Primer-enzyme Combinations Research Articles

Creation of gene pools with amplified fragment length polymorphis markers for development of winter oilseed rape (Brassica napus L.) hybrid cultivars

The genetic diversity of two winter oilseed rape (Brassica napus L.) collections was investigated using amplified fragment length polymorphism marker technology. The first collection of 78 genotypes was analyzed using 11 primer-enzyme combinations and PCR products were resolved by 13.35% denaturing polyacrylamide gels. The second collection was investigated using seven fluorescent-labeled AFLP primer combinations and PCR products were separated using capillary electrophoresis. A total of 262 polymorphic AFLP markers were obtained for the first collection and 423 polymorphic markers for the second. On the basis of all markers, principal component analysis was performed for both collections separately. The unweighted pair group method with arithmetic method based on the coefficient of dissimilarity separated the genotypes in collection 1 into two clusters and those in the second collection into three distinct clusters. Resynthesized lines formed a cluster that was clearly distinct from 42 winter oilseed rape parental lines in the cytoplasmic male sterility ogura system. Analysis of molecular variance showed that 79% and 81% of the detected variation was found to be within the groups (in the two collections, respectively), while the variation between groups contributed, respectively, to only 21% and 19% of the variance. Our results indicate that AFLP technology can be useful for the creation of a gene pool of parental components of winter oilseed rape hybrid cultivars. Additionally, de novo resynthesized Brassica napus lines provide a significant opportunity for enrichment of the gene pool of winter rapeseed.

Characterization of strawberry cultivars by SSR and CAPS markers

This study was performed to reveal the value of genetic relationship among 34 Turkish and foreign strawberry cultivars and genotypes using CAPS and SSR DNA molecular markers. Seventeen primer-enzyme combinations in CAPS and 12 SSR primer pairs in SSR analysis were used. In CAPS analysis, 60 bands were produced and 52 of them were polymorphic. In SSR analysis, 150 alleles were generated and 145 of them were polymorphic. In the analysis of combined CAPS and SSR data, genetic similarity ranged between 0.59 and 0.91. 'Call Giant-2' and 'Call Giant-3' (0.91) were the closest cultivars, while Genotype 8 and Call Giant-4 were the most distant genotypes. As a result, SSR was better than CAPS from the point of view of polymorphism rates and polymorphism information contents, whereas CAPS was better than SSR from the point of view of resolving power values, in the characterization of strawberry cultivars and genotypes. The result of this study may allow us to design a cultivar breeding program in strawberry.

Organellar Genome Diversity in <i>Saccharum</i> and <i>Erianthus</i> spp. revealed by PCR-RFLP

The organellar genome diversity in Saccharum and Erianthus species was analysed by chloroplast deoxyribonucleic acid (cpDNA) and mitochondrial deoxyribonucleic acid (mtDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Six different chloroplast primers ( psbC - trnS , trnL , psaA , clpP , matK and ccsA ); and ten mitochondrial primers ( nad 1, nad 4/1-2, nad 4/2-3, nad 5/1-2, nad 5/4-5, 18S-5SrRNA , coxI , matR , cob and mttb ) were used to amplify the genes/intergenic spacers of 8 different members of Saccharum complex namely S . officinarum , S. robustum , S. spontaneum , S. barberi , E. arundinaceus, E. ciliaris , E. elegans and CoC 671 ( S . officinarum hybrid). The amplified PCR-products were digested with ten different restriction enzymes namely Alu I, BamH I, Bgl II, Dra I, EcoR I, Hae III, Hind III, Hinf I and Pst I, Taq I. Our results suggest that although monomorphic bands were observed with the PCR using chloroplast and mitochondrial primers; there exists restriction fragment length polymorphism in these genes/intergenic spacers. Out of the sixty primer-enzyme combinations studied, thirty primer-enzyme combinations revealed cpPCR-RFLP while out of hundred primer-enzyme combinations studied fifty-seven primer-enzyme combinations revealed mtPCR-RFLP in sugarcane. Differentiation in Saccharum and Erianthus species was found in the psbC - trnS region of the chloroplast digested using enzyme Hae III and also in the trnL region digested using enzyme Taq I. One new finding in our work was the mtPCR-RFLP revealed by nad 4/2-3region restriction digested using enzyme Alu I which was able to differentiate Saccharum species and Erianthus species. Our results may add to the knowledge about organellar genome diversity in sugarcane and may be useful for identification of sugarcane hybrids.

