Polymorphic ISSR Research Articles

GENETIC CHARACTERIZATION OF RELICT ENDEMIC LIQUIDAMBAR ORIENTALIS (ALTINGIACEAE) POPULATIONS

Liquidambar orientalis, a relict endemic species, is distributed in the south-west of Turkey. The natural spread areas of the species have been decreasing since the early 1900s. This poses a great threat to the future and genetic diversity of the species. In this study, the genetic diversity of a total of 190 individuals in ten populations was examined with ten polymorphic ISSR primers. Data analysis was performed using POPGENE ver. 1.32 and GenAlEx 6.503 softwares. According to the results of the analysis, a total of 271 loci were identified. For the ten populations, the rate of polymorphic loci ranged from 21.03% to 34.69%. The mean number of observed alleles (Na) and mean number of effective alleles (Ne) were found as 1.98 ± 0.13 and 1.47 ± 24 0.37, respectively. Nei's genetic diversity values (h) ranged from 0.07 ± 0.15 to 0.12 ± 0.18. According to the results of Molecular Analysis of Variance (AMOVA) and POPGENE ver. 1.32 analysis, it was determined that the main contribution to the genetic variance originated from among the populations. In addition, the gene flow level (NM) was found to be low with a value of 0.27 per generation.

EFFECTIVENESS OF ISSRS TO DETERMINE THE GENETIC RELATIONSHIPS BETWEEN THIRTY SPECIES FROM ELEVEN GENERA OF BAMBOO

The identification of bamboo is traditionally dependent on vegetative characters due to its long and unpredictable flowering cycle. Molecular techniques are suited and consistent for resolving the genetic relationship between bamboo species and that was studied among 30 species from 11 genera using ISSRs markers. Thirteen ISSR primers revealed 305 polymorphic loci among the accessions Polymorphic information content and ISSR primer index ranged from 0.26 to 0.40 and 4.77 to 9.91 respectively. The Neighbour-Joining dendrogram of 30 species resulted in two major groups. all species of Bambusa were grouped in cluster I along with three species of Dendrocalamus, the remaining species of Dendrocalamus were distributed in cluster II along with nine other bamboo genera. This study supports the monophyletic origin of Bambusa and the paraphyletic origin of the genus Dendrocalamus.

Comparison between the field plants and their micropropagated plants of four Mentha spp. using molecular markers and phytochemical analyses

The efficient micropropagation procedures among four Mentha species (M. longifolia, M. spicata, M. viridis, and M. piperita) were established. In addition, the shoots were produced in 3.0 mg/l BA. M. longifolia had the highest percentage of regeneration (70%), whereas M. piperita recorded that the lowest value (47%). The micropropagated plants and field plants were subjected to phytochemical analyses. The gas chromatography and spectrophotometer results indicated that the active ingredients were changed in the regenerated plants. Nevertheless, the results of molecular markers showed that the thirteen SRAP primer combinations had 80 scorable bands; of them, 58 were polymorphic (72%). Nine ISSR markers showed a total of 60 scorable bands; of them, 44 were polymorphic (73%). Twelve out of the 102 polymorphic SRAP and ISSR markers were found to be genotype unique specific markers.

Study of genetic diversity using molecular markers in sunflower (Helianthus annuus)

Considering the significance of sunflower (Helianthus annuus L.) cultivation under diverse agro-climatic conditions, present study was done to recognize assorted parental lines utilizing RAPD and ISSR markers to lessen environmental effects or test blunders at Rajasthan College of Agriculture, MPUA & T, Udaipur, Rajasthan during 2016. In the present study, RAPD primers produced 334 polymorphic band and ISSR primers produced 160 polymorphic bands. RAPD primers detected more polymorphic loci (56.88%) than the ISSR primers (52.24%). The dendrograms constructed by RAPD, ISSR primers and pooled data were associated with each other, indicating the efficacy of both marker systems in genetic diversity study of sunflower. Based on RAPD, ISSR and pooled data analysis, the restorer line EC 623023, R 16 and seed parental line CMS 234B, PET 2-7-1B were found to be genetically most diverse and fall into different groups. Therefore, their genetic origin seems to be different and could be efficiently used in sunflower breeding programs.

