Non-coding Regions Of Chloroplast DNA Research Articles

Phylogeographic study based on intraspecific sequence variation of chloroplast DNA for the conservation of genetic diversity in the Japanese endangered species Primula sieboldii

To gather information on intraspecific phylogeography for use in conservation programs for the endangered species Primula sieboldii in Japan, we analyzed sequence variation in five noncoding regions of chloroplast DNA. Twenty-two distinct haplotypes were recognized in total. The distribution of most haplotypes was geographically confined, but one haplotype was widely distributed throughout northern Japan, and several haplotypes were found in geographically distant regions. Three major clades were revealed by phylogenetic analysis of the haplotypes. Clade I was distributed in Kyushu and central Honshu, clade II in western Honshu and Hokkaido, and clade III from central Honshu to Hokkaido. According to analysis of molecular variance, 59.9% of the total cpDNA variation existed among regions, 32.5% among populations within regions, and 7.6% within populations. Therefore, if genetic conservation of the species is valued, transplanting of P. sieboldii among regions should be avoided. Multiple lineages often existed even in geographically narrow areas (e.g., within a 20-km range), so transplantation between adjacent populations in restoration activities should be carefully designed so as not to change the gene pool of local populations significantly. Also, the geographical distribution of cpDNA haplotypes may allow us to confirm the origin of plants collected for commercial purposes.

Phylogenetic and evolutionary relationships between Elymus humidus and other Elymus species based on sequencing of non-coding regions of cpDNA and AFLP of nuclear DNA.

Species of the genus Elymus are closely related to some important cereal crops and may thus serve as potential alien genetic resources for the improvement of these crops. E. humidus is indigenous to Japan and is well adapted to a humid climate. However, the phylogenetic and evolutionary relationships between E. humidus and other Elymus species are unclear. To elucidate these relationships, we examined the sequences of three non-coding regions of chloroplast DNA (cpDNA) and the amplified fragment length polymorphism (AFLP) variation of nuclear DNA in E. humidus and other related species. A total of 15 sequence mutations from the three non-coding regions, trnL-trnF, trnF- ndhJ(C), and atpB-rbcL, covering approximately 1,800 bp, were detected in the Elymus species. A phylogenic tree resulting from the cpDNA sequence data revealed that all the species containing the St nuclear genome (St, StH, StY, and StHY) formed a well-supported clade that is remote from the Hordeum species (H). This result strongly supports the finding that Pseudoroegneria is the maternal genome donor to the genus Elymus. In addition, E. humidus showed the closest relationship with the cpDNA genome of the Pseudoroegneria species. The AFLP analysis detected 281 polymorphic bands with 11 AFLP primer combinations. The AFLP result showed that E. humidus is relatively closer to E. tsukushiensis. However, the cpDNA sequencing results indicated that E. humidus and E. tsukushiensis have different cytoplasmic origins. Our results suggest that the evolutionary process between E. humidus and E. tsukushiensis is not monophyletic, although the two species have similar morphological characters and adaptability.

A rapid method for identifying the origin of North AmericanPhragmites populations using RFLP analysis

The common reed,Phragmites australis, is a common feature in wetlands across North America. Recent studies have suggested that the widespread invasions of this species may be due to the introduction of a non-native strain from EurAsia. Since native population types is needed to facilitate management of ulations are also found, a method for distinguishing population persist in many areas where introduced populations are also found, a method for distinguishing population types is needed to facilitate management of the species. A restriction fragment length polymorphism (RFLP) assay was developed to distinguish native, non-native, and Gulf Coast type populations ofPhragmites from each other. Two amplified non-coding chloroplast DNA regions are each cut with one restriction enzyme, allowing the distinction of native, non-native and Gulf Coast haplotypes from each other. When used together, these cut sites provide a low cost, rapid way of determining the origin ofPhragmites populations.

Allopolyploidization and evolution of species with reduced floral structures in Lepidium L. (Brassicaceae).

