Inter-simple Sequence Repeat Variation Research Articles

Molecular and Morpho‐Agronomic Characterization among Introduced Bristle Oat Collection Using ISSR Fingerprinting

Core Ideas The genetic diversity of Avena strigosa was examined in order to clarify its phylogenic relationships and analyze the domestication of these well‐adapted ecotypes in the Tunisian environment. The analysis of molecular variance revealed that the 91% of the variation was within groups of accessions according to their geographic origins and only 9% appeared among groups. Three analyses revealed a scattered distribution, independent of the geographic origin. Knowledge about the genetic variation of bristle oat (Avena strigosa Schreb.) cultivars is helpful for the development of conservation strategies to molecularly assist selection to improve the introduced germplasm in the Tunisian environment. The present study examines the genetic diversity among 22 accessions of A. strigosa and clarifies its phylogenic relationships to analyze the domestication of these well‐adapted ecotypes. Twenty‐two accessions of diploid A. strigosa sampled from 22 various geographical locations were analyzed with 20 universal inter‐simple sequence repeat (ISSR) primers. Twelve morpho‐agronomic traits were scored and all data were compiled to establish the repartition of the total ISSR variation and to analyze the genetic structure of the accessions under study. Out of 275 bands, 252 were recorded as polymorphic. The analysis of molecular variance revealed that 91% of the variation was within groups of accessions according to their geographic origins. In addition, three analyses (Bayesian, principal component analysis, and unweighted pair group method with arithmetic mean) revealed a confused distribution, independent of the geographic origin. Therefore, it may be concluded that the bristle oat accessions are interrelated in spite of their agronomic divergence. Avena strigosa may be a potential forage crop that is suited to marginal Tunisian environments.

Genetic diversity and population structure of the Chinese Fungus Metarhizium rileyi causing green muscardine in silkworm

Green muscardine caused by Metarhizium rileyi affects sericulture, and is typically enzootic and occurs frequently at low incidence. We collected 152 M. rileyi isolates from silkworm cadavers in eight sericulture areas in seven provinces of China, and four strains from other Lepidoptera larvae in Qianshan(QS) County, Anhui province. Nine microsatellite inter-simple sequence repeat (ISSR) primers produced 91 distinct and reproducible bands, revealing a high level (90.11%) of DNA polymorphism. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) cluster analysis divided the populations into four groups, with isolates from Qianshan County forming a single branch. All the 156 M. rileyi isolates were heterogenic and polyphyletic and did not displayed typical regional distribution except strains from Qianshan country. PCA analysis of the nine populations of M. rileyi revealed similar phylogenies among accessions. Genetic differentiation index (Gst) among eight enzootic populations was 0.3789 and gene flow (Nm) was 0.4098, suggesting the low gene flow maintained a high degree of differentiation. Gst between the enzootic population of Qianshan County and other seven populations exceeded the threshold of severe differentiation, with moderate differentiation between the remaining seven enzootic populations. Analysis of molecular variance (AMOVA) showed most ISSR variation (61%) among isolates occurred within populations. No significant correlation was observed between geographical and genetic distance. According to cluster analysis based on single enzootic population, every enzootic population showed dominance, namely mainly constituted of strains with high genetic similarity. These data indicated that the green muscardine in each local silkworm population was predominantly caused by a native group of M. rileyi. Furthermore, Gst and Nm of M. rileyi from silkworm and other Lepidoptera larvae in Qianshan County were 0.1174 and 1.8791, respectively, suggesting strains isolated from different hosts in Qianshan County do not show obvious host specificity. This demonstrated that host transfer may take place in silkworm and other insects.

English

Malaysia is a genetically diverse hotspot for wild relatives of Mangifera and other Anacardiaceae members. The genus Bouea is interesting in that it has small sweet-sour edible fruits. This genus consists of two species, namely Kundang (Bouea macrophylla) and Remia (B. oppositifolia) which are of economic value. In this study, we highlighted the molecular evidences using inter simple sequence repeats (ISSR) markers to understand its genetic relatedness among accessions. A total of 52 ISSR have been screened on selected accession of Kundang and Remia from Peninsular Malaysia. Ten of the 52 primers for Kundang generated 89 scorable bands, where 45 bands (50.2%) were polymorphic. The simple matching coefficient of similarity provided similarity values ranging from 0.659 to 0.955 while relationship among the different accessions was distributed among three main divergent clusters. For Remia, 10 of the 52 primers tested gave 89 scorable bands, where 54 bands (59.5%) were polymorphic. The simple matching coefficient of similarity values ranged from 0.591 to 0.977 while the cluster analysis separated 23 accessions into two well-defined clusters. These studies indicate that there was considerable ISSR variation among the accessions reflecting variation among the accessions, which were morphologically indistinguishable. DNA polymorphism detected by ISSR analysis offers a useful molecular marker for the identification of different accessions of Kundang and Remia.   Key words: Biodiversity, Bouea, genetic diversity, molecular marker, ISSR.

Genetic diversity of Chimonanthus grammatus populations determined with inter-simple sequence repeats (ISSR) analysis: Implications for conservation

Chimonanthus grammatus is an endangered and national-protected medicinal plant species with highly restricted geographical distribution and disjunctly locates in the border area of Anyuan and Huichang county of Jiangxi province, China. In this study, inter-simple sequence repeats (ISSR) markers were used to analyzed genetic diversity of seven populations of C. grammatus, collected from the area of Anyuan (4 populations) and Huichang (3 population) county of Jiangxi province, China. Our results showed that ten primers produced 74 discernible bands (39 polymorphic), with a low genetic diversity at species level percentages of polymorphic bands (PPB = 52.7%, H = 0.157, I = 0.241) and at population level (PPB = 35.7%,H = 0.119, I = 0.177). A high population differentiation (Gst = 0.249) was detected among seven populations and significant association were found between genetic and geographical distances (r = 0.712, p < 0.001), suggesting that gene flow was restricted geographically. Analysis of molecular variance (AMOVA) also revealed that 26.4% of the ISSR variation resided among populations, while 73.6% resided within populations, and 21.8% of the ISSR variation resided among Anyuan and Huichang groups, while 78.2% resided within groups. Factors, such as gene flow, genetic drift and evolutionary history, might have important influence on genetic structure and diversity of C. grammatus populations. An understanding of the genetic diversity and population structure of C. grammatus can provide insight into the conservation and management of this species. Key words: Chimonanthus grammatus, genetic diversity, inter-simple sequence repeats (ISSR), conservation.

Genetic variation and cultivar identification in Cymbidium ensifolium

As a popular flowering species with many cultivars, Cymbidium ensifolium (L.) is commercially important in horticulture. However, so far little has been known about genetic diversity and conservation genetics of this species. Understanding of the genetic variation and relationships in cultivars of C. ensifolium is a prerequisite for development of future germplasm conservation and cultivar improvement. Here we report assessment of genetic variations in C. ensifolium cultivars using the DNA fingerprinting technique of inter-simple sequence repeats (ISSR). A total of 239 ISSR loci were identified and used for evaluation of genetic variation with a selection of 19 ISSR primers. Among these ISSR loci, 99.16% were polymorphic with wide genetic variation as shown by Nei’s gene diversity (H = 0.2431) among 85 tested cultivars. ISSR fingerprinting profiles showed that each cultivar had its characteristic DNA pattern, indicating unequivocal cultivar identification at molecular level. Eighteen cultivar-specific ISSR markers were identified in seven cultivars. The cultivar Sijiwenhan was confirmed as hybrid by four ISSR primers. Several cultivars with same name but different geographical origins were distinguished based on their ISSR profiles. A dendrogram generated with ISSR markers could group 73 of 85 cultivars into four major clusters. Further analysis of ISSR variation revealed that about 69% of total genetic variation in this species is due to genetic divergence inside geographical groups. Our results suggest that both germplasm collection and in situ conservation are important for future planning of C. ensifolium species conservation.

Association between chemical and genetic variation in Calophyllum inophyllum, a medicinally important tree of the Western Ghats of India

The objective of the present work is to study the chemical variation in Calophyllum inophyllum growing along the Western Ghats of India. Contents of dipyranocoumarins (inophyllums) in C. inophyllum were determined to assess whether they could be used as a taxonomic marker for C. inophyllum. This study also aims to establish inter simple sequence repeat (ISSR) markers that can be used to study genetic variation within the species and explore correlation between ISSR and chemical markers. The contents of dipyranocoumarins were estimated in seeds collected from 20 locations. Leaves from plants at the same 20 locations were assayed for ISSR variation. A dendrogram based on Nei’s genetic distance as well as principal component analysis based on dipyranocoumarins and ISSR variation clustered plants from these 20 locations into three groups that indicated close relationship among ISSR, dipyranocoumarins contents, and geographical position (variation) of locations. Based on this study, two locations of elite plants were identified.

Genetic diversity of cultivated flax (Linum usitatissimum L.) and its wild progenitor pale flax (Linum bienne Mill.) as revealed by ISSR markers

Little is known about the genetic diversity of pale flax (Linum bienne Mill.), the wild progenitor of cultivated flax (L. usitatissimum L.), and ex situ germplasm of pale flax was scarce. Effort was made to collect 34 pale flax accessions and five landrace accessions of cultivated flax in Turkey. The inter simple sequence repeat (ISSR) technique was applied to characterize this set of flax germplasm, along with one Turkish cultivar, one Russian cultivar, five winter and four dehiscent type accessions of cultivated flax. Twenty-four ISSR primer pairs detected a total of 311 DNA fragments, of which 298 bands were polymorphic across 493 flax samples (roughly 10 samples per accession). These polymorphic bands had frequencies ranging from 0.002 to 0.998 and averaging 0.38. Accession-specific ISSR variation (Fst values) ranged from 0.469 to 0.514 and averaged 0.493. There was 49.3% ISSR variation resided among these 50 accessions, 35.9% harbored among landrace, winter, dehiscent types of cultivated flax and pale flax, and 38.2% present among 34 pale flax accessions. Pale flax displayed more ISSR variation than landraces and dehiscent type, but less than winter type, of cultivated flax. Clustering 493 individual plants revealed that these assayed plants were largely grouped according to their plant types and that pale flax was genetically more close to the dehiscent type, followed by the winter type and landrace, of cultivated flax. Pale flax collected within the geographic range of 180 km displayed a significant spatial genetic autocorrelation. Genetic distances among the pale flax accessions were significantly associated with their geographic distances and elevation differences. These findings are significant for understanding flax domestication and its primary gene pool.

ISSR variation within and among wild Brassica juncea populations: implication for herbicide resistance evolution

There is a need to understand whether weed genetic diversity is the same among different populations, especially between those exposed to herbicide selection and other without exposure history. Inter-simple sequence repeat (ISSR) were used to assess level and patterns of genetic diversity in wild Brassica juncea (L.) Czern. et Coss. populations. A total of 93 plants from 24 wild populations in China were analysed by eight primers resulting in 86 highly reproducible ISSR bands. The analysis of molecular variance (AMOVA) with distances among individuals corrected for the dominant nature of ISSRs showed that most of the variation (54.09%) occurred among populations, and the remaining 45.91% variance was attributed to differences among individuals within populations. The high differentiation was, perhaps, due to limited gene flow (Nm < 1.0) of this species. Though highest gene diversity was observed in resistant B. juncea population, the overall distribution of diversity across China was not geographic dependent. High F ST value (0.541) corroborated AMOVA partitioning and provided significant evidence for population differentiation in wild B. juncea. UPGMA cluster analyses, based on Nei’s genetic distance, revealed grouping pattern geographically. Based on these results, the factors affect weed population genetic diversity and implication for herbicide resistance evolution were discussed in the context of transgenic crops advent and increasing herbicide usage in China.

Assessing genetic diversity of Elymus sibiricus (Poaceae: Triticeae) populations from Qinghai-Tibet Plateau by ISSR markers

Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population structure in eight populations of Elymus sibiricus L. from the southeast of Qinghai-Tibet Plateau of China. Of the 100 primers screened, 13 produced highly reproducible ISSR bands. Using these primers, 193 discernible DNA fragments were generated with 149 (77.2%) being polymorphic, indicating considerable genetic variation at the species level. In contrast, there were relatively low levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 44.04 to 54.92%. The mean gene diversity ( H E) was estimated to be 0.181 within populations (range 0.164–0.200), and 0.274 at the species level. A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (33.1%), Shannon's index analysis (34.5%), Bayesian method (33.2%) and AMOVA analysis (42.5%). No significant statistical differences (analysis of molecular variance [AMOVA], P = 0.08) in ISSR variation were found between the sample collection regions. However, among populations (42.5% of the variance) and within populations (57.5% of the variance), there were significant differences ( P < 0.001). Populations shared high levels of genetic identity. This pattern of genetic variation was different from that for most of inbreeding Triticeae species reported. In addition, a geographical pattern of population differentiation, where the populations from south and north of sampling sites were clearly separated from each other, was revealed by both the cluster and principal coordinates analyses. Generally, the result of this study indicates that E. sibiricus contains high molecular variation in its populations. The implications of these results for the conservation of the species are discussed.

Genetic structure of endangered Emmenopterys henryi Oliv. based on ISSR polymorphism and implications for its conservation

Inter-simple sequence repeat (ISSR) markers were used to determine the genetic variation and genetic differentiation of nine populations of Emmenopterys henryi Oliv., an endangered plant endemic to China. Relatively low genetic diversity was detected at population level (the percentage of polymorphic loci P=22.56%, the number of alleles per locus A=1.183+/-0.045, the effective number of alleles per locus A(E)=1.007+/-0.345, Nei's gene diversity h=0.071+/-0.017, Shannon information index I=0.104+/-0.025). However, the genetic diversity at species level was relatively high (P=56.05%; A=1.561+/-0.498, A(E)=1.325+/-0.371, h=0.191+/-0.199, I=0.287+/-0.284). Analysis of molecular variance showed that most of the ISSR variation (68.03%) in E. henryi occurred among populations. The estimated Nm from F (ST )was 0.235. It indicated that the fragmentation and isolation of populations might result from specific evolutionary history and anthropogenic activity. Consequently, genetic drift might play an important role in determining the genetic structure of E. henryi. Conservation strategies for this endangered species are proposed based on the genetic data.

Palaeoclimate‐induced range shifts may explain current patterns of spatial genetic variation in renosterbos (Elytropappus rhinocerotis, Asteraceae)

The impact of Pleistocene climatic fluctuations on the distributions of plant species in the Greater Cape Floristic Region is largely unknown. We used a molecular fingerprinting tool, inter‐simple sequence repeat (ISSR) PCR to examine the spatial distribution of genetic variation in the common and widespread shrub Elytropappus rhinocerotis. We wished to test the hypothesis that refugia for the species were located in areas which were buffered from marked variations in precipitation between glacial and interglacial periods. Populations from less protected areas, in contrast, should have suffered size reductions or extinctions during the dry Holocene optimum. We detected a large amount of genetic variation in the species, which was apportioned largely amongst individuals within populations rather than amongst populations or regions, as expected for an outcrossing and well‐dispersed plant species. However, there was significant spatial structure and an uneven distribution of diversity across the range. Geographic distance is a very poor predictor of genetic distance between localities, especially towards the east of the range. This may be due to range alteration over the time‐scale reflected by ISSR polymorphism. Inter‐SSR variation declined from south to north in the western arm of the range, consistent with the prediction of Holocene aridification starting first and being most extreme in the north. Areas shown by the marker to harbour populations with high levels of variability include most parts of the eastern arm of the range, and the Kamiesberg highlands. Possible explanations for the observed patterns of ISSR variation are discussed.

Genetic variation in the endangered and endemic species Changium smyrnioides (Apiaceae)

Changium smyrnioides H. Wolff (Apiaceae) is an endangered plant endemic to Eastern China. Levels of genetic variation and patterns of population structure in C. smyrnioides were investigated using inter-simple sequence repeat (ISSR) markers. Of the 100 primers screened, 18 produced highly reproducible ISSR bands. Using these primers, 196 discernible DNA fragments were generated with 166 (84.7%) being polymorphic, indicating considerable genetic variation at the species level. In contrast, there were relatively low levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 29.6% to 36.2%. Genetic diversity within populations ranged from 0.10 to 0.13, averaging 0.24 at the species level. A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis (52.9%), Shannon’s index analysis (54.3%) and AMOVA analysis (52.3%). No significant statistical differences (analysis of molecular variance [AMOVA], P=0·6) in ISSR variation was found between regions. However, among populations (52.3% of the variance) and within populations (47.7% of the variance), there were significant differences ( P<0.001). Restricted gene flow ( Nm=0.446) among populations may result from several factors, such as low seed dispersal, isolation of populations and/or self-pollination. Limited gene flow among populations is a plausible reason for the high genetic differentiation observed for this species.

ISSR variation in a worldwide collection of velvetleaf (Abutilon theophrasti) and the genetic background of weedy strains mingled in grains imported into Japan

Velvetleaf seeds have been found in imported grains in Japan. The plants from these seeds show a weedy growth habit, such as that seen in the noxious velvetleaf which has recently emerged in Japan and noted in our previous study. To elucidate the genetic background of the velvetleaf strains found in imported grains we evaluated the genetic variation of six strains and an additional 39 worldwide velvetleaf accessions using the intersimple sequence repeat (ISSR) polymorphism. Cluster analysis based on the ISSR polymorphism emitted two major clusters which corresponded well with classification by growth habit, that is, crop type and weed type. Five of the six strains from the imported grains formed a small cluster. All six strains united into one major cluster containing the weed type accessions, while all of the old Japanese accessions formed another major cluster containing the crop type accessions. The genetic difference between imported weedy velvetleaf and old Japanese accessions suggests that imported velvetleaves are the source of the recent outbreak of the noxious velvetleaf in Japan.

Intersimple sequence repeat (ISSR) variation in Lactoris fernandeziana (Lactoridaceae), a rare endemic of the Juan Fernández Archipelago, Chile

AbstractSixteen populations and 89 individuals of Lactoris fernandeziana were examined for variation in intersimple sequence repeat (ISSR) banding patterns. The species is a rare endemic of Masatierra Island in the Juan Fernández Archipelago, and is the only member of the endemic family Lactoridaceae. Five populations showed a single genotype whereas the other 11 populations had from two to 16 multilocus genotypes. Over 73% of the ISSR diversity occurred across populations, with only about 27% within populations. Diversity among populations results from the presence of different subsets of loci within each population rather than unique loci within populations; only two populations displayed novel loci, with one and three in each. Levels of differentiation at ISSR loci among populations are not correlated with geographic distance on Masatierra; rather, the pattern of variation is mosaic. The presence of differentiated local populations is concordant with the geitonogamous breeding system of the species and suggests low levels of long distance pollen or seed dispersal. The mosaic pattern of ISSR variation on Masatierra may result, in part, from drift and inbreeding in small populations following fragmentation of a once more continuous distribution of Lactoris with the formation of canyons by erosion. Also, the generation of new ISSR loci by mutation could occur with rare, sporadic gene flow among populations accounting for the mosaic pattern of variation and the paucity of unique alleles within populations. The ISSR results for Lactoris suggest that studies of morphological, ecological and physiological features may elucidate differentiation among populations of L. fernandeziana. Field studies have demonstrated that plants occur both in the dense forest understory and in the full sunlight in forest openings.

Inter-simple sequence repeat (ISSR) diversity within Monarda fistulosa var. brevis (Lamiaceae) and divergence between var. brevis and var. fistulosa in West Virginia

Inter-simple sequence repeat (ISSR) banding patterns were used to examine genetic diversity within and among populations ofMonarda fistulosa var.brevis, a rare taxon restricted to several populations in limestone glades and barrens in eastern West Virginia and Virginia. More than 34% of the total ISSR diversity in var.brevis occurred among populations, which is high when compared to the few other rare species that have been examined for ISSR variation. Prior studies demonstrated that var.brevis is morphologically distinct from the more widespread var.fistulosa, and that the differences are maintained when the two varieties are grown together in a uniform environment. The present study utilizing ISSR markers indicated that the two varieties are distinct, though quite similar genetically, and this is concordant with prior investigations documenting their morphological and habitat differences. However, the ISSR results suggest that the two varieties have diverged relatively recently and/or there is a low level of gene flow between them.

Molecular fingerprinting evidence (ISSR, Inter-Simple Sequence Repeats) for a wild status of Olea europaea L. (Oleaceae) in the Eurosiberian North of the Iberian Peninsula

Although the olive tree ( Olea europaea L.) is one of the best indicators of the Mediterranean region, there exist some isolated populations of this species in the Eurosiberian North of the Iberian Peninsula. To elucidate the wild or feral status of these populations, we studied a total of 46 populations of the Olea europaea complex. mat K sequences and Inter-Simple Sequence Repeats (ISSR) variation were analysed to infer relationships among the six subspecies of the Olea europaea complex and within O. europaea subsp. europaea. Based on mat K. sequences, populations of sspp. europaea (Mediterranean basin), cerasiformis (Madeira), guanchica (Canary Islands), maroccana (Agadir Mountains) and laperrinei (the Sahara) form one group separate from subsp. cuspidata (South and East of Africa, Arabia, Asia). In the parsimony-based (Heuristic) and distance-based (UPGMA and Neighbor Joining) analyses of ISSR variation, populations of the cultivated olive (var. europaea) form two groups which are intermingled with the populations of the wild olive (var. sylvestris ). The four olive populations from the Eurosiberian North of the Iberian Peninsula included in the analysis are closely related to each other and to other Iberian wild populations. This is interpreted as strong evidence for their wild rather than feral status in this area.