Responses of pepper to waterlogging stress

One of the effective ways to address the effects of abnormal climate change on plant is to find germplasms that have better resistance to adverse environments. In this paper, we studied the responses of 5 pepper species Capsicum annuum L. (CA), C. baccatum L. (CB), C. chinense Jacquin. (CC), C. frutescens L. (CF) and C. pubescens Ruiz & Pavon (CP) as well as a wild pepper C. baccatum var. baccatum (CBY) to waterlogging stress. The results showed that warterlogging treatment greatly decreases photosynthetic pigment content, net photosynthetic rate (P _N) and stomatal conductance (g _s), and dramatically increases proline content and water-use efficiency (WUE) in all tested pepper, suggesting that pepper has weak resistance to waterlogging stress. The results also showed that changes of the above parameters vary in different species. CP had the smallest decreases in photosynthetic pigment content, P _N, and g _s and greatest increases in proline content and WUE. By contrast, CC had the greatest decreases in photosynthetic pigment content, P _N, and g _s and smallest increases in proline content and WUE, indicating that different species had different resistance to adverse environment and species CP and CC had the strongest and the weakest resistances, respectively. In addition, the study also demonstrated that wild pepper CBY had better resistance to adverse environment than all the tested species, indicating loss of the stress resistance genes during the process of domestication. Taking together, our study strongly suggests that pepper species should crossbreed with other species and wild pepper to expand genetic diversity, enlarge genetic distance, promote production, and improve the resistance to adverse environments.     

干旱对辣椒光合作用及相关生理特性的影响

采用盆栽实验研究了干旱对5个栽培种Capsicum annuum L.(CA),Capsicum baccatum L.(CB),Capsicum chinenseJacquin.(CC),Capsicum frutescens L.(CF)Capsicum pubescens Ruiz&Pavon(CP)和1个野生种Capsicum baccatum var.baccatum(CBY)的光合作用及相关生理特性的影响。结果发现,干旱条件下辣椒的光合色素下降幅度不大,复水后光合速率恢复较快,非光化学淬灭参数和保护酶活性大幅度上升,气孔导度和蒸腾速率显著下降,WUE和qN明显上升,这表明辣椒对干旱有较强的适应力;同时发现不同栽培种间的抗旱能力存在一定的差异,CB的光合色素、光合速率和气孔导度下降幅度最小,非光化学淬灭参数和水分利用率上升幅度最大,这表明CB比其他栽培种的抗旱能力强;研究同时发现,野生种Capsicum baccatum var.baccatum(CBY)的耐旱能力比5个栽培种都强。研究结果表明,辣椒育种应加强栽培种间以及栽培种与野生种之间的基因交流,达到提高产量和品种的抗逆能力的目的。     

Evaluation of six candidate DNA barcoding loci in Ficus (Moraceae) of China

Ficus, with about 755 species, diverse habits and complicated co‐evolutionary history with fig wasps, is a notoriously difficult group in taxonomy. DNA barcoding is expected to bring light to the identification of Ficus but needs evaluation of candidate loci. Based on five plastid loci (rbcL, matK, trnH‐psbA, psbK‐psbI, atpF‐atpH) and a nuclear locus [internal transcribed spacer (ITS)], we calculated genetic distances and DNA barcoding gaps individually and in combination and constructed phylogenetic trees to test their ability to distinguish the species of the genus. A total of 228 samples representing 63 putative species in Ficus (Moraceae) of China were included in this study. The results demonstrated that ITS has the most variable sites, greater intra‐ and inter‐specific divergences, the highest species discrimination rate (72%) and higher primer universality among the single loci. It is followed by psbK‐psbI and trnH‐psbA with moderate variation and considerably lower species discrimination rates (about 19%), whereas matK, rbcL and atpF‐atpH could not effectively separate the species. Among the possible combinations of loci, ITS + trnH‐psbA performed best but only marginally improved species resolution over ITS alone (75% vs. 72%). Therefore, we recommend using ITS as a single DNA barcoding locus in Ficus.     

Nucleotide polymorphism pattern and multiple maternal origin in Thinopyrum intermedium inferred by trnH-psbA sequences

Thinopyrum intermedium is an important species with potential utilization value in breeding of wheat. In this study, the non-coding intergenic region of trnH-psbA was investigated to assess the genetic diversity and infer the maternal origin within T. intermedium accessions. Eleven haplotypes were distinguished among the thirty-five accessions of T. intermedium. They showed a relatively low nucleotide diversity (π) of 0.00473 ± 0.00037 and a moderately high haplotype diversity (H_d) of 0.733 ± 0.061. In the phylogenetic analysis, all accessions of T. intermedium were positioned into two clades, which corresponded to the different diploid donors. These results suggested that there were two phylogenetically divergent maternal donors in T. intermedium.     

