atpB-rbcL Intergenic Spacer Research Articles

Genetic variability and phylogenetic relationships of Begonia multangula based on atpB-rbcL non-coding spacer of cpDNA sequences

Abstract. Warseno T, Efendi M, Chasani AR, Daryono BS. 2022. Genetic variability and phylogenetic relationships of Begonia multangula based on atpB-rbcL non-coding spacer of cpDNA sequences. Biodiversitas 23: 5491-5501. Begonia (Begoniaceae) belongs to Section Platycentrum-Sphenanthera Group which has a wide distribution from Sumatra to the Lesser Sunda Islands and Sulawesi. Information on B. multangula genetic variability and intraspecies relationships based on molecular characters is critical for developing appropriate strategies in conservation biology, breeding activities, and many other applied fields. The genetic variability and interspecific relationships of B. multangula in Indonesia were investigated using sequence data from the atpB-rbcL intergenic spacer (IGS) cpDNA regions. The atpB-rbcL IGS fragment was amplified using atpB-1 as the forward primer and rbcL-1 as the reversed primer. Genetic variations were found in the length of the sequence and nucleotide divergences in the atpB-rbcL IGS region. The genetic distance between 822 fixed sites ranged from 0 to 0.61%. Eighteen of the 822 sites (99.27%) analyzed were invariable, six sites (0,73%) were variable consisting of 4 singleton variable sites and two parsimony informative sites, and twelve sites were gaps. The phylogenetic relationships generated by B. multangula based on the atpB-rbcL IGS sequence analysis indicated the genetic variation and divided it into two clades. However, the clustering pattern of B. multangula specimens resulting from molecular analysis based on atpB-rbcL IGS sequences did not show the geographic clustering grouping pattern.

Genetic variability and phylogenetic relationships of Begonia multangula based on atpB-rbcL non-coding spacer of cpDNA sequences

Abstract. Warseno T, Efendi M, Chasani AR, Daryono BS. 2022. Genetic variability and phylogenetic relationships of Begonia multangula based on atpB-rbcL non-coding spacer of cpDNA sequences. Biodiversitas 23: 5491-5501. Begonia (Begoniaceae) belongs to Section Platycentrum-Sphenanthera Group which has a wide distribution from Sumatra to the Lesser Sunda Islands and Sulawesi. Information on B. multangula genetic variability and intraspecies relationships based on molecular characters is critical for developing appropriate strategies in conservation biology, breeding activities, and many other applied fields. The genetic variability and interspecific relationships of B. multangula in Indonesia were investigated using sequence data from the atpB-rbcL intergenic spacer (IGS) cpDNA regions. The atpB-rbcL IGS fragment was amplified using atpB-1 as the forward primer and rbcL-1 as the reversed primer. Genetic variations were found in the length of the sequence and nucleotide divergences in the atpB-rbcL IGS region. The genetic distance between 822 fixed sites ranged from 0 to 0.61%. Eighteen of the 822 sites (99.27%) analyzed were invariable, six sites (0,73%) were variable consisting of 4 singleton variable sites and two parsimony informative sites, and twelve sites were gaps. The phylogenetic relationships generated by B. multangula based on the atpB-rbcL IGS sequence analysis indicated the genetic variation and divided it into two clades. However, the clustering pattern of B. multangula specimens resulting from molecular analysis based on atpB-rbcL IGS sequences did not show the geographic clustering grouping pattern.

<strong><em>Impatiens bullatisepala</em> (Balsaminaceae), a new species from Guizhou, China</strong>

Impatiens bullatisepala (Balsaminaceae), a new species supported by morphological and phylogenetic evidence from Fanjing Mountain, Guizhou province in China, is described here. It is morphologically similar to I. davidii but can be distinguished by its dorsally ridged lateral sepals with sunk reticulate veins and bullate projections on abaxial surface, 2–2.5 cm deep saccate lower sepal with ca. 0.8 cm long narrowly triangular tip at the mouth, and broadly ovate dorsal petal. Phylogenetic analyses of a combined dataset of nuclear ITS and plastid atpB-rbcL intergenic spacer DNA sequences furtherly confirmed its novelty.