Identification of native Dendrobium species in Thailand by PCR-RFLP of rDNA-ITS and chloroplast DNA

The PCR-restriction fragment length polymorphism (PCR-RFLP) approach was successfully developed to identify 25 native Dendrobium species in Thailand. PCR-RFLP of the rDNA-ITS with six restriction enzymes and three chloroplast (cp) DNA regions with five primer-enzyme combinations produced 24 types of DNA patterns altogether. Twenty-three out of the 25 species determined in this study were found to belong to unique classes and were successfully differentiated. Two species, D. crumenatum and D. formosum, possessing the same DNA pattern, however, were identified after cutting the chloroplast DNA fragment amplified by psbC-trnS primer with MboI enzyme. An effective procedure for identifying each Dendrobium species was developed. PCR-RFLP of the rDNA-ITS with TaqI, which is the most informative enzyme, was used for the early detection of 16 Dendrobium species. To identify the remaining Dendrobium species, PCR- RFLP analysis was performed using one more primer-enzyme combination. Our study provides a rapid, simple, and reliable identification method for these Dendrobium species.

English

Fifty-four Cymbidium kanran cultivars from China, Japan and South Korea were examined and analyzed by using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) markers to determine their molecular diversity and relationships. In chloroplast (cp) PCR-RFLP analyses, genetic differences were revealed in 4 of 6 primer sets (66.67%) and 19 of 72 primer-enzyme combinations (26.38%), 116 polymorphic bands were detected. For mitochondrion (mt) PCR-RFLP markers, genetic differences were revealed in 2 of 8 primer sets (25%) and 55 polymorphic bands (53.49%) were detected with 7 restriction primer-enzyme combinations. According to the PCR-RFLP marker profiling data, all the cultivars were classified into four groups. The group 1 has included three subclusters that two of them were generally consistent with geography classii¬cation. Subcluster 1 and Subcluster 2 were mainly composed of Chinese cultivars. Comparably, Subcluster 3 was composed of two cultivars which originated from Japan separately. Group 2 comprised Huangchengzhiyue and Xiongnu originated from Japan. Group 3 was included in seven Chinese cultivars and five Japanese cultivars. Group 4 was composed of eight Chinese cultivars and four Japanese cultivars. Therefore, we demonstrated that the PCR-RFLP technique could provide a powerful tool for cultivar identification and establishment of genetic relationships of cultivars in cymbidium kanran. Key words: Cymbidium kanran, chloroplast (cp) polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), mitochondrion (mt) PCR-RFLP.

Characterization and mapping of QTL used in breeding of Scots pine (Pinus sylvestris L.)

This paper reports the construction a map b ased on Amplified Fragment Length Polymorphic DNA (AFLP) in Scots pine (Pinus sylvestris L.). The main purpose of map construction was its application to quantitative traits loci (QTL) mapping for breeding traits economically important in Scots pine breeding program such as tree height and diameter at breast height, number of needles and their length, width, and area. Genomic DNA of needles and haploid megagamethophytes from seeds originating from a sing le tree were amplified with 25 AFLP primer-enzyme combinations with three or four selective nucleotides. Sixteen of them generated easily readable patterns and revealed a poly morphism. Each analyzed marker was tested for the expected 1 : 1 seg regation ratio using χ 2 – test and only 6 were significant with (α ≤ 0.05). The total map size equaled 291,7 cM an d all markers were distributed within one linkage group. For all traits only one QTL associated with tree height (H) was detected.

Assessment of Genetic Diversity in Zingiberaceae Through Nucleotide Binding Site-Based Motif-Directed Profiling

Functional motif-directed profiling was performed with 15 nucleotide binding site (NBS) primer-enzyme combinations to identify and elucidate the phylogenetic relationships among 15 genotypes of the family Zingiberaceae. We retrieved 167 polymorphic bands (24.85 %), with an average of 11.13 bands per primer. Mean polymorphism rates were detected using MseI (26 %), RsaI (21 %), and AluI (28 %) as restriction enzymes. The polymorphism information content (PIC) for each NBS primer-enzyme combination ranged from 0.48 to 0.76 with a mean value of 0.65. The 38 NBS profiling markers had PIC values ranging from 0.3 to 0.6 and exhibited good power to discriminate between genotypes. Comparison of NBS profiling with microsatellite data for the same set of genotypes exhibited a correlation value of 0.78, P ≤ 0.001. Our study suggests that genetic variability assessment could be more efficient if it targeted genes that exhibit functionally relevant variation, rather than random markers.