Understanding the pattern of speciation in a group of plants is critical for understanding its morphological evolution. Lepidium is the genus with the largest variation in floral structure in Brassicaceae, a family in which the floral ground plan is remarkably stable. However, flowers in more than half of Lepidium species have reduced stamen numbers, and most of these also have reduced petals. The species with reduced flowers are geographically biased, distributed mostly in the Americas and Australia New Zealand. Previous phylogenetic studies using noncoding regions of chloroplast DNA and rDNA internal transcribed spacer were incongruent in most New World species relationships. These data, combined with the presence of many polyploid Lepidium species, implied a reticulate history of the genus but did not provide enough information to infer the evolutionary pattern of flower structures. To address this question more thoroughly, sequences of the first intron of a single copy nuclear gene, PISTILLATA, were determined from 43 species. Phylogenetic analysis of the PI intron suggests that many species in the New World have originated from allopolyploidization, and that this is correlated with floral reduction. Interspecific hybrids were generated to understand why allopolyploidization is associated with reduced flowers. The phenotypes of F1 flowers indicate allelic dominance of the absence of lateral stamens, suggesting that propagation of dominant alleles through interspecific hybridization could account for the abundance of the allopolyploid species without lateral stamens.

Nunatak survival of the high Alpine plant Eritrichium nanum (L.) Gaudin in the central Alps during the ice ages.

Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and sequence analysis of noncoding regions of chloroplast DNA were used to investigate 37 populations of Eritrichium nanum covering its total distribution area, the European Alps. There was no haplotypic variation within the populations, and most haplotypes were restricted to single sites or to neighbouring populations, suggesting low levels of long distance gene flow via seeds. The present geographical distribution of haplotypes probably reflects an ancient geographical pattern within two regions in the intensely glaciated western and eastern central Alps identified as genetic hotspot areas. These two regions contained seven of the total of 11 haplotypes, including many of the most derived ones. The divergent haplotypes formed closely related groups, which supported a separate evolution of these haplotypes in these two regions and, more importantly, gave strong evidence for the in situ survival of these populations on nunataks within the western and eastern central Alps during Pleistocene glaciation. This result is in concordance with a previous study on E. nanum using nuclear markers. Only one haplotype was common and widespread throughout the distributional range of E. nanum. At the same time, it was the evolutionarily basal-most and all other haplotypes were best described as its descendants. This haplotype is hypothesized to be genetically identical to a Tertiary Alpine colonizing ancestor, whose distribution was secondarily fragmented and infiltrated by derived haplotypes originating through local mutations.

The influence of adjacent nucleotides on the pattern of nucleotide substitution in mitochondrial introns of angiosperms.

It has been known that in noncoding regions of the chloroplast genome, the pattern of nucleotide substitution is influenced by the two nucleotides flanking the substitution site. In a GC-rich environment, a bias toward transition was observed, whereas in an AT-rich environment, a bias toward transversion was observed. In this study, the influence of the two adjacent neighbors on the substitution pattern was observed in the first intron of the mitochondrial nad4 gene, although the AT content of this intron is only 48%. The proportion of transversions increases from 0.32 to 0.75 as the A + T content (number of A's + T's) of the two nearest neighbors increases from 0 to 2. This trend was also observed in another mitochondrial group I intron with an AT content of 64%. In addition, a similar, though weaker, effect was observed in vertebrate pseudogenes. So this effect is present in all three types of genomes. Furthermore, in contrast to the situation in the noncoding regions of chloroplast DNA, where most nucleotide substitutions occurred in the categories with an A + T content of either 1 or 2, nucleotide substitutions in the mitochondrial first nad4 intron occurred more evenly in three categories of different A + T contents. This might be due largely to the difference in the AT content (0.48 vs. 0.72) between the mitochondrial first nad4 intron and the chloroplast DNA regions studied.

Phylogeny and classification of Naucleeae s.l. (Rubiaceae) inferred from molecular (ITS, rBCL, and tRNT‐F) and morphological data

Parsimony analyses of the tribe Naucleeae sensu lato (s.l.) using the noncoding internal transcribed spacer (ITS) regions of nuclear rDNA, the protein-coding rbcL and noncoding trnT-F regions of chloroplast DNA, and morphological data were performed to construct new intratribal classification, test the monophyly of previous subtribal circumscriptions, and evaluate the generic status of Naucleeae s.l. Fifty-two ITS, 45 rbcL, and 55 trnT-F new sequences are published here. Our study supports the monophyly of the subtribes Anthocephalidae, Mitragynae, Uncariae all sensu Haviland and Naucleinae sensu Ridsdale. There was no support for Cephalanthidae sensu Haviland and Adininae sensu Ridsdale. Naucleeae can be subdivided into six highly supported and morphologically distinct subtribes, Breoniinae, Cephalanthinae, Corynantheinae, Naucleinae, and Mitragyninae, Uncarinae, plus one, Adininae, which is poorly supported. The relationships among these subtribes were largely unresolved. We maintain the following 22 genera: Adina, Adinauclea, Breonadia, Breonia, Burttdavya, Cephalanthus, Gyrostipula, Haldina, Janotia, Ludekia, Metadina, Mitragyna, Myrmeconauclea, Nauclea, Neolamarckia, Neonauclea, Ochreinauclea, Pausinystalia, Pertusadina, Sarcocephalus, Sinoadina, and Uncaria. Pseudocinchona is reestablished. Corynanthe is restricted to C. paniculata and Hallea is reincluded in Mitragyna. Our results were inconclusive for assessing the relationships among Adina, Adinauclea, Metadina, and Pertusadina due to lack of resolution.

A study of the phylogeny of Brassica rapa, B. nigra, Raphanus sativus, and their related genera using noncoding regions of chloroplast DNA

There are two evolutionary lineages in the genus Brassica: the rapa/oleracea lineage and the nigra lineage. Using nuclear DNA sequences such as the intergenic spacer between 5S rRNA genes and the internal transcribed spacer between 18S and 25S rRNA genes, we and others had previously demonstrated that Raphanus sativus is closely related to the nigra lineage. In the present study, we sequenced the chloroplast noncoding region between trnT and trnF and that between trnD and trnT in seven species and showed that R. sativus is more closely related to the rapa/oleracea lineage than to the nigra lineage. The conflicting results from nuclear DNA and chloroplast DNA support the hypothesis that Raphanus was derived from a hybridization between the rapa/oleracea and the nigra lineages. We estimated the date of this hybridization event to be 60% of the divergence time between the two Brassica lineages. In addition, the pattern and rate of nucleotide substitution were studied. There were more transversions than transitions in these noncoding regions, which have a high AT content. Furthermore, the proportion of transversions among the substitutions at a site increases with increasing A + T content of its two adjacent nucleotides. An influence of immediate 5 ′ pyrimidine on substitution pattern is also observed when both adjacent bases in the two DNA strands are A or T. The rate of nucleotide substitution in the trnL group I intron is only about one third of the rate in the nearby intergenic spacers in the trnT– trnF fragment. The rate of nucleotide substitution in the rapa/oleracea lineage is at least 1.5 times that in the nigra lineage.

Phylogeographical structure revealed by chloroplast DNA variation in Japanese Beech (Fagus crenata Blume)

Intraspecific genetic variation in three non-coding chloroplast DNA (cpDNA) regions (trnT-L and trnL-F spacers, and trnL intron) of Japanese beech (Fagus crenata Blume) was investigated. This species is a major constituent of the typical cool-temperate deciduous forests in Japan. Twenty-one F. crenata populations from throughout Japan, and four F. japonica populations, a close relative of F. crenata, were examined. Seven haplotypes were distinguishable in F. crenata based on nucleotide substitutions and indels. Pairwise nucleotide diversities among haplotypes ranged from 0.0000 to 0.0042 for F. crenata, including F. japonica. The geographical distribution of cpDNA haplotypes was found to be highly structured in F. crenata. Four haplotypes predominated: haplotypes FC1 and FC4 are prevalent on the Pacific Ocean coast, haplotype FC6 is prevalent on the Japan sea coast from the San-in district to Hokkaido, whilst haplotype FC3 is restricted to northern Kyushu and the western-most part of Honshu. Two haplotypes (FC5 and FC7) are restricted to single populations and one haplotype (FC2) is a derivative of FC1. Each of these haplotypes, except FC2, are thought to be derived from different glacial refugia. Phylogenetic analysis showed that neither F. crenata nor F. japonica was monophyletic for the haplotypes, suggesting either ancestral polymorphism or ancient introgression between the lineages of these two Fagus species.

