Zhong-Hu Li

Comparative Transcriptome and Chloroplast Genome Analyses of Two Related Dipteronia Species

Dipteronia (order Sapindales) is an endangered genus endemic to China and has two living species, D.sinensis and D. dyeriana. The plants are closely related to the genus Acer, which is also classified in the order Sapindales. Evolutionary studies on Dipteronia have been hindered by the paucity of information on their genomes and plastids. Here, we used next generation sequencing to characterize the transcriptomes and complete chloroplast genomes of both Dipteronia species. A comparison of the transcriptomes of both species identified a total of 7814 orthologs. Estimation of selection pressures using Ka/Ks ratios showed that only 30 of 5435 orthologous pairs had a ratio significantly >1, i.e., showing positive selection. However, 4041 orthologs had a Ka/Ks < 0.5 (p < 0.05), suggesting that most genes had likely undergone purifying selection. Based on orthologous unigenes, 314 single copy nuclear genes (SCNGs) were identified. Through a combination of de novo and reference guided assembly, plastid genomes were obtained; that of D. sinensis was 157,080 bp and that of D. dyeriana was 157,071 bp. Both plastid genomes encoded 87 protein coding genes, 40 tRNAs, and 8 rRNAs; no significant differences were detected in the size, gene content, and organization of the two plastomes. We used the whole chloroplast genomes to determine the phylogeny of D. sinensis and D. dyeriana and confirmed that the two species were highly divergent. Overall, our study provides comprehensive transcriptomic and chloroplast genomic resources, which will be valuable for future evolutionary studies of Dipteronia.

Comparative Analysis of the Complete Chloroplast Genome of Four Endangered Herbals of Notopterygium

Notopterygium H. de Boissieu (Apiaceae) is an endangered perennial herb endemic to China. A good knowledge of phylogenetic evolution and population genomics is conducive to the establishment of effective management and conservation strategies of the genus Notopterygium. In this study, the complete chloroplast (cp) genomes of four Notopterygium species (N. incisum C. C. Ting ex H. T. Chang, N. oviforme R. H. Shan, N. franchetii H. de Boissieu and N. forrestii H. Wolff) were assembled and characterized using next-generation sequencing. We investigated the gene organization, order, size and repeat sequences of the cp genome and constructed the phylogenetic relationships of Notopterygium species based on the chloroplast DNA and nuclear internal transcribed spacer (ITS) sequences. Comparative analysis of plastid genome showed that the cp DNA are the standard double-stranded molecule, ranging from 157,462 bp (N. oviforme) to 159,607 bp (N. forrestii) in length. The circular DNA each contained a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs). The cp DNA of four species contained 85 protein-coding genes, 37 transfer RNA (tRNA) genes and 8 ribosomal RNA (rRNA) genes, respectively. We determined the marked conservation of gene content and sequence evolutionary rate in the cp genome of four Notopterygium species. Three genes (psaI, psbI and rpoA) were possibly under positive selection among the four sampled species. Phylogenetic analysis showed that four Notopterygium species formed a monophyletic clade with high bootstrap support. However, the inconsistent interspecific relationships with the genus Notopterygium were identified between the cp DNA and ITS markers. The incomplete lineage sorting, convergence evolution or hybridization, gene infiltration and different sampling strategies among species may have caused the incongruence between the nuclear and cp DNA relationships. The present results suggested that Notopterygium species may have experienced a complex evolutionary history and speciation process.

The complete chloroplast genome sequence of Acer morrisonense (Aceraceae)

Abstract Acer morrisonense (Aceraceae) is an important forest tree species and is endemic to Taiwan area. In this study, we determined the complete nucleotide sequence of A. morrisonense chloroplast genome (cpDNA) by using next-generation sequencing. The cpDNA was 157 197 bp in size, contained a pair of 26 728 bp inverted repeat (IR) regions, which were separated by a large single copy region (LSC) and a small single copy (SSC) region of 85 655 and 18 086 bp, respectively. The cpDNA contained 134 genes, including 86 protein-coding genes (78 PCG species), 8 ribosomal RNA genes (four rRNA species), and 40 transfer RNA genes (31 tRNA species). The most of gene species occur as a single copy, while 21 gene species occur in double copies. The overall AT content of A. morrisonense chloroplast genome were 62.2%, while the corresponding values of the LSC, SSC, and IR regions were 64.0%, 67.9%, and 57.3%, respectively. The phylogenetic analysis suggested that the cpDNA of A. morrisonense is closely related to that of congeneric A. buergerianum subsp. ningpoense.

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).

Molecular Evolution of Chloroplast Genomes of Orchid Species: Insights into Phylogenetic Relationship and Adaptive Evolution