A molecular phylogeny of the genus Drimia (Asparagaceae: Scilloideae: Urgineeae) in India inferred from non-coding chloroplast and nuclear ribosomal DNA sequences

The evolutionary history of the medicinally important bulbous geophyte Drimia (subfamily: Scilloideae) has long been considered as a matter of debate in the monocot systematics. In India the genus is represented by a species complex, however, the taxonomic delimitation among them is ill-defined till date. In the present study, a comprehensive phylogenetic relationship among Indian species of this genus has been inferred for the first time based on chloroplast DNA trnL intron, rps16-trnK intergenic spacer, atpB-rbcL intergenic spacer and ribosomal DNA ITS1-5.8S-ITS2 sequences, leaf morphology, anatomy, stomatal characteristics and pollen exine ornamentations. The present findings revealed the monophyletic origin of the Indian members of Drimia and grouped them into two possible lineages (clade- I and II). The phylogenetic tree based on cpDNA concatenated sequences further resolved the clade-I into two distinct subclades (I and II) and clarified the intraspecies relationship among the studied members. The present study suggested a strong relationship between the molecular phylogeny and the morphological characteristics of the species studied. A possible trend of evolution of two important traits: ‘type of palisade cells’ in leaf and ‘pollen exine patterns’ among the members of Drimia in India was also suggested.

PLACEMENT OF Syzygium boerlagei (Merr.) Govaerts (MYRTACEAE) CONFIRMED WITH ATPB-RBCL INTERGENIC SPACER

A molecular analysis was conducted to determine whether Eugenia boerlagei Merr. (Myrtaceae) belongs to genus Eugenia or Syzygium based on sequences of cpDNA fragments namely atpB-rbcL intergenic spacer. The study used seven specimens of Syzygium sect. Jambosa, three of Syzygium sect. Syzygium, two of Eugenia s.s. and one of Eugenia boerlagei Merr. with Baeckea ovalifolia and B. tuberculata as the outgroup. The results show that Eugenia boerlagei is appropriately placed under the genus Syzygium.

Molecular phylogenetic data and seed coat anatomy resolve the generic position of some critical Chenopodioideae (Chenopodiaceae - Amaranthaceae) with reduced perianth segments.

The former Chenopodiumsubgen.Blitum and the genus Monolepis (Chenopodioideae) are characterised in part by a reduced (0–4) number of perianth segments. According to recent molecular phylogenetic studies, these groups belong to the reinstated genera Blitum incl. Monolepis (tribe Anserineae) and Oxybasis (tribe Chenopodieae). However, key taxa such as C.antarcticum, C.exsuccum, C.litwinowii, C.foliosumsubsp.montanum and Monolepisspathulata were not included and so their phylogenetic position within the Chenopodioideae remained equivocal. These species and additional samples of Blitumasiaticum and B.nuttallianum were incorporated into an expanded phylogenetic study based on nrDNA (ITS region) and cpDNA (trnL-trnF and atpB-rbcL intergenic spacers and rbcL gene). Our analyses confirm the placement of C.exsuccum, C.litwinowii and C.foliosumsubsp.montanum within Blitum (currently recognised as Blitumpetiolare, B.litwinowii and B.virgatumsubsp.montanum, respectively); additionally, C.antarcticum, currently known as Oxybasisantarctica, is also placed within Blitum (reinstated here as B.antarcticum). Congruent with previous studies, two of the three accepted species of Monolepis – the type species M.trifida (= M.nuttalliana) as well as M.asiatica – are included in Blitum. The monotypic genus Carocarpidium described recently with the type C.californicum is not accepted as it is placed within Blitum (reinstated here as B.californicum). To date, few reliable morphological characters have been proposed that consistently distinguish Blitum (incl. two Monolepis species) from morphologically similar Oxybasis; however, two key differences are evident: (1) the presence of long-petiolate rosulate leaves in Blitum vs. their absence in Oxybasis and (2) a seed coat structure with the outer wall of the testa cells lacking stalactites (‘non-stalactite seed coat’) but with an obvious protoplast in Blitum vs. seed coat with the outer walls of the testa cells having stalactites (‘stalactite seed coat’) and a reduced protoplast in Oxybasis. Surprisingly, the newly sequenced North American Monolepisspathulata nested within the tribe Dysphanieae (based on ITS and trnL-trnF + rbcL + atpB-rbcL analyses).The phylogenetic results, as well as presence of the stalactites in the outer cell walls of the testa and lack of the rosulate leaves, confirm the distinctive nature of Monolepisspathulata from all Blitum and, therefore, the recent combination Blitumspathulatum cannot be accepted. Indeed, the morphological and molecular distinctive nature of this species from all Dysphanieae supports its recognition as a new monotypic genus, named herein as Neomonolepis (type species: N.spathulata). The basionym name Monolepisspathulata is also lectotypified on a specimen currently lodged at GH. Finally, while Micromonolepispusilla is confirmed as belonging to the tribe Chenopodieae, its position is not fully resolved. As this monotypic genus is morphologically divergent from Chenopodium, it is retained as distinct but it is acknowledged that further work is required to confirm its status.