Genetic relationships among wild and cultivated blackberries (Rubus caucasicus L.) based on amplified fragment length polymorphism markers

Rubus is a large genus of flowering plants in the rose family, Rosaceae, subfamily Rosoideae. The blackberries, as well as various other Rubus species with mounding or rambling growth habits, are often called brambles. Little information is available on the genetic diversity of wild-grown blackberries. The objective of this study was to determine the genetic relationships among nine promising (high-yield capacity, free of pest and diseases, better fruit traits) wild blackberry (Rubus caucasicus L.) selections and the well-known cultivar, “Chester” by using amplified fragment length polymorphism (AFLP) markers. Genotypes were evaluated with three selective primer-enzyme combinations, producing a total of 223 AFLP fragments with 53% polymorphism ratio. Clustering of genotypes using unweighted pair-group method of arithmetic average cluster analysis clearly separated groups of wild blackberry genotypes while the variety “Chester” was clustered independently. Wild selections represented a distinct germplasm source on the basis of the estimated genetic distance among them. Genetic diversity data from this study will be helpful in using and exploiting the wild genetic material for breeding purposes as well as for further research.

Profile of Belgian Pediatric Crohn's Disease (CD) Patients: Associations Between Variables at Diagnosis

GCAP. CONCLUSIONS: The present method could reveal a trend of different intestinal microflora between UC patients' family and UC patients. We believe that a further study including more factors for analyses is warranted to better understand the significance of intestinal microflora in the immunopathogenesis of IBD. REFERENCES 1. Osborn, AM, et al. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ Microbiol 2000;2:3950. 2. Nagashima K, et al. Application of new primer-enzyme combinations to terminal restriction fragment length polymorphism profiling of bacterial populations in human feces. Appl Environ Microbiol 2003; 69:1251-62. 3. Suzuki Y, et al. Selective granulocytes and monocyte adsorptive apheresis as a first-line treatment for steroid naive patients with active ulcerative colitis. Dig Dis Sci 2004;49:565-71.

Molecular marker-based prediction of hybrid performance in maize using unbalanced data from multiple experiments with factorial crosses

In hybrid breeding, the prediction of hybrid performance (HP) is extremely important as it is difficult to evaluate inbred lines in numerous cross combinations. Recent developments such as doubled haploid production and molecular marker technologies have enhanced the prospects of marker-based HP prediction to accelerate the breeding process. Our objectives were to (1) predict HP using a combined analysis of hybrids and parental lines from a breeding program, (2) evaluate the use of molecular markers in addition to phenotypic and pedigree data, (3) evaluate the combination of line per se data with marker-based estimates, (4) study the effect of the number of tested parents, and (5) assess the advantage of haplotype blocks. An unbalanced dataset of 400 hybrids from 9 factorial crosses tested in different experiments and data of 79 inbred parents were subjected to combined analyses with a mixed linear model. Marker data of the inbreds were obtained with 20 AFLP primer-enzyme combinations. Cross-validation was used to assess the performance prediction of hybrids of which no or only one parental line was testcross evaluated. For HP prediction, the highest proportion of explained variance (R (2)), 46% for grain yield (GY) and 70% for grain dry matter content (GDMC), was obtained from line per se best linear unbiased prediction (BLUP) estimates plus marker effects associated with mid-parent heterosis (TEAM-LM). Our study demonstrated that HP was efficiently predicted using molecular markers even for GY when testcross data of both parents are not available. This can help in improving greatly the efficiency of commercial hybrid breeding programs.

Comparison of Genetic Structure Assessed by Amplified Fragment Length Polymorphism and Retrotransposon-based Sequence-specific Amplification Polymorphism for Portuguese Populations of Pinus pinea L.

AbstractIn order to assess genetic diversity within and among populations of Pinus pinea L. (stone pine), seven Portuguese populations originating from three Provenance Regions were selected and genotyped using two marker systems. We compared the genetic variation of these populations using retrotransposon-based sequence-specific amplification polymorphism (SSAP) and amplified fragment length polymorphism (AFLP). In total, 105 trees were screened with three primer enzyme combinations (PEC), producing 232 SSAP and 132 AFLP loci. Where SSAP yielded approximately twice-the number of polymorphic fragments compared to AFLP. Differentiation was slightly higher for SSAP, than for AFLP (FST = 0.105 for SSAP and 0.074 for AFLP), and both significantly different from zero, P < 0.01. The levels of average genetic diversity within-population found with the two types of marker were not significantly different between SSAPs and AFLPs (26.6% and 22.8%, respectively). The populations that displayed the highest and lowest genetic diversity scores were the same for both markers, and only two populations had significantly different He estimates. The neighbor-joining tree based on the Nei’s genetic distance displayed some geographic pattern. With the AFLP markers the populations grouped according to the provenance regions where they were sampled, resulting in one well supported cluster with the Southern populations, but with SSAP the pattern was not so coherent. In this study SSAP generated more polymorphic fragments and higher estimates of genetic diversity than AFPL did, due, probably, to the higher mutation rate of retrotransposition relative to base mutation. Nevertheless, congruence was found between estimates obtained with both markers, which is very interesting, for, in general, SSAP markers have lower costs compared to AFLPs, and they might be an interesting alternative marker system, when higher resolution is requested.

