviations from Hardy–Weinberg equilibrium was only detected in a single locus (Ek1073). These informative microsatellite markers can be readily used for genetic analyses of this threatened plant.

Isolation and characterization of polymorphic microsatellite loci in the endangered plant Dipteronia sinensis (Sapindaceae).

UNLABELLED PREMISE OF THE STUDY Microsatellite markers were developed in Dipteronia sinensis to investigate the population genetics of this endangered plant. • METHODS AND RESULTS Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 19 microsatellite loci were developed in D. sinensis and evaluated for their variability in 29 samples from a natural population. For the 15 polymorphic loci, the number of alleles ranged from nine to 33, while the observed and expected heterozygosities ranged from 0.3793 to 0.9655 and from 0.6029 to 0.9609, respectively. Their cross-taxa transferability was also investigated in Acer miaotaiense, A. palmatum, and A. pictum subsp. mono, and 10 to 15 loci proved amplifiable in these species. • CONCLUSIONS These microsatellite markers could be employed to investigate the population genetics of D. sinensis and may potentially be applicable to other related species.