Population genetic structure and connectivity of a riparian selfing herb Caulokaempferia coenobialis at a fine-scale geographic level in subtropical monsoon forest

BackgroundRivers and streams facilitate movement of individuals and their genes across the landscape and are generally recognized as dispersal corridors for riparian plants. Nevertheless, some authors have reported directly contrasting results, which may be attributed to a complex mixture of factors, such as the mating system and dispersal mechanisms of propagules (seed and pollen), that make it difficult to predict the genetic diversity and population structure of riparian species. Here, we investigated a riparian self-fertilizing herb Caulokaempferia coenobialis, which does not use anemochory or zoochory for seed dispersal; such studies could contribute to an improved understanding of the effect of rivers or streams on population genetic diversity and structure in riparian plants. Using polymorphic ISSR and cpDNA loci, we studied the effect at a microgeographic scale of different stream systems (a linear stream, a dendritic stream, and complex transverse hydrological system) in subtropical monsoon forest on the genetic structure and connectivity of C. coenobialis populations across Dinghu Mountain (DH) and Nankun Mountain (NK).ResultsThe results indicate that the most recent haplotypes (DH: H7, H8; NK: h6, h7, h11, h12) are not shared among local populations of C. coenobialis within each stream system. Furthermore, downstream local populations do not accumulate genetic diversity, whether in the linear streamside local populations across DH (H: 0.091 vs 0.136) or the dendritic streamside local populations across NK (H: 0.079 vs 0.112, 0.110). Our results show that the connectivity of local C. coenobialis populations across DH and NK can be attributed to historical gene flows, resulting in a lack of spatial genetic structure, despite self-fertilization. Selfing C. coenobialis can maintain high genetic diversity (H = 0.251; I = 0.382) through genetic differentiation (GST = 0.5915; FST = 0.663), which is intensified by local adaptation and neutral mutation and/or genetic drift in local populations at a microgeographic scale.ConclusionWe suggest that streams are not acting as corridors for dispersal of C. coenobialis, and conservation strategies for maintaining genetic diversity of selfing species should be focused on the protection of all habitat types, especially isolated fragments in ecosystem processes.

Genetic diversity analysis of sweet potato [Ipomoea batatas (L.) Lam.] germplasms through RAPD and ISSR markers

Sweet potato [Ipomoea batatas (L.) Lam.] germplasms are unique as they are heterozygous, vegitatively as well as sexually propagated and polyploid in nature. Forty-four germplasms from local farmer’s field and from various centres of India were determined using 15 RAPD and 15 ISSR markers to evaluate their genetic relatedness. Average polymorphisms for RAPD and ISSR markers were found to be 86.72 and 81.64%, respectively. The OPM series primer produced 145 total bands with 138 polymorphic loci and ISSR markers gave 116 total bands with 101 polymorphic loci. The clusters were made using the UPGMA method using both the RAPD and ISSR data and group diverse yet similar germplasm in two different clusters. This gives ample information to use them in further improvement programme.

A longitudinal study on morpho-genetic diversity of pathogenic Rhizoctonia solani from sugar beet and dry beans of western Nebraska

BackgroundRoot and stem rot caused by Rhizoctonia solani is a serious fungal disease of sugar beet and dry bean production in Nebraska. Rhizoctonia root rot and crown rot in sugar beet and dry bean have reduced the yield significantly and has also created problems in storage. The objective of this study was to analyze morpho-genetic diversity of 38 Rhizoctonia solani isolates from sugar beet and dry bean fields in western Nebraska collected over 10 years. Morphological features and ISSR-based DNA markers were used to study the morphogenetic diversity.ResultsFungal colonies were morphologically diverse in shapes, aerial hyphae formation, colony, and sclerotia color. Marker analysis using 19 polymorphic ISSR markers showed polymorphic bands ranged from 15 to 28 with molecular weight of 100 bp to 3 kb. Polymorphic loci ranged from 43.26–92.88%. Nei genetic distance within the population ranged from 0.03–0.09 and Shannon diversity index varied from 0.24–0.28. AMOVA analysis based on ΦPT values showed 87% variation within and 13% among the population with statistical significance (p < 0.05). Majority of the isolates from sugar beet showed nearby association within the population. A significant number of isolates showed similarity with isolates of both the crops suggesting their broad pathogenicity. Isolates were grouped into three different clusters in UPGMA based cluster analysis using marker information. Interestingly, there was no geographical correlation among the isolates. Principal component analysis showed randomized distribution of isolates from the same geographical origin. Identities of the isolates were confirmed by both ITS-rDNA sequences and pathogenicity tests.ConclusionIdentification and categorization of the pathogen will be helpful in designing integrated disease management guidelines for sugar beet and dry beans of mid western America.