The architecture of the chloroplast psbA-trnH non-coding region in angiosperms

The psbA-trnH intergenic region is among the most variable regions in the angiosperm chloroplast genome. It is a popular tool for plant population genetics and species level phylogenetics and has been proposed as suitable for DNA barcoding studies. This region contains two parts differing in their evolutionary conservation: 1) the psbA 3′UTR (untranslated region) and 2) the psbA-trnH intergenic non-transcribed spacer. We compared the sequence and RNA secondary structure of the psbA 3′ UTR across angiosperms and found consensus motifs corresponding to the stem portions of the RNA stem-loop structures and a consensus TTAGTGTATA box. The psbA-trnH spacer exhibited patterns that can be explained by the independent evolution of large inversions in the psbA 3′UTR and mutational hot spots in the remaining portion of the psbA-trnH spacer. We conclude that a comparison of chloroplast UTRs across angiosperms offer clues to the identity of putative regulatory elements and information about selective constraints imposed on the chloroplast non-coding regions.     

Variation in the psbC gene region of gymnosperms and angiosperms as detected by a single restriction site polymorphism

 This paper reports on a PCR-RFLP analysis in a chloroplast DNA region consisting of coding and intergenic spacer sequences of trnS and the adjacent psbC gene. This region was PCR-amplified in 62 woody plant species, predominantly tree species, that represent a broad systematic range in both gymnosperms and dicotyledonous angiosperms. The amplification products were digested by the restriction endonuclease HaeIII (GG↓CC). Fourteen different restriction patterns occurred, 5 of which characterised representatives of the gymnosperms, and 9 angiosperm representatives. A single restriction site polymorphism revealed most of the species to share restriction patterns. Groups formed which showed relationships to plant systematic units. This phenomenon is discussed with regard to the psbC gene and the GGCC motif for tracing species’ relationships on a high taxonomic level of gymnosperms and angiosperms. Received: 5 June 1996/Accepted: 20 September 1996     

DNA barcoding and phylogeny in neotropical species of the genus Spondias

The genus Spondias belongs to the Anacardiaceae family, with about 18 species, having significant economic and social importance and with some species used in the agricultural industry, however, problems are encountered when trying to identify phylogenetic relationships among the species. The use of DNA barcoding is of importance to this group, allowing species identification at the molecular level and in determining the phylogenetic relationships within the group. The objective of this study is to obtain DNA barcoding and to determine the phylogenetic relationships among the species. For this, DNA from six species of the genus was extracted and amplified by PCR using sequences from the rbcL and matK genes and the trnH-psbA spacer gene, followed by sequencing using the Sanger method. The results show that the matK and rbcL genes cannot be used for DNA barcoding, because their discriminatory level between species is low. On the other hand, trnH-psbA shows a high level of discrimination, allowing most of the species to be identified. However it is not possible to separate Spondias venulosa and Spondias tuberosa. Phylogenetic analysis shows that Spondias mombim and S. tuberosa are distinct “umbucajá” clades, suggesting a non-hybrid origin for “umbucajá”.     

A molecular phylogeny of the genus Gagea (Liliaceae) in Germany inferred from non-coding chloroplast and nuclear DNA sequences

The systematics of the mainly yellow flowered Gagea species complex (Liliaceae) has long been considered difficult because only a few phenotypic features within this genus and as a result of hypothesized interspecific hybridisation. A molecular phylogenetic study of seven Gagea species (G. bohemica, G. lutea, G. minima, G. pomeranica, G. pratenis, G. spathacea and G. villosa) from Germany has been undertaken, based on plastid DNA sequences (trnL(UAA)-trnF(GAA), psbA-trnH) and on the nuclear ribosomal internal transcribed spacer (ITS). Sequence divergence within the Gagea species ranges up to 15.5% for psbA-trnH, 22.0% for trnL-trnF and 23.7% for ITS (ITS1 + 5.8S rRNA + ITS2). Two subspecies of Gagea bohemica: G. bohemica subsp. saxatilis and G. bohemica subsp. bohemica are characterized by trnL-trnF data and morphological features. Analysis of the ITS region shows that G. pomeranica represents a hybrid of G. pratensis and G. lutea. Lloydia serotina was initially used as an outgroup species, but it was placed within the investigated Gagea species in the psbA-trnH and the trnL-trnF phylogenetic tree.     