Phylogenetic positions of seven poorly known species of Ferula (Apiaceae) with remarks on the phylogenetic utility of the plastid trnH-psbA, trnS-trnG, and atpB-rbcL intergenic spacers

Ferula L. is one of the most species-rich and taxonomically difficult genera of Apiaceae. In this study, we obtained nrDNA ITS sequences of seven poorly known species of Ferula (Ferula anatolica, Ferula sp. (tentatively identified as F. candelabrum by collectors), F. drudeana, F. huber-morathii, F. marmarica, F. talassica, and F. tunetana) and explored their phylogenetic positions using 148 ITS sequences of the subtribe Ferulinae from GenBank. Five of these newly sequenced species fall into three groups, corresponding to clades recognized in earlier molecular studies. Ferula sp. are added to clade, which is mostly composed of Central Asian species. This placement showed that identification as F. candelabrum was erroneous. The second clade, which is mostly composed of Mediterranean taxa, includes two species from North Africa: F. marmarica and F. tunetana. Despite the well-supported monophyly of this clade, the relationships inside this group need to be revised, as broadly distributed F. communis is paraphyletic with respect to other species. Ferula drudeana and F. huber-morathii, two narrow endemics from Turkey, are placed in the Central Asian clade. Two species, F. anatolica and F. talassica, do not fall into any of the recognized clades. In addition, we examined the sequence variation of three potentially highly variable pDNA regions, the trnH-psbA, trnS-trnG, and atpB-rbcL intergenic spacers, for a subset of 18 specimens. The resulting pDNA and ITS based phylogenetic trees were incongruent, as supported by significant ILD tests. The cause of this incongruence can be manifold, including hybridization, a lack of a phylogenetic signal, and homoplastic substitutions. Our analyses suggest that only trnS-trnG can be added to the list of pDNA markers used for phylogenetic studies of Ferula, as it has the highest number of parsimony informative characters and is easy to amplify from degraded material.

Three new species of Impatiens (Balsaminaceae) from Myanmar

Three new species of Impatiens (Balsaminaceae) from Myanmar are here described: I. decurva Ruchis. & S.B. Janssens, I. hartnolliae Hook. f. ex Ruchis. & Suksathan, and I. oblongata Ruchis. &Van der Niet. The 5-lobed short fusiform fruit of all three species suggests that they are members of subgen. Impatiens sect. Uniflorae Hook. f. & Thomson. For I. decurva and I. oblongata, subgenus membership was corroborated by phylogenetic analyses of a combined dataset of nuclear ITS and plastid atpB-rbcL intergenic spacer DNA sequences. This was not possible for I. hartnolliae, which is only known from a single herbarium specimen.

DNA Barcodes Confirm the Taxonomic and Conservation Status of a Species of Tree on the Brink of Extinction in the Pacific.

The taxonomic status of a single island, narrow range endemic plant species from Palau, Micronesia (Timonius salsedoi) was assessed using DNA barcode markers, additional plastid loci, and morphology in order to verify its conservation status. DNA barcode loci distinguished T. salsedoi from all other Timonius species sampled from Palau, and were supported by sequence data from the atpB-rbcL intergenic spacer region. Timonius salsedoi was only known from two mature individual trees in 2012. Due to its extremely narrow range and population size, it had previously been recommended to be listed as Critically Endangered Status under three separate IUCN Criteria. In 2014 a second survey of the population following a typhoon revealed that the only two known trees had died suggesting that this species may now be extinct. Comprehensive follow up surveys of suitable habitat for this species are urgently required.

Molecular phylogenetic relationships of the Andean genus Aylostera Speg. (Cactaceae, Trichocereeae), a new classification and a morphological identification key

The Andes represent a hotspot of diversity within Cactaceae. Owing to its small globular growth and its diverse flower colours the Andean genus Aylostera Speg. has drawn the attention of many cactus enthusiasts. Based on molecular data, the genus was recently split from Rebutia s.l. In contrast to the large number of taxa described within Aylostera, a comprehensive phylogenetic reconstruction is still warranted. The aim of our study was to reconstruct a phylogeny of Aylostera and to review its taxonomy based on morphological characters. We sequenced the chloroplast DNA markers atpB-rbcL and trnS-trnG intergenic spacers and scored amplified fragment lengths polymorphisms (AFLPs). We reconstructed phylogenetic trees and analysed results of AFLP neighbour-nets, Bayesian clustering and Principal Coordinate Analysis based on this genetic data. Our data demonstrated that Aylostera is monophyletic and is best recognized as a separate genus because it is only distantly related to the clade containing the type species of Rebutia. Within Aylostera we detected three clades—the Digitorebutia clade, the Aylostera sensu stricto clade and the A. einsteinii clade—which are characterized by unique combinations of morphological character states. The species composition of these clades was largely congruent between the molecular markers but AFLP data indicated some admixture. Based on genetic and morphological characters, we propose a new classification of Aylostera and present a key to species recognizing nine species.

Phylogenetic Analyses of Molecular Data and Reconstruction of Morphological Character Evolution in Asian <I>Impatiens</I> section <I>Semeiocardium</I> (Balsaminaceae)

Phylogenetic analyses have revealed extensive evolutionary lability of morphological characters in the large genus Impatiens. This potentially complicates taxonomic classifications, especially if these are based on analyses which include a limited number of characters and taxa. In this study we focus on the systematics of previously poorly represented Impatiens from Southeast Asia, a hotspot of species diversity, using an expanded taxon sampling. Specifically we implement phylogenetic analyses of DNA sequences from the nuclear ITS and plastid atpB-rbcL intergenic spacer and reconstruct the evolution of several morphological characters to study Semeiocardium, a taxon which has been recognized as a distinct genus, subgenus, and section in previous taxonomic treatments. Both Bayesian and Parsimony analyses demonstrated that Impatiens subg. Semeiocardium is not monophyletic. Thirteen representatives are part of a large clade, which also includes many other Southeast Asian taxa. Connate lateral united petals are a synapomorphy of this clade, while a four-locular ovary and connate outer lateral sepals are not. Impatiens muscicola and I. santisukii, which were previously classified under I. subg. Semeiocardium, do not belong to the clade with connate lateral united petals or to I. sect. Semeiocardium. Due to ambiguous combinations of morphological characters we could not determine whether sampled species belonged to I. sect. Semeiocardium. In addition to the characters that were used to diagnose I. sect. Semeiocardium, we identify connate lateral united petals as a taxonomically useful character to distinguish an additional monophyletic clade in I. sect. Semeiocardium. Further research is needed to identify diagnostic characters for the sister clade comprising I. stenosepala and its allies.

Phylogenetic Relationships in the Genus Rosa Revisited Based on rpl16, trnL-F, and atpB-rbcL Sequences

Three chloroplast DNA (cpDNA) sequences [the rpl16 intron, the trnL-F, and atpB-rbcL intergenic spacer (IGS)] were employed to study phylogenetic relationships in the genus Rosa. Phylogenetic analyses using these three concatenated sequences were performed using maximum parsimony (MP) and Bayesian inference (BI) methods. Both analyses results suggest that the molecular phylogeny conforms closely to the conventional classification of botanical sections. Morphological similarities between R. sects. Synstylae and Chinenses, and R. sects. Rosa and Carolinae are corroborated on the molecular level in our analyses. Four taxa from R. sect. Pimpinellifoliae are further divided into two small clades, which reflect the morphological characters for these species on a molecular level. Whereas three accessions of R. foetida from R. sect. Pimpinellifoliae form a separate clade. R. ×fortuniana forms a clade with R. laevigata based on its maternal inheritance of cpDNA. R. ×cooperii is hypothesized to be a hybrid with seed parent from R. sects. Synstylae and Chinenses. And R. roxburghii should be classified as a section within the R. subgen. Rosa, rather than being treated as its own subgenus, based on molecular analyses.

