Interspecific polymorphism at non-coding regions of chloroplast,mitochondrial DNA and rRNA IGS region in Elymus species

Several published universal primers for amplification of non-coding regions of chloroplast, mitochondrial and ribosomal (rRNA) IGS region were tested whether they can amplify respective regions in Elymus species. PCR-RFLP analysis of the chloroplast, mitochondral DNA, and rRNA IGS region of the genus Elymus was used to determine if the method could be employed to detect inter-specific variation in this genus. Published universal primers for amplification of trnK [tRNA-Lys (UUU) exon 1]-trnK [tRNA-Lys (UUU) exon2], and mitochondrial nad1 exon B-nadl exon C intron successfully amplified the respective regions in Elymus species. However, the primers for amplification of chloroplast trnD-trnT intron and rRNA IGS failed to amplify the respective region in Elymus species. New primer pairs were designed and successfully amplified the cpDNA trnD-trnT intron and rRNA IGS region in Elymus species. The amplification products were digested with seven restriction enzymes. The results showed that the investigated regions of chloroplast and mitochondrial genomes are variable in most of the tested taxa and contain multiple variable regions. These regions should serve as useful molecular markers in phylogenetic studies of closely related species, at least at the interspecific level in Elymus. It is likely that further studies, including larger sample sizes, more regions of these genomes and/or more powerful methods for the detection of cpDNA and mt DNA variation will reveal additional variation for this genus. Highly inter- and intra-specific polymorphisms for rRNA IGS region were detected, suggesting the IGS will be a useful molecular marker for population studies of Elymus species.     

Chloroplast-specific universal primers and their uses in plant studies

Universal (consensus) primers are those primers that have the ability to amplify the targeted region of DNA across a broad range of individuals in a certain group of organisms. In plants, such universal primers have been designed to target regions in the nuclear, mitochondrial or chloroplast genome. Among these three genomes, the chloroplast genome is the most suited for the design of consensus primers due to the lower rate of evolution and hence conservation of gene order and sequence of the genome among the different plant species compared to the other two genomes. Several molecular studies in plants have developed and used chloroplast-specific universal primers. In this review, I present some examples of the nuclear DNA-specific universal primers and discuss the features of the chloroplast DNA that make it the most suited for the design of such primers. I then refer to all chloroplast-specific primers developed so far and provide some examples of molecular studies and applications that made use of them.     

Analysis of SSR dynamics in chloroplast genomes of Brassicaceae family

Simple sequence repeats (SSRs) are present abundantly in most eukaryotic genomes. They affect several cellular processes like chromatin organization, regulation of gene activity, DNA repair, DNA recombination, etc. Though considerable data exists on using nuclear SSRs to infer phylogenetic relationships, the potential of chloroplast microsatellites (cpSSR), in this regard, remains largely unexplored. In the present study we probe various nucleotide repeat motifs (NRMs) / types of SSRs present in chloroplast genomes (cpDNA) of 12 species belonging to Brassicaceae family. NRMs show a non-random distribution in coding and non-coding compartments of cpDNA. As expected, trinucleotide repeats are more common in coding regions while other repeat motifs are prominent in non-coding DNA. Total numbers of SSRs in coding region show little variation between species while considerable variation is exhibited by SSRs in non-coding regions. Finally, we have designed universal primers that yield polymorphic amplicons from all 12 species. Our analysis also suggests that amplicon length polymorphism shows no significant relationship with sequence based phylogeny of SSRs in cpDNA of Brassicaceae family.     

Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals

Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH-psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DETl, COPl, PGICl, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugioniun (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DETl primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.     

PCR-RFLP analysis of cpDNA in the genus Abies

 We used PCR-RFLP analysis of the chloroplast DNA of the genus Abies (family Pinaceae), to determine if the method could be employed to detect inter-specific variation in this genus and to study how the variation was distributed in different regions of the genome. Ten different chloroplast DNA regions, consisting of coding and non-coding DNA sequences, were amplified with specific primers in ten different Abies taxa. The amplification products were digested with several restriction enzymes. The results showed that the chloroplast genome is highly variable in most of the investigated taxa and contains multiple variable regions that appear to be distributed throughout the whole genome. Species-diagnostic markers were found for four of the ten investigated species. Unexpectedly, intra-specific variation was also detected in four species. It is likely that further studies, including larger sample sizes and/or more powerful methods for the detection of chloroplast DNA variation, will reveal additional variation for this genus. Received: 2 September 1998 / Accepted: 17 September 1998     

Molecular characterization of reciprocal crosses of Aerides vandarum and Vanda stangeana (Orchidaceae) at the protocorm stage

Aerides vandarum and Vanda stangeana are two rare and endangered vandaceous orchids with immense floricultural traits. The intergeneric hybrids were synthesized by performing reciprocal crosses between them. In vitro germination response of the immature hybrid embryos was found to be best on half-strength Murashige and Skoog medium supplemented with 20% (v/v) coconut water/liquid endosperm from tender coconut. Determination of hybridity was made as early as the immature seeds or embryos germinated in vitro, using randomly amplified polymorphic DNA (RAPD) markers. Out of 15 arbitrarily chosen decamer RAPD primers, two were found to be useful in amplification of polymorphic bands specific to the parental species and their presence in the reciprocal crosses. However, a decisive profile that can identify the reciprocal crosses could not be provided by RAPD. Amplification of the trnL-F non-coding regions of chloroplast DNA of the parent species and hybrids aided easy identification of the reciprocal crosses from the fact that maternal inheritance of chloroplast DNA held true for these intergeneric hybrids. Subsequent restriction digestion of the polymerase chain reaction (PCR) amplified trnL-F non-coding regions of chloroplast DNA also consolidated the finding. Such PCR-based molecular markers could be used for early determination of hybridity and easy identification of the reciprocal crosses.     

Are nuclear loci ideal for barcoding plants? A case study of genetic delimitation of two sister species using multiple loci and multiple intraspecific individuals

Considerable debate remains as to which DNA region should be used to barcode plants. Several different chloroplast (cp) DNA regions (rbcL, matK, and trnH–psbA) and nuclear ribosomal internal transcribed regions (ITS) have been suggested as suitable barcodes in plants. Recently, low-copy nuclear loci were also suggested to be potentially ideal barcode regions. The aim of the present study was to test the effectiveness of these proposed DNA fragments and five additional low-copy loci (CHS, DET1, COP1, PGIC1, and RPS2; comprising both coding and non-coding regions) in barcoding closely related species. We examined the divergences within and between two species of Pugionium (Brassicaceae). We failed to find any interspecific variation from three cpDNA fragments with which to discriminate the two species. However, a single base mutation in the internal transcribed spacer (ITS) could discriminate between the two species consistently. We found more variations among all individuals of the two species using each of the other five low-copy nuclear loci. However, only alleles from one locus (DET1) of the five low-copy loci related to flowering regulations was able to distinguish the sampled individuals into two species. We failed to amplify the corresponding fragments out of Brassicaceae using the designed DET1 primers. We further discussed the discrimination power of different loci due to incomplete lineage sorting, gene flow, and species-specific evolution. Our results highlight the possibility of using the nuclear ITS as a core or complementary fragment to barcode recent diverged species.     

Chloroplast DNA markers reveal a geographical divide across Argentinean southern beech Nothofagus nervosa (Phil.) Dim. et Mil. distribution area

  Nothofagus nervosa is one of the most important species of the temperate forests of southern South America. On the eastern slope of the Andes Mountains (Argentina) it has a very small and narrow natural distribution area which follows the valleys of the numerous west-east lake basins. Re-colonisation after the last glaciation is assumed to have originated from refugia located in the western pacific coast or even in free interglacial patches of the Andes Mountains. Sixteen pairs of primers were used to amplify 16 non-coding regions of chloroplast DNA in 11 populations from over the entire distribution area in Argentina. After restriction analysis two polymorphic fragments were found which defined two haplotypes and allowed a differentiation among populations. A clear geographic divide occurs separating populations distributed south and north of a west-east mountain chain. This suggests that after the last glaciation the species spread out from at least two different refugia. Hypotheses about the possible location of these refugia are discussed. Received: 28 October 1997 / Accepted: 19 March 1998     

Taxonomic individuality of Leonurus cardiaca and Leonurus quinquelobatus in view of morphological and molecular studies

The main goal of this study was to determine the number and taxonomic rank of taxa belonging to the complex Leonurus cardiaca agg. in Poland. Based on statistical analysis of selected features, two morphological forms of this plant were distinguished. In order to determine their genetic polymorphism and the relationships between them, the nuclear, mitochondrial and chloroplast genomes were analysed with the use of RAPD and PCR–RFLP markers. 39 RAPD primers produced a total of 234 nuclear DNA fragments, of which 128 were polymorphic and distributed almost equally between two forms. It was found that 87 % of the compared pairs of RAPD profiles differ from each other. Five chloroplast and two mitochondrial primer pairs were used to amplify non-coding regions of organelle genomes. Restriction analysis revealed uniformity of mtDNA and occurrence of two cpDNA haplotypes, corresponding to naked and hairy forms of L. cardiaca agg. The obtained results justifies the recognition of these forms as separate species L. cardiaca s. s. L. and L. quinquelobatus Gilib., respectively. The distribution of both species in Poland is given in the paper.     

叶绿体基因infA-rpl36区域在小麦族物种中的序列变异分析

利用小麦叶绿体基因组中infA-rpl36区域的序列设计引物, 对小麦族(Triticeae)的12个二倍体和多倍体的物种进行了PCR扩增和序列测定, 获得了长度为584～603 bp的12条DNA序列。序列分析表明, 供试物种在infA-rpl36基因间隔区的核苷酸变异明显高于基因编码区。基因编码区核苷酸序列同源性高达97%, 表明了目标片段具有高度的保守性。但在5个物种的infA编码区出现了较大的插入、缺失突变, 导致推导的氨基酸序列也发生了很大的变化, 证实了infA基因是叶绿体基因组中最活跃的基因之一, 而rpl36基因的变异较小, 说明不同叶绿体基因的进化速度是不同的。基于测定序列建立的种系树分析发现, 多倍体物种中间偃麦草(Thinopyrum intermedium)具有多种不同的细胞质起源, 与核基因组一样在进化上较为复杂。