Molecular identification of species in Prunus sect. Persica (Rosaceae), with emphasis on evaluation of candidate barcodes for plants

Abstract Species of Prunus L. sect. Persica are not only important fruit trees, but also popular ornamental and medicinal plants. Correct identification of seedlings, barks, or fruit kernels is sometimes required, but no reliable morphological characters are available. Nowadays, the technique of DNA barcoding has the potential to meet such requirements. In this study, we evaluated the suitability of 11 DNA loci (atpB‐rbcL, trnH‐psbA, trnL‐F, trnS‐G, atpF‐H, rbcL, matK, rpoB, rpoC1, nad1, and internal transcribed spacer [ITS]) as candidate DNA barcodes for peaches, using samples from 38 populations, covering all the species in sect. Persica. On the whole, the primers worked well in this group and sequencing difficulties were met only in the case of ITS locus. Five loci (rbcL, matK, rpoB, rpoC, and nad1) have very low variation rates, whereas atpB‐rbcL, atpF‐H, trnH‐psbA, trnL‐F and trnS‐G show more variability. The most variable loci, atpB‐rbcL and trnH‐psbA, can distinguish three of the five species. Two two‐locus combinations, atpB‐rbcL+trnL‐F and atpB‐rbcL+atpF‐H, can resolve all five species. We also find that identification powers of the loci are method‐dependent. The NeighborNet method shows higher species identification power than maximum parsimony, neighbor joining, and unweighted pair group method with arithmetic mean methods.     

Phylogeography of Pulsatilla vernalis (L.) Mill. (Ranunculaceae): chloroplast DNA reveals two evolutionary lineages across central Europe and Scandinavia

Aim The aim of this study was to test hypotheses regarding some of the main phylogeographical patterns proposed for European plants, in particular the locations of glacial refugia, the post‐glacial colonization routes, and genetic affinities between southern (alpine) and northern (boreal) populations. Location The mountains of Europe (Alps, Balkans, Carpathians, Central Massif, Pyrenees, Scandinavian chain, Sudetes), and central European/southern Scandinavian lowlands. Methods As our model system we used Pulsatilla vernalis, a widely distributed European herbaceous plant occurring both in the high‐mountain environments of the Alps and other European ranges and in lowlands north of these ranges up to Scandinavia. Based on a distribution‐wide sampling of 61 populations, we estimated chloroplast DNA (cpDNA) variation along six regions using polymerase chain reaction–restriction fragment‐length polymorphisms (PCR–RFLPs) (trnH–trnK, trnK–trnK, trnC–trnD, psbC–trnS, psaA–trnS, trnL–trnF) and further sequencing of trnL–trnF and trnH–psbA. In addition, 11 samples of other European species of Pulsatilla were sequenced to survey the genus‐scale cpDNA variation. Results Eleven PCR–RFLP polymorphisms were detected in P. vernalis, revealing seven haplotypes. They formed two distinct genetic groups. Three haplotypes representing both groups dominated and were widely distributed across Europe, whereas the others were restricted to localized regions (central Alps, Tatras/Sudetes mountains) or single populations. Sequencing analysis confirmed the reliability of PCR–RFLPs and homology of haplotypes across their distribution. The chloroplast DNA variation across the section Pulsatilla was low, but P. vernalis did not share haplotypes with other species. Main conclusions The genetic distinctiveness of P. vernalis populations from the south‐western Alps with respect to other Alpine populations, as well as the affinities between the former populations and those from the eastern Pyrenees, is demonstrated, thus providing support for the conclusions of previous studies. Glacial refugia in the Dolomites are also suggested. Isolation is inferred for the high‐mountain populations from the Tatras and Sudetes; this is in contrast to the case for the Balkans, which harboured the common haplotype. Specific microsatellite variation indicates the occurrence of periglacial lowland refugia north of the Alps, acting as a source for the post‐glacial colonization of Scandinavia. The presence of different fixed haplotypes in eastern and western Scandinavia, however, suggests independent post‐glacial colonization of these two areas, with possible founder effects.     