Molecular identification of new varieties ofNierembergia linariaefolia (Graham), a native Argentinean ornamental plant

Six Nierembergia linariaefolia clones were selected for ornamental traits during a native germplasm development program. For fingerprinting diagnosis, 13 anchored inter-simple sequence repeat (ISSR) primers and 6 amplified fragment length polymorphism (AFLP) primer-enzyme combinations were used. Both markers revealed high levels of polymorphism, enabling genetic discrimination of the accessions analyzed by using 443 informative ISSRs and 541 AFLP markers. Both molecular techniques are suitable for monitoring genetic diversity in Nierembergia linariaefolia and, under our experimental conditions, they showed correlation coefficients of 0.629 for similarity matrices and of 0.649 in the cophenetic matrices. These results suggest that ISSRs are a good choice for DNA analysis in N. linariaefolia when simple manipulation and a low budget are required.

Prediction of single-cross hybrid performance in maize using haplotype blocks associated with QTL for grain yield

Marker-based prediction of hybrid performance facilitates the identification of untested single-cross hybrids with superior yield performance. Our objectives were to (1) determine the haplotype block structure of experimental germplasm from a hybrid maize breeding program, (2) develop models for hybrid performance prediction based on haplotype blocks, and (3) compare hybrid performance prediction based on haplotype blocks with other approaches, based on single AFLP markers or general combining ability (GCA), under a validation scenario relevant for practical breeding. In total, 270 hybrids were evaluated for grain yield in four Dent x Flint factorial mating experiments. Their parental inbred lines were genotyped with 20 AFLP primer-enzyme combinations. Adjacent marker loci were combined into haplotype blocks. Hybrid performance was predicted on basis of single marker loci and haplotype blocks. Prediction based on variable haplotype block length resulted in an improved prediction of hybrid performance compared with the use of single AFLP markers. Estimates of prediction efficiency (R(2)) ranged from 0.305 to 0.889 for marker-based prediction and from 0.465 to 0.898 for GCA-based prediction. For inter-group hybrids with predominance of general over specific combining ability, the hybrid prediction from GCA effects was efficient in identifying promising hybrids. Considering the advantage of haplotype block approaches over single marker approaches for the prediction of inter-group hybrids, we see a high potential to substantially improve the efficiency of hybrid breeding programs.

Prediction of single-cross hybrid performance for grain yield and grain dry matter content in maize using AFLP markers associated with QTL

Prediction methods to identify single-cross hybrids with superior yield performance have the potential to greatly improve the efficiency of commercial maize (Zea mays L.) hybrid breeding programs. Our objectives were to (1) identify marker loci associated with quantitative trait loci for hybrid performance or specific combining ability (SCA) in maize, (2) compare hybrid performance prediction by genotypic value estimates with that based on general combining ability (GCA) estimates, and (3) investigate a newly proposed combination of the GCA model with SCA predictions from genotypic value estimates. A total of 270 hybrids was evaluated for grain yield and grain dry matter content in four Dent x Flint factorial mating experiments, their parental inbred lines were genotyped with 20 AFLP primer-enzyme combinations. Markers associated significantly with hybrid performance and SCA were identified, genotypic values and SCA effects were estimated, and four hybrid performance prediction approaches were evaluated. For grain yield, between 38 and 98 significant markers were identified for hybrid performance and between zero and five for SCA. Estimates of prediction efficiency (R (2)) ranged from 0.46 to 0.86 for grain yield and from 0.59 to 0.96 for grain dry matter content. Models enhancing the GCA approach with SCA estimates resulted in the highest prediction efficiency if the SCA to GCA ratio was high. We conclude that it is advantageous for prediction of single-cross hybrids to enhance a GCA-based model with SCA effects estimated from molecular marker data, if SCA variances are of similar or larger importance as GCA variances.