Cryptic invasion by a non-native genotype of the common reed, Phragmites australis, into North America.

Cryptic invasions are a largely unrecognized type of biological invasion that lead to underestimation of the total numbers and impacts of invaders because of the difficulty in detecting them. The distribution and abundance of Phragmites australis in North America has increased dramatically over the past 150 years. This research tests the hypothesis that a non-native strain of Phragmites is responsible for the observed spread. Two noncoding chloroplast DNA regions were sequenced for samples collected worldwide, throughout the range of Phragmites. Modern North American populations were compared with historical ones from herbarium collections. Results indicate that an introduction has occurred, and the introduced type has displaced native types as well as expanded to regions previously not known to have Phragmites. Native types apparently have disappeared from New England and, while still present, may be threatened in other parts of North America.

Distribution of chloroplast DNA variation in British oaks (Quercus robur and Q. petraea): the influence of postglacial colonisation and human management

Variation in the non-coding region of chloroplast DNA (cpDNA) was studied to determine the route and pattern of postglacial recolonisation of native oak throughout mainland Britain. In total, 1076 mature oak trees of either Quercus petraea (Matt.) Liebl. or Quercus robur L. from 224 British ancient woodland sites were analysed, and represent the largest and most saturating molecular analysis of British oak undertaken to date. The majority of oaks (98%) possessed one of the three cpDNA haplotypes from lineage B that are commonly found in Spain and western regions of France. Thus, our findings strongly support the hypothesis that most native British Q. robur and Q. petraea originate from a Pleistocene refugium in the Iberian Peninsula. A haplotype previously thought to be the result of a de novo postglacial mutation in East Anglia, Native and Non-native in British Forestry. Proceedings of a Discussion Meeting, March 31–April 2, 1995. University of Warwick, pp. 38–55] was found at several sites in eastern Britain, including major population clusters in East Anglia, Tyneside and northeast Scotland. We propose that this haplotype was introduced to Britain via normal postglacial colonisation processes. Less than 2% of oak trees sampled possessed haplotypes from lineages which originate from more eastern refugia (Italy and Balkans). Whilst human-mediated translocation may be responsible for the occurrence of these haplotypes in Britain, single long distance dispersal events (of between 300 and 500km from France) cannot be excluded for at least one case. The majority of populations sampled (including those where both species are present) were fixed for a single haplotype (69%). However, significant areas of mixed haplotype woods occurred in the Welsh Marches and Scotland and the proportion of population fixation was lower and intra-population diversity higher for Q. robur (GST=0.661; hS=0.224) than for Q. petraea (GST=0.884; hS=0.089). The distribution of cpDNA variation in British oak populations is discussed and interpreted in the light of hypothesised recolonisation routes and dynamics, inter-specific hybridisation and chloroplast capture, and human impacts due to the management of ancient woodland.

Chloroplast DNA Variation and Biogeography in the Genus Rorippa Scop. (Brassicaceae)

Abstract: Phylogenetic relationships and biogeography of 25 Rorippa species were studied using sequences of two non‐coding regions of chloroplast DNA (trnL intron, trnL/F spacer). Our results indicate a close relationship between European (R. islandica ssp. islandica, R. pyrenaica) and North American (R. curvipes, R. sinuata) mountain species. The polyploid European lowland species R. amphibia, R. palustris and R. sylvestris are much younger than the mountain species and have their closest relatives in western Asia and Siberia. Different colonization routes of the southern hemisphere are discussed for Rorippa. Australasia was colonized at least twice, most likely via the Malayan route. A molecular clock approach dates the first colonization to the end of Pliocene or early Pleistocene. R. gigantea reached Australia later in the Pleistocene. Our data provide evidence for an amphitropical disjunction between the South American (R. philippiana) and North American (R. curvisiliqua) species. Long‐distance dispersal via migrating birds is the most likely explanation for this intercontinental disjunction. Two of the analysed African species (R. nudiuscula, R. madagascariensis) have their closest relative (R. austriaca) in eastern Europe and western Asia. The lack of sequence divergence among these species indicates a colonization event probably not earlier than 100 000 years ago.

A set of primers for amplification of noncoding regions of chloroplast DNA in the grasses

AbstractTwelve primer pairs, which amplify noncoding regions of chloroplast DNA, were designed for grass species. Their universality was tested on the six major grass subfamilies, and several were sequenced to examine their variability in Phragmites australis. The primers amplified well for most species tested and the majority showed intraspecific variation.

Sequence variation of non-coding regions of chloroplast DNA of soybean and related wild species and its implications for the evolution of different chloroplast haplotypes

Soybean chloroplast DNAs (cpDNAs) are classified into three types (I, II and III) based on RFLP profiles. Type I is mainly observed in cultivated soybean (Glycine max), while type II and type III are frequently found in both cultivated and wild soybean (Glycine soja), although type III is predominant in wild soybean. In order to evaluate the diversity of cpDNA and to determine the phylogenetic relationship among different chloroplast types, we sequenced nine non-coding regions of cpDNA for seven cultivated and 12 wild soybean accessions with different cpDNA types. Eleven single-base substitutions and a deletion of five bases were detected in a total of 3849 bases identified. Five mutations distinguished the accessions with types I and II from those with type III, and seven were found in the accessions with type III, independently of their taxa. Four species of the subgenus Glycine shared bases that were identical to those with types I and II at two of the five mutation sites and shared bases that were identical to those with type III at the remaining three sites. Therefore, the different cpDNA types may not have originated monophyletically, but rather may have differentiated from a common ancestor in different evolutionary directions. A neighbor-joining tree resulting from the sequence data revealed that the subgenus Soja connected with Glycine microphylla which formed a distinct clade from Clycine clandestina and the tetraploid cytotypes of Glycine tabacina and Glycine tomentella. Several informative length mutations of 54 to 202 bases, due to insertions or deletions, were also detected among the species of the genus Glycine.

Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA

Genetic diversity and relationships of 40 accessions of Ipomoea, representing ten species of series Batatas, were examined using ISSR markers and restriction-site variation in four non-coding regions of chloroplast DNA. A total of 2071 ISSR fragments were generated with 15 primers in these accessions and, on average, 52 bands per accession were amplified. Most of the primers contained dinucleotide repeats. The ISSR fragments were highly polymorphic (62.2%) among the 40 accessions studied. Restriction analysis of chloroplast (cp) DNA revealed 47 informative restriction-site and length mutations. Phylogenetic analyses of ISSR and cpDNA datasets generally revealed similar relationships at the interspecific level, but the high polymorphism of ISSRs resulted in a better separation of intraspecific accessions. However, the combined ISSR and cpDNA dataset appeared to be appropriate in resolving both intra- and interspecific relationships. Of the species examined, I. trifida was found to be the most closely related to cultivated sweetpotato, the hexaploid I.batatas, while I. ramosissima and I. umbraticola were the most distantly related to I. batatas within the series. Ipomoea triloba, hitherto considered to be one of the ancestors of sweetpotato, was only distantly related to sweetpotato based on ISSR similarity index.

Comparison of Sequence of rbcL and Non-coding Regions of Chloroplast DNA and ITS2 Region of rDNA in Genus Humulus.

The divergence among hop cultivars, as well as wild hops (Humulus lupulus var. lupulus, neomexicanus and cordifolius) and related species (H.japonicus) was studied using the sequence difference of the partial rbcL fragment, the noncoding regions, the trnL intron and spacer between the trnL3' exon and trnF, of chloroplast DNA and the ITS2, one of the nuclear ribosomal internal transcribed spacer regions. There were no differences within the varieties of H.lupulus and within H.japonicus. A few nucleotide substitutions were observed between the varieties of H.lupulus in the 1260 bp nucleotide sequence of rbcL or the 853 bp total of the noncoding regions of choroplast DNA. There were no nucleotide changes in the 232 bp ITS2, even though this region showed a higher substitution rate when H.lupulus and H.japonicus were compared. H.japonicus showed 13 substitutions in rbcL, 9 in the noncoding regions and 24 in the ITS2 region compared with H.lupulus var.lupulus. Synonymous substitution at tne 3rd codon position of rbcL enabled to estimate the divergence time of the two species at 3.74 million years ago. This study which revealed a low variability within H.lupulus suggests that variation, which could be utilized in hop breeding is limited.

Intraspecific variation in Cannabis sativa L. based on intergenic spacer region of chloroplast DNA.

We analyzed the nucleotide sequences of the non-coding region of chloroplast DNA: the intergenic spacer between trnL (UAA) 3'exon and trnF (GAA). Two kinds of sequence, "type-1" and "type-2," were detected in 33 populations of Cannabis sativa. The length of the "type-1" fragment was 354 bp. In contrast, the "type-2" fragment from 3 populations was 353 bp long, with only one base deletion compared to "type-1." The fragment length from Humulus lupulus was 353 bp with a 1-bp deletion, and ten 1-bp substitutions compared to the sequences from C. sativa "type-1." Furthermore, we could clearly identify differences between C. sativa and H. lupulus using single-strand conformation polymorphism of PCR products (PCR-SSCP) analysis.

Amplification of oak DNA from ancient and modern wood.

A polymorphic noncoding region of chloroplast DNA (cpDNA) was successfully amplified by the polymerase chain reaction (PCR) from various oak wood samples, including recent and more ancient (about 600-years-old) samples from different oak species. Adaptation of DNA isolation and amplification protocols was necessary to obtain this result. Polymorphisms useful to distinguish species or geographical origin of these samples could be scored through sequencing. These polymorphisms include one substitution and two microsatellite-type polymorphisms, due to a variable number of A/T repeats. Identical results were obtained independently in two separate laboratories.

Further Analysis of Intraspecific Sequence Variation of Chloroplast DNA in Primula cuneifolia Ledeb. (Primulaceae): Implications for Biogeography of the Japanese Alpine Flora

Ledeb. (Primulaceae), we analyzed intraspecific variation of the nucleotide sequences of non-coding regions of chloroplast DNA: the intergenic spacers between trnT (UGU) and the trnL (UAA) 5′exon, the trnL (UAA) 3′exon and trnF (GAA), and atpB and rbcL. In 20 populations of P. cuneifolia, 22 nucleotide substitutions and five insertions/deletions were inferred, and their genetic distances ranged from 0.001 to 0.008. Eight distinct haplotypes could be recognized and each haplotype was found to be geographically structured. Three major clades (the Northern, Hokkaido and Southern clades) were revealed in phylogenetic analyses of the haplotypes. The haplotypes of the Northern clade had a wider distribution area in the populations of Mt. Rausu and Rishiri Island of eastern and northern Hokkaido in Japan, northward to Unalaska Island in the Aleutians, and those of the Hokkaido clade were distributed in the populations of central Hokkaido and Mt. Iwaki of the northern Honshu in Japan; in addition, those of the Southern clade were observed only in the populations of the central Honshu. It was shown that the genetic diversifications of the Southern clade were higher than those of the Northern and Hokkaido clades. Furthermore, it was shown that the topology within the Southern clade was hierarchical, and the haplotypes of the Southern populations in the clade were derivative. From these results, we concluded that the cpDNA haplotypes of the three clades in P. cuneifolia arose and assumed the present distribution areas through several cycles of glacial advance and retreat in the Pleistocene.

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.