Three New Species of <I>Mormolyca</I> (Orchidaceae: Maxillariinae) with an Updated Molecular Phylogenetic Analysis

Abstract Three Neotropical orchid species, Mormolyca dodsonii, Mormolyca cruentata, and Mormolyca prunina are described on the basis of molecular and morphological characters. Phylogenetic relationships are estimated using sequence data from five plastid loci (rpoC1, matK gene and flanking trnK intron, atpB-rbcL intergenic spacer, and the 3′ portion of ycf1) plus the nuclear ribosomal internal and external transcribed spacers (ITS, ETS), and a portion of the low-copy nuclear gene phyC. Phylogenetic analyses confirm the monophyly of Mormolyca and clarify relationships within the genus: an early divergent paraphyletic grade of species diverging from the line leading to a major clade of two distinctive subclades. The three new species are contained within these subclades: M. dodsonii and M. cruentata clustering with the subclade characterize by flowers with a labellum pad of short glandular papillae; whereas M. prunina groups with the clade that includes all species with glossy labella.

Epimedium tianmenshanensis (Berberidaceae), a new species from central China

A distinctive, previously undescribed species of Epimedium (Berberidaceae), which we propose naming E. tianmenshanensis, was found during a floristic survey in Hunan, China. It is similar to E. baojingense and E. franchetii, but has smaller flowers and petals with a short, blunt spur. We determined the phylogenetic position of the new species using DNA sequences of nuclear ITS and two chloroplast regions (atpB-rbcL intergenic spacer and matK). Molecular evidence indicates that E. tianmenshanensis occupies a distinct group related to E. baojingense.

Molecular phylogenetics of <i>mormolyca</i> (orchidaceae: maxillariinae) based on combined molecular data sets

The Neotropical orchid genus Mormolyca Fenzl, as currently circumscribed, encompasses a diverse group of ca. 27species. Many of these were included traditionally in Maxillaria sect. Rufescens, when similarity of floral morphology was considered foremost in their classification rather than the evolutionary history of the taxa. In order to begin revising species delimitation and clarifying the evolution and biology of the genus, we present a phylogenetic hypothesis using sequence data from five plastid loci (rpoC1, matK gene and flanking trnK intron, atpB-rbcL intergenic spacer, and the 3’ portion of ycf1) and the nuclear ribosomal internal and external transcribed spacers (ITS, ETS). Resulting trees using both Bayesian and parsimony inference are congruent with each other, and generally well resolved. Based on current level of sampling across Maxillariinae, these molecular data support the monophyly of Mormolyca and shed light on the interspecific phylogenetic patterns within the genus. These include an early divergent paraphyletic grade of Mormolyca species successively sister to a clade with at least two definable subclades within. The latter are characterized by two different flower morphologies that are likely related to their pollination systems. Although not all relationships within the genus are fully resolved or supported, these results offer a first glimpse into the phylogeny of a small group of epiphytic orchids characterized by an unusually high level of variable vegetative characters, floral fragrance profiles, and pollination systems.

Phylogeny and Biogeography of <I>Dendropanax</I> (Araliaceae), an Amphi-Pacific Disjunct Genus Between Tropical/Subtropical Asia and the Neotropics

Dendropanax (Araliaceae) is a genus of about 80 species disjunctly distributed in tropical to subtropical Asia and the Neotropics, showing an amphi-Pacific tropical disjunction. The phylogeny of the genus was constructed by sampling 95 accessions representing 33 species of Dendropanax and 43 closely related taxa using sequences of the nuclear ribosomal internal transcribed spacer (ITS) region and six plastid regions (the ndhF gene, the trnL-trnF region, the rps16 intron, the atpB-rbcL intergenic spacer, the rpl16 intron, and the psbA-trnH intergenic spacer). Phylogenetic analyses of the combined plastid and ITS data suggested that the monophyly of Dendropanax was not supported because the Asian D. lancifolius - D. hainanensis clade did not group with the main Dendropanax clade. Nevertheless, the maximally parsimonious trees (MPTs) from the analysis constraining Dendropanax as a monophyletic group were only one step longer than the unconstrained MPTs. The New and Old World Dendropanax species except D. lancifolius and D. hainanensis each formed a robustly supported clade, and the two clades were sister to each other. Based on the biogeographic analyses and fossil-calibrated Bayesian dating, Dendropanax was hypothesized to have originated in the Old World and migrated into the New World via the North Atlantic land bridges in the early Tertiary. The amphi-Pacific intercontinental disjunction of Dendropanax was dated to be 41.83 mya with a 95% high posterior density [HPD] interval of 28.46–56.15 mya.