Prospects for hybrid breeding in winter triticale: II. Relationship between parental genetic distance and specific combining ability

AbstractSignificant relative midparent heterosis (MPH%) for grain yield in triticale (×Triticosecale Wittm.) has generated interest in the development of hybrid cultivars. The objectives of this study were to (i) examine the association between parental genetic distance (GD) and specific combining ability (SCA), (ii) investigate the existence of genetically distant heterotic groups in elite germplasm, and (iii) draw conclusions for future hybrid breeding in winter triticale. Genetic distance between 61 lines was estimated, based on 93 polymorphic simple sequence repeat (SSR) marker loci and 10 AFLP (amplified fragment length polymorphism) primer‐enzyme combinations (PEC). Agronomic data of 206 F1 crosses and their 61 parental lines grown in six German environments were published recently in a companion study. Correlations were made between SCA for grain yield, number of spikes/m2, 1000‐kernel weight and number of kernels per spike with GD estimates of the 56 female and five male parents (testers). Principal co‐ordinate analyses (PCoA) based on SSR data revealed no distinct subgroups in the germplasm. Correlations between GD and SCA were low for all traits (|r| ≤ 0.31), which hampers the prediction of SCA from molecular data. A multi‐stage procedure is recommended for future hybrid breeding in triticale by applying a pragmatic approach for the grouping of germplasm following the early history of hybrid breeding of maize.

Identification of Molecular Markers for Selected Wood Properties of Norway Spruce Picea abies L. (Karst.) II. Extractives Content

Abstract We describe the development of a SCAR-marker linked to low extractives content of Norway Spruce (Picea abies L [Karst.]) derived from AFLPs. In these analyses 57 different primer enzyme combinations were used in a bulked segregant analysis approach comparing individuals with high and low extractives content. A total of 14 polymorphic AFLP markers were detected between the pools. Five markers were selected for further analyses to verify their linkage to extractives content based on individuals used for pool constitution. One AFLP marker, found to be significant linked to low extractives content was converted into a SCAR marker for further validation. For this marker, a monomorphic band was obtained by using sets of nested primers or restriction site specific primers (RSS) which include the AFLP-restriction recognition site. The separation of the marker from unlinked size homologous marker-alleles was realized by a SSCP-approach. Validation of the marker on different full-sib families confirmed the usability to separate the classes for low and high extractives content of Picea abies.

Characterization of Microbial Communities Found in the Human Vagina by Analysis of Terminal Restriction Fragment Length Polymorphisms of 16S rRNA Genes

To define and monitor the structure of microbial communities found in the human vagina, a cultivation-independent approach based on analyses of terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes was developed and validated. Sixteen bacterial strains commonly found in the human vagina were used to construct model communities that were subsequently used to develop efficient means for the isolation of genomic DNA and an optimal strategy for T-RFLP analyses. The various genera in the model community could best be resolved by digesting amplicons made using bacterial primers 8f and 926r with HaeIII; fewer strains could be resolved using other primer-enzyme combinations, and no combination successfully distinguished certain species of the same genus. To demonstrate the utility of the approach, samples from five women that had been collected over a 2-month period were analyzed. Differences and similarities among the vaginal microbial communities of the women were readily apparent. The T-RFLP data suggest that the communities of three women were dominated by a single phylotype, most likely species of Lactobacillus. In contrast, the communities of two other women included numerically abundant populations that differed from Lactobacillus strains whose 16S rRNA genes had been previously determined. The T-RFLP profiles of samples from all the women were largely invariant over time, indicating that the kinds and abundances of the numerically dominant populations were relatively stable throughout two menstrual cycles. These findings show that T-RFLP of 16S rRNA genes can be used to compare vaginal microbial communities and gain information about the numerically dominant populations that are present.

Multiple origin of metallicolous populations of the pseudometallophyte Arabidopsis halleri (Brassicaceae) in central Europe: the cpDNA testimony

The population structure of the pseudo-metallophyte herb, Arabidopsis halleri, was studied using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) on chloroplast DNA (cpDNA). The history of metallicolous (M) populations showing increased zinc tolerance was investigated. Eight primer-enzyme combinations out of 72 tested were applied to a total of 625 individuals from 28 widespread populations, 14 of them being M. Eleven distinct chlorotypes were found: five were common to nonmetallicolous (NM) and M populations, whereas six were only observed in one edaphic type (five in NM and one in M). No difference in chlorotype diversity between edaphic types was detected. Computed on the basis of chlorotype frequencies, the level of population differentiation was high but remained the same when taking into account levels of molecular divergence between chlorotypes. Isolation by distance was largely responsible for population differentiation. Geographically isolated groups of M populations were more genetically related to their closest NM populations than to each other. Our results suggest that M populations have been founded separately from distinct NM populations without suffering founding events and that the evolution towards increased tolerance observed in the distinct M population groups occurred independently.