Orchidaceae is the 3rd largest family of angiosperms, an evolved young branch of monocotyledons. This family contains a number of economically-important horticulture and flowering plants. However, the limited availability of genomic information largely hindered the study of molecular evolution and phylogeny of Orchidaceae. In this study, we determined the evolutionary characteristics of whole chloroplast (cp) genomes and the phylogenetic relationships of the family Orchidaceae. We firstly characterized the cp genomes of four orchid species: Cremastra appendiculata, Calanthe davidii, Epipactis mairei, and Platanthera japonica. The size of the chloroplast genome ranged from 153,629 bp (C. davidi) to 160,427 bp (E. mairei). The gene order, GC content, and gene compositions are similar to those of other previously-reported angiosperms. We identified that the genes of ndhC, ndhI, and ndhK were lost in C. appendiculata, in that the ndh I gene was lost in P. japonica and E. mairei. In addition, the four types of repeats (forward, palindromic, reverse, and complement repeats) were examined in orchid species. E. mairei had the highest number of repeats (81), while C. davidii had the lowest number (57). The total number of Simple Sequence Repeats is at least 50 in C. davidii, and, at most, 78 in P. japonica. Interestingly, we identified 16 genes with positive selection sites (the psbH, petD, petL, rpl22, rpl32, rpoC1, rpoC2, rps12, rps15, rps16, accD, ccsA, rbcL, ycf1, ycf2, and ycf4 genes), which might play an important role in the orchid species’ adaptation to diverse environments. Additionally, 11 mutational hotspot regions were determined, including five non-coding regions (ndhB intron, ccsA-ndhD, rpl33-rps18, ndhE-ndhG, and ndhF-rpl32) and six coding regions (rps16, ndhC, rpl32, ndhI, ndhK, and ndhF). The phylogenetic analysis based on whole cp genomes showed that C. appendiculata was closely related to C. striata var. vreelandii, while C. davidii and C. triplicate formed a small monophyletic evolutionary clade with a high bootstrap support. In addition, five subfamilies of Orchidaceae, Apostasioideae, Cypripedioideae, Epidendroideae, Orchidoideae, and Vanilloideae, formed a nested evolutionary relationship in the phylogenetic tree. These results provide important insights into the adaptive evolution and phylogeny of Orchidaceae.

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.

Isolation and characterization of polymorphic microsatellite loci primers for Cupressus funebris (Cupressaceae)

Cupressus funebris Endl. (Cupressaceae) is an endemic conifer species in central and eastern China. In order to investigate the genetic diversity within and between populations and design the effective conservation strategies, we aimed to develop microsatellite markers for this species in the present study. We isolated and characterized 12 novel polymorphic microsatellite loci for this species through the combined biotin capture method. Polymorphism of each locus was further assessed through 62 individuals from three geographically distant populations. The number of alleles per locus ranged from 5 to 12. The observed and expected heterozygosity per locus ranged from 0.115 to 0.396, and 0.204 to 0.542, respectively. One locus (Cf07) showed significant deviation from Hardy–Weinberg equilibrium and no Linkage disequilibrium was detected after Bonferroni corrections. These microsatellite markers will be useful for population genetics studies of this conifer species.

The complete chloroplast genome of the Taiwan red pine Pinus taiwanensis (Pinaceae)

Abstract The complete nucleotide sequence of the Taiwan red pine Pinus taiwanensis Hayata chloroplast genome (cpDNA) is determined in this study. The genome is composed of 119,741 bp in length, containing a pair of very short inverted repeat (IRa and IRb) regions of 495 bp, which was divided by a large single-copy (LSC) region of 65,670 bp and a small single-copy (SSC) region of 53,080 bp in length. The cpDNA contained 115 genes, including 74 protein-coding genes (73 PCG species), 4 ribosomal RNA genes (four rRNA species) and 37 tRNA genes (22 tRNA species). Out of these genes, 12 harbored a single intron, and one (rps12) contained a couple of introns. The overall AT content of the Taiwan red pine cpDNA is 61.5%, while the corresponding values of the LSC, SSC and IR regions are 62.2%, 60.6% and 63.6%, respectively. A maximum parsimony phylogenetic analysis suggested that the genus Pinus, Picea, Abies and Larix were strongly supported as monophyletic, and the cpDNA of P. taiwanensis is closely related to that of P. thunbergii.

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 Chloroplast Genomics of Dipsacales Species: Insights Into Sequence Variation, Adaptive Evolution, and Phylogenetic Relationships

In general, the chloroplast genomes of angiosperms are considered to be highly conserved and affected little by adaptive evolution. In this study, we tested this hypothesis based on sequence differentiation and adaptive variation in the plastid genomes in the order Dipsacales. We sequenced the plastid genomes of one Adoxaceae species and six Caprifoliaceae species, and together with seven previously released Dipsacales chloroplasts, we determined the sequence variations, evolutionary divergence of the plastid genomes, and phylogeny of Dipsacales species. The chloroplast genomes of Adoxaceae species ranged in size from 157,074 bp (Sinadoxa corydalifolia) to 158,305 bp (Sambucus williamsii), and the plastid genomes of Caprifoliaceae varied from 154,732 bp (Lonicera fragrantissima var. lancifolia) to 156,874 bp (Weigela florida). The differences in the number of genes in Caprifoliaceae and Adoxaceae species were largely due to the expansion and contraction of inverted repeat regions. In addition, we found that the number of dispersed repeats (Adoxaceae = 37; Caprifoliaceae = 384) was much higher than that of tandem repeats (Adoxaceae = 34; Caprifoliaceae = 291) in Dipsacales species. Interestingly, we determined 19 genes with positive selection sites, including three genes encoding ATP protein subunits (atpA, atpB, and atpI), four genes for ribosome protein small subunits (rps3, rps7, rps14, and rps15), four genes for photosystem protein subunits (psaA, psaJ, psbC, and pabK), two genes for ribosome protein large subunits (rpl22 and rpl32), and the clpP, infA, matK, rbcL, ycf1, and ycf2 genes. These gene regions may have played key roles in the adaptation of Dipsacales to diverse environments. In addition, phylogenetic analysis based on the plastid genomes strongly supported the division of 14 Dipsacales species into two previously recognized sections. The diversification of Adoxaceae and Caprifoliaceae was dated to the late Cretaceous and Tertiary periods. The availability of these chloroplast genomes provides useful genetic information for studying taxonomy, phylogeny, and species evolution in Dipsacales.