Inter simple sequence repeat markers to assess genetic diversity of the desert date (Balanites aegyptiaca Del.) for Sahelian ecosystem restoration

Drought and desertification are the major environmental constraints facing the Sahelian agro-ecosystems for decades. Assessing genetic diversity of native tree species is critical to assist ecosystems restoration efforts. Here we describe genetic diversity and structure of seven Balanites aegyptiaca L. natural populations distributed across the Sahelian-Saharan zone of Mauritania using 16 polymorphic ISSR primers. These generated 505 polymorphic bands. Polymorphism information content (PIC) varied from (0.13–0.29) with an average 0.23, marker index (MI) averaged 7.3 (range 3.3–10.3) and resolving power (RP) ranged from (4.53–14.6) with an average 9.9. The number of observed alleles (Na) ranged from (0.62–1.39), Effective number of alleles (Ne) varied from (1.26–1.37), Shannon’s information index (I) ranged from (0.25–0.36). AMOVA analysis showed that 80% of the genetic variation was fined within populations, which is supported by a low level of genetic differentiation between population (GST = 0.21) and an overall estimate of gene flow among populations (Nm = 1.9). The dendrogram based on Jaccard's similarity coefficient and the structure analysis divided the seven populations into two main clusters in which two populations from the Saharan zone were grouped. Our results provide baseline data for genetic conservation programs of this Sahelian neglected crop and with an important econ-ecological role.

Morphological, cytological, and molecular variations induced by gamma rays in ground-grown chrysanthemum ‘Pinkling’

In this study, we analyzed the morphological, cytological, and molecular changes induced by different doses of 60Co-γ radiation on root cuttings of the ground-grown chrysanthemum ‘Pinkling’ plants whose survival and height were decreased with increasing radiation dose. A variety of mutants were observed among irradiated ‘Pinkling’ plants; floral mutations included alteration of floral types, term ligulate floret number, capitulum diameter, and floral colour. Meiotic observation of mutants showed that the frequency of chromosome aberrations increased with radiation dose. The mutants were further analyzed using ISSR markers and on the basis of polymorphic DNA banding patterns, floral mutants were genotypically distinguished. Eighteen polymorphic ISSR primers produced 110 scorable bands, of which 93 (84.5%) were polymorphic. The percentage of polymorphism ranged from 60% to 100%. Jaccard’s dissimilarity coefficients varied from 0.337 to 1.000, which attested to the high genetic variability induced by irradiation. It is concluded that gamma ray irradiation induces a sufficient frequency of mutations and that ISSR analysis may offer a useful molecular marker for the detection of mutants in chrysanthemum. The present findings provide valuable information for future breeding of ‘Pinkling’.

Assessment of the genetic diversity and relationships of a collection of Mentha spp. in Tunisia using morphological traits and ISSR markers

ABSTRACT The taxonomic identity of Mentha species is difficult as it displays variations at morphochemical characters. The aim of this investigation was to study Mentha spp. diversity in 60 Tunisian accessions using morphological traits and molecular markers. Thirty-three botanical descriptors were analysed. The numerical taxonomic analysis identified 58 accessions and defined seven taxonomic groups including M. aquatica, M. longifolia, M. piperita, M. pulegium, M. rotundifolia, M. spicata and M. spicata var. crispa ‘moroccan’. Thereafter, genetic diversity of these accessions was investigated using ISSR markers. Nine highly polymorphic ISSR primers were selected to amplify DNA of each accession. Based on UPGMA method, these accessions could be grouped into two major clusters. M. pulegium group occupied a relatively basal position in the dendrogram and formed a single cluster. The second large cluster contained the accessions of the section Mentha, clustered into five subgroups. Molecular separation of the Mentha suggested that hybridisation among species may result in convergence and/or divergence of different species. Our study has shown (1) a high morphological and molecular diversity in the Tunisian accessions and (2) that combining morphological and molecular data is crucial to a proper identification, allowing the correct use of these accessions in future breeding programmes.