Species-diagnostic markers in Larix spp. based on RAPDs and nuclear, cpDNA, and mtDNA gene sequences, and their phylogenetic implications

Genetic markers from the nuclear, chloroplast, and mitochondrial genomes were developed to distinguish unambiguously among four larch species [Larix laricina (Du Roi) K. Koch, Larix decidua (Mill.), Larix kaempferi (Lamb.) Sarg., and Larix sibirica (Ledeb.)] used in intensive forestry in eastern North America. Nine random amplified polymorphic DNA (RAPD) fragments had good diagnostic value, and 3 out of 12 nuclear genes were found to harbor fixed interspecific polymorphisms implicating a total of 17 single nucleotide polymorphisms (SNPs) and 2 indels. The sequencing of five mtDNA introns (cox1-intron1, matR-intron1, nad1-intron b/c, nad3-intron1, and nad5-intron1) and four cpDNA regions (matK, trnL-intron, trnT–trnL and trnL–trnF intergenic spacers) resulted in the identification of 14 sites with fixed interspecific differences among the four species. Including the ten Larix species, one polymorphic site per 47 nucleotide sites sampled was observed for nuclear genes, one per 283 sites for cpDNA, and one per 374 sites for mtDNA. The phylogeny of the genus could be estimated from variation among the ten species detected in two cpDNA intergenic regions and four mtDNA introns. There was congruence between cpDNA and mtDNA phylogenies with three large groups delineated: the North American, North Eurasian, and South Asian taxa. The position of L. sibirica differed between organelle genomes. It was regrouped with South Asian species on the cpDNA tree, but with its North Eurasian congenerics on the mtDNA tree. To simplify the detection of diagnostic DNA sequence polymorphisms among the four main Larix species, cleaved amplified polymorphic sequence (CAPS) assays were developed from the polymorphisms identified in the various genomes. Seventeen primer–enzyme combinations were tested, and six were selected for their high level of informativeness. These new species-specific diagnostic markers should be useful for the certification of larch breeding materials and hybrid stocks used in intensive forestry in the northern hemisphere.

Genetic diversity of Miyamasukashi‐yuri (Lilium maculatum Thunb. var. bukosanense), an endemic and endangered species at Mount Buko, Saitama, Japan

Abstract Lilium maculatum var. bukosanense, Miyamasukashi‐yuri in Japanese, grows endemically in Saitama Prefecture, Japan, and is listed in the Saitama Prefecture Red Data Book as a critically endangered plant. Articles about its genetic diversity and population genetic structure have not been reported. Before it is lost, population genetic studies can provide information for efficient in situ conservation as well as for possible future use in breeding programs. Therefore, we carried out a population genetic study of natural L. maculatum var. bukosanense using chloroplast DNA (cpDNA) and inter‐simple sequence repeat (ISSR) markers. In this study, fresh leaves of 46 L. maculatum var. bukosanense individuals were collected from two populations in Mount Buko. By polymerase chain reaction (PCR)‐restriction fragment length polymorphism analysis of cpDNA, we estimated the maternal origin of individuals sampled. Sixteen cpDNA primer pairs were tested, and only four of them produced clear bands. Twenty‐four combinations from these PCR fragments combined with six restriction enzymes were assayed; however, only 18 of them could be digested, producing two to four fragments. All individuals of L. maculatum var bukosanense analyzed were virtually identical for all primer–enzyme combinations. These findings suggest that the maternal origin of the 46 individuals analyzed is close. In addition, we analyzed ISSR markers to provide mainly biparental inheritance. PCR products of five anchored simple sequence repeat primers showed polymorphism, indicating that the individuals sampled were not clones, and thus it appears that they could have been reproduced from seeds. The primers yielded a total of 30 polymorphic bands (i.e. an average of six polymorphic bands per primer). The mean genetic distance and genetic identity values between the populations were 0.032 and 0.97, respectively. These findings suggest that all individuals from both populations are genetically similar. The individuals growing at 1000 m have greater genetic variation than those individuals from 650 m and this could possibly be attributed to altitude. A significant correlation between gene diversity and population size is also reported. From the results presented here some strategies for its in situ and ex situ conservation might be considered.