Low‐copy nuclear primers and ycf1 primers in Cactaceae

To increase the number of variable regions available for phylogenetic study in the Cactaceae, primers were developed for a portion of the plastid ycf1 gene and intron-spanning regions of two low-copy nuclear genes (isi1, nhx1). • Primers were tested on several families within Caryophyllales, focusing on the Cactaceae. Gel electrophoresis indicated positive amplification in most samples. Sequences of these three regions (isi1, nhx1, ycf1) from Harrisia exhibited variation similar to or greater than two plastid regions (atpB-rbcL intergenic spacer and rpl16 intron). • The isi, nhx, and ycf1 primers amplify phylogenetically useful information applicable to the Cactaceae and other families in the Caryophyllales.

Inferring Multiple Refugia and Phylogeographical Patterns in Pinus massoniana Based on Nucleotide Sequence Variation and DNA Fingerprinting

Background Pinus massoniana, an ecologically and economically important conifer, is widespread across central and southern mainland China and Taiwan. In this study, we tested the central–marginal paradigm that predicts that the marginal populations tend to be less polymorphic than the central ones in their genetic composition, and examined a founders' effect in the island population.Methodology/Principal FindingsWe examined the phylogeography and population structuring of the P. massoniana based on nucleotide sequences of cpDNA atpB-rbcL intergenic spacer, intron regions of the AdhC2 locus, and microsatellite fingerprints. SAMOVA analysis of nucleotide sequences indicated that most genetic variants resided among geographical regions. High levels of genetic diversity in the marginal populations in the south region, a pattern seemingly contradicting the central–marginal paradigm, and the fixation of private haplotypes in most populations indicate that multiple refugia may have existed over the glacial maxima. STRUCTURE analyses on microsatellites revealed that genetic structure of mainland populations was mediated with recent genetic exchanges mostly via pollen flow, and that the genetic composition in east region was intermixed between south and west regions, a pattern likely shaped by gene introgression and maintenance of ancestral polymorphisms. As expected, the small island population in Taiwan was genetically differentiated from mainland populations.Conclusions/SignificanceThe marginal populations in south region possessed divergent gene pools, suggesting that the past glaciations might have low impacts on these populations at low latitudes. Estimates of ancestral population sizes interestingly reflect a recent expansion in mainland from a rather smaller population, a pattern that seemingly agrees with the pollen record.

Development of PCR primer sets for intron 1 of the low‐copy gene LEAFY in Davalliaceae

Primers were designed for amplifying intron 1 of the single-copy nuclear LEAFY gene for species of Davalliaceae. New primer sets were designed and successfully amplified for intron 1 of the LEAFY gene in 13 species representing the five genera of Davalliaceae. The orthology of these sequences was further confirmed by phylogenetic analyses. Site variation in LEAFY intron 1 sequences across genera of the Davalliaceae and among accessions of the Humata repens complex were 18% and 8%, respectively. Such variation was greater than that for the cpDNA atpB-rbcL intergenic spacer region across the same taxa and accessions. Using our newly designed primers, intron 1 of the LEAFY gene could be amplified for all species tested. In addition, this single-copy, biparentally inherited, and quickly evolving region showed considerable potential for addressing infraspecific-level questions.

Application of high-resolution melting for variant scanning in chloroplast gene atpB and atpB-rbcL intergenic spacer region of Crucifer species

High-resolution melting (HRM) analysis is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In this study, a novel HRM assay was carried out to detect SNPs in the chloroplast gene atpB which encodes the beta subunit of the ATP synthase and atpB upstream intergenic region. The polymorphisms of the two fragments in intertribal samples from the Cruciferae family and within the species of Brassica napus were detected. Based on this results, we found that HRM were able to determine over 90% of the variants which included single or multiple variants and insertion-deletion polymorphisms (INDELs) and rendered possible genotyping of more closely spaced polymorphisms, although there were several false positives (FPs) and misclassification. Six haplotypes were identified in the intertribal materials. The analysis of 90 B. napus found five variation types and the variations were all located in the intergenic region. In conclusion, HRM analysis is a closed tube assay that is easy to perform and is a more effective approach to identify variant of chloroplast genes. This study will facilitate further functional investigations into the role of chloroplast genes in photosynthesis, phylogeny and molecular evolution.