Conservation genetics and geographic patterns of genetic variation of the endangered officinal herb Fritillaria pallidiflora

Fritillaria pallidiflora is an endangered officinal herb distributed in the Tianshan Mountains of northwestern China. We examined its phylogeography to study evolutionary processes and suggest implications for conservation. Six haplotypes were detected based on three chloroplast non‐coding spacers (psbA‐trnH, rps16, and trnS‐trnG); genetic variation mainly occurred among populations and SAMOVA groups. This species is distributed in different deep valleys, and we speculate that these fragmented habitats cause gene flow barriers among populations and groups. We also speculate that during glacial periods, extremely low temperatures and aridity caused additional range shrinkage and fragmentation, factors consequently resulting in significant intraspecific differentiation in allopatric regions. For setting a conservation management plan, we identified the Lucaogou region as the most important area, and we designated two ESUs for separate management.     

The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

山麦冬及其近缘种DNA条形码研究

通过分析山麦冬及其近缘种cpDNA trnL-F、psbA-trnH间隔区序列特点,探讨trnL-F、psbA-trnH序列作为山麦冬及其近缘种DNA条形码的潜力。分别对两者进行PCR扩增后,并进行纯化测序。其中山麦冬及其近缘种9个物种18个样品的trnL-F序列长度为355~356 bp,在山麦冬属内序列完全一致,但在沿阶草属中存在特异变异位点,可对麦冬与沿阶草两物种进行鉴别。而psbA-trnH序列长度为543~544 bp,仅在麦冬中存在特异鉴别位点。结果表明,trnL-F、psbA-trnH序列由于进化速率低、保守性强,仅适合麦冬类植物属间鉴别的DNA条形码,而在属下水平的应用有一定的局限。     

Phylogeography of a species complex of lowland Neotropical rain forest trees (Carapa,Meliaceae)

Aim Many tropical tree species have poorly delimited taxonomic boundaries and contain undescribed or cryptic species. We examined the genetic structure of a species complex in the tree genus Carapa in the Neotropics in order to evaluate age, geographic patterns of diversity and evolutionary relationships, and to quantify levels of introgression among currently recognized species. Location Lowland moist forests in the Guiana Shield, the Central and Western Amazon Basin, Chocó and Central America. Methods Genetic structure was analysed using seven nuclear simple sequence repeats (nuSSR), five chloroplast SSRs (cpSSR), and two chloroplast DNA (cpDNA) intergenic sequences (trnH–psbA and trnC–ycf6). Bayesian clustering analysis of the SSR data was used to infer population genetic structure and to assign 324 samples to their most likely genetic cluster. Bayesian coalescence analyses were performed on the two cpDNA markers to estimate evolutionary relationships and divergence times. Results Two genetic clusters (nu_guianensis and nu_surinamensis) were detected, which correspond to the Neotropical species C. guianensis (sensu latu) and C. surinamensis. Fourteen cpDNA haplotypes clustered into six haplogroups distributed between the two nuclear genetic clusters. Divergence between the haplogroups was initiated in the Miocene, with some haplotype structure evolving as recently as the Pleistocene. The absence of complete lineage sorting between the nuclear and chloroplast genomes and the presence of hybrid individuals suggest that interspecific reproductive barriers are incomplete. NuSSR diversity was highest in C. guianensis and, within C. guianensis, cpDNA diversity was highest in the Central and Western Amazon Basin. Regional genetic differentiation was strong but did not conform to an isolation‐by‐distance process or exhibit a phylogeographical signal. Main conclusions The biogeographical history of Neotropical Carapa appears to have been influenced by events that took place during the Neogene. Our results point to an Amazonian centre of origin and diversification of Neotropical Carapa, with subsequent migration to the Pacific coast of South America and Central America. Gene flow apparently occurs among species, and introgression events are supported by inconsistencies between chloroplast and nuclear lineage sorting. The absence of phylogeographical structure may be a result of the ineffectiveness of geographical barriers among populations and of reproductive isolation mechanisms among incipient and cryptic species in this species complex.     

The phylogeographic history of the self-pollinated herb Tacca chantrieri (Dioscoreaceae) in the tropics of mainland Southeast Asia

The geological and climatic oscillations influenced the geographic distribution and demography of most present-day species, but few studies have investigated evolutionary history of species adapted to the tropical regions of Southeast Asia. Here, using sequence datasets obtained from three chloroplast DNA fragments (trnH-psbA, trnS-trnG, and trnL-F) from 320 individuals belonging to 24 natural populations, we investigated the phylogeographical history of Tacca chantrieri, which inhabits Southeast Asian tropical forests. Although relatively high level of differentiation among the populations were observed, mismatch distribution and neutrality tests showed no evidence of recent demographic population expansion. Phylogenetic inference exhibited two identified population groups showing a disjunctive distribution of dominant haplotypes. The split in cpDNA was largely consistent with the Tanaka line and Red River geographically. Molecular clock estimations revealed that the two lineages diverged during Pleistocene approximately 1.16 Ma. Therefore, the disjunct distribution of T.chantrieri could be explained by both the vicariance caused by Red River as well as ecological barriers caused by the different monsoon climates (Southwest monsoon vs. Southeast monsoon) that developed during the Pleistocene. The Tanaka line can be considered as a climatically driven barrier that influenced present-day plant dispersal.     

Phylogeography and population genetics of Acanthophyllum squarrosum complex (Caryophyllaceae) in the Irano-Turanian region

Acanthophyllum squarrosum and two closely related species, A. heratense and A. laxiusculum (Caryophyllaceae), form a complex that covers parts of subalpine steppes of the Irano-Turanian (IT) region. In this study, we explored the genetic structure and phylogeography of this complex based on partial sequences of two chloroplasts (psbA–trnH and rpl32–trnL (UAG)) and two nuclear (EST24 and nrITS) DNA regions. We analysed 80 individuals from eight populations and detected 12 chloroplast haplotypes, 16 and eight nuclear alleles in EST24 and nrITS sequences, respectively. Phylogenetic trees and haplotype networks did not show distinct genetic groups in the complex and this could be explained by incomplete lineage sorting or introgression between species. Divergence time analysis revealed a Quaternary origin for A. squarrosum complex at approximately 1.8 million years ago (Mya) and the neutrality test results indicated that this complex experienced a recent population expansion. AMOVA analysis of the chloroplast regions showed a significant genetic differentiation among populations and low genetic differentiation within populations, but opposite results were found with nuclear markers, implying introgression between A. squarrosum complex populations.     

Molecular phytogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes

We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes — frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.     

Comparative analysis of the constituents of two cultivars of Dioscorea dumetorum (Kunth) Pax. and their molecular barcoding

The edible and wild cultivars of Dioscorea dumetorum (Kunth) Pax. (Family: Dioscoreaceae) are commonly used as close substitutes in herbal markets due to their edibility and medicinal importance. Comparative study of the cultivars was done using chemical analysis of their methanol tuber extracts and by utilizing the DNA barcode regions, ribulose-1, 5-bisphosphate carboxylase subunits (rbcL) and the non-coding intergenic spacer gene (trnH-psbA) for nucleotide sequences comparison of cultivars. Of the 54 compounds identified from the methanol extract of the edible and wild cultivars using gas chromatography-mass spectrometry (GC-MS), 12 are similar, while 42 are remarkably different. In particular n-hexadecanoic (palmitic) acid had a higher percentage area in the edible cultivar (31.16%) compared to the wild cultivar (0.26%). The cultivars were successfully amplified using the universal primers gene rbcL and trnH-psbA. Their nucleotide sequences showed a slight variation when aligned with CodonCode Aligner V.9.0.1. They were identified by the Basic Local Alignment Search Tool (BLAST) through comparison with Genbank data, which shows close similarities with submitted data. The use of molecular barcoding in identifying the cultivars of Dioscorea dumetorum as well as the chemical composition of their tubers may form an important marker in the compilation of future herbal pharmacopoeia for its proper identification.     

Principal features of the cpDNA evolution in Taraxacum (Asteraceae, Lactuceae): a conflict with taxonomy

Phylogenetic relationships based on the chloroplast genome of Taraxacum were studied. Representative samples of 44 sections or species groups and a number of isolated species were analyzed. On the basis of the sequence variation in psbA–trnH and in trnL–trnF, mutations associated with RFLPs were monitored. Five RFLPs without homoplasy were recognized and used to reconstruct four main cpDNA groups (haplotypes); Group I is ancestral and, contrary to the information in the primary sequences, the RFLPs were not distinct from those of the outgroup species of Agoseris and Prenanthes. This group corresponds to dandelions believed to be ancestral on the basis of morphological data and previous studies of the chloroplast genome. A comparison of parsimony analysis of morphological and chloroplast data showed an overall lack of congruence. The conflict can most probably be accounted for as a consequence of reticulation.