Genetic diversity analysis and molecular characterization of grain amaranth genotypes using inter simple sequence repeat (ISSR) markers

BackgroundGrain amaranth (Amaranthus spp.) has been cultivated since ancient times in some countries in the world and it is one of the oldest food crops. At present, the crop has gained more importance in the plains of India, especially in parts of Gujarat and Maharashtra. Grain amaranth exhibits an incredible extent of morphological diversity and an extensive adaptability to diverse eco-geographical conditions. Hence, the aim of the recent research was to evaluate the genetic diversity of 19 genotypes from four diverse species of Amaranthus from India using ISSR markers.ResultsThe set of 11 polymorphic ISSR primers produced a total of 114 amplicons, among which 98 amplicons were polymorphic. The mean number of polymorphic amplicons per primer was 8.91. Overall, the size of PCR-amplified DNA fragments ranged from 200 to 3702 bp. The average percent polymorphism was 87.15%, and the average PIC value was 0.853, which indicates good selection of primers in the present study for the assessment of genetic diversity. The unique amplicon (marker)-producing primers were also found which can be used for identification of genotypes. The dendrogram grouped 19 grain amaranth genotypes into two major clusters. The groups formed on the principle component analysis (PCA) plot resembles with the results of the dendrogram although some genotypes have been diverted on the PCA plot.ConclusionsThe technique may be used to obtain reasonably precise information on the genetic relationship among grain amaranth genotypes. Such information may be useful for selecting the diverse parents and monitoring the genetic diversity periodically in the breeder’s working collection of grain amaranth.

Молекулярно-генетический анализ Malus sieversii – сравнение Джунгарской популяции in situ и ex situ

Malus sieversii (Ledeb.) M. Roem. is recognized as the ancestor of the cultivated apple tree. It has a valuable gene pool, and in the last decades, it becomes endangered in Kazakhstan. The genetic diversity of 13 clone-varieties of Sievers apple tree (Malus sieversii) of Dzungarian population from the introduction collection of the Main Botanical Garden in Almaty (Kazakhstan) and 31 samples from three natural populations were assessed using eight polymorphic ISSR markers. The phylogenetic relationship of the clone-varieties with the natural population was estimated using ribosomal (ITS) and chloroplast DNA (rps16 intron, trnL intron, trn L-trn F). The data revealed a high genetic diversity of various clones and samples of Sievers apple tree of the Dzungarian populations. As a result of phylogenetic and cluster analysis the studied clones and samples of M. sieversii from natural populations formed a cluster, samples of supposedly hybrid origin formed 2 clusters, and no clustering was detected depending on geographical distance. The data demonstrate that there is an active information exchange between the populations.

Analysis of genetic variation using ISSR and the development of SCAR marker in synthetic autotetraploids of Vigna mungo (L.) Hepper

Autopolyploidy is one of the foremost mechanisms by which additional numbers of genome copies are acquired resulting in genetic and evolutionary novelty to the organisms. As such, it is considered to be a pathway for adaptation and has played a significant role in plant speciation. Autopolyploids are characterized by increased genome flexibility, allowing them to adapt and persist across heterogeneous landscapes in the long run. Genomic redundancy and polysomic inheritance are the hallmarks of doubled genome(s). In view of the significance of autopolyploidy in plant evolution, plant breeding and crop improvement, we sought to investigate the genetic variation/s associated with genome multiplication by exploiting artificial somatic autopolyploids in Vigna mungo. ISSR was employed to detect and identify any genomic DNA variation in colchitetraploids of V. mungo of three different generations, viz. C0, C1 and C2, in comparison to their putative diploids plants. Our data suggests a random, ‘short-term’ rapid change of the genome that may have occurred immediately after polyploidization. The population of autopolyploids demonstrated some degree of genetic heterozygosity in comparison to their diploid counterparts. Further, a polymorphic ISSR DNA loci was identified among the colchitetraploids which was translated into SCAR marker for identification of colchitetraploids versus the putative diploids of V. mungo.

Van Gölü Havzası Fasulye Genotipleri (Phaseolus vulgaris L.) Arasındaki Genetik Çeşitliliğin Fenotipik ve Moleküler Yöntemlerle Belirlenmesi

Van Gölü havzasının değişik yörelerinden toplanmış olan 95 fasulye genotipinin genetik akrabalık ilişkileri hem fenotipik hem de moleküler yöntemlerle incelenmiştir. Fenotipik yöntemde, 71 adet morfolojik özellik incelenmiş ve bunlar arasında yüksek korelasyon gösterenler değerlendirme dışı bırakılarak fasulye genotiplerine ait 61 adet ölçüm veya gözlemden yararlanılmış; moleküler yöntemde ise 28 primerden elde edilen 219 adet polimorfik ISSR belirteci ve 10 primerden elde edilen 76 adet polimorfik RAPD belirteci kullanılmıştır. Fasulye genotipleri arasındaki genetik akrabalık dereceleri, fenotipik ve moleküler veriler kullanılarak elde edilen değişik matrislerden (Öklid ve Jaccard katsayı matrisleri) dendrogram oluşturularak incelenmiştir. Fenotipik olarak incelenen genotiplerin %69,5‘inin Güney Amerika (Andean) ve %30,5‘inin Orta Amerika (Mesoamerican) orijinli olduğu ve genotipler arasında yüksek genetik çeşitliliğin olduğu saptanmıştır. Fenotipik ve moleküler verilerin birlikte incelenmesi sonucunda, Güney Amerika ve Orta Amerika orijinli genotiplerin; bodur ve sırık genotiplerin; beyaz, diğer tek renkli ve çok renkli tohumlara sahip genotiplerin ayrı kümelenmelerde yer aldıkları gözlenmiştir.

Relationship between chemotypic and genetic diversity of natural populations of Artemisia herba-alba Asso growing wild in Tunisia

A total of 80 individuals collected from eight populations growing wild in different geographic zones were considered to assess the intraspecific variability of essential oil composition, genetic diversity and population structure of Artemisia herba-alba.The essential oil composition varied significantly between populations. Essential oil profiles were classified into four chemotypes (trans-sabinyl acetate, α-thujone/trans-sabinyl acetate, camphor and α-thujone/camphor/β-thujone). Despite significant correlation between the amount of some essential oil compounds and a set of climatic data, the global chemical divergence among populations was not related to their bioclimatic and geographic appurtenances.A high level of genetic diversity within populations was revealed either with RAPD and ISSR markers (Na = 1.67, PPL = 66.5%, H = 0.26, I = 0.38 and Na = 1.7, PPL = 69.8%, H = 0.26, I = 0.38, respectively). The level of genetic diversity varied across populations and chemotypes. Populations from the α-thujone/trans-sabinyl acetate chemotype exhibited the highest genetic diversity as revealed by the RAPD markers. However, populations from α-thujone/camphor/β-thujone chemotype showed the important genetic variation determined by ISSR markers. A significant genetic differentiation among populations and among chemotypes was detected.The combined analysis showed a significant correlation (r = 0.484, p = .032) between the chemical and molecular markers. The PCA, performed on percentages of major oil compounds and the frequencies of polymorphic RAPD and ISSR bands, divided populations according to their chemotypic classification.Taking into consideration the current situation of A. herba-alba populations and their endangered habitats, these results are of value in order to ensure the in-situ and ex-situ conservation of this medicinal species.

ISSR marker-assisted determination of the genetic polymorphism of an original sterile form of winter rape

The aim and tasks of the study. To identify the genetic differences in descendants of an original sterile form of winter rape bred at the Plant Production Institute nd a VYa Yuryev and based on the Ogura- and Polima-type male sterility using microsatellite analysis.Materials and methods. The study was conducted under laboratory conditions in the Plant Production Institute nd a VYa Yuryev of NAAS in 2014-2016. PCR with ISSR markers was carried out in a horizontal Hoefer HE 100 SuperSub unit (USA). Seven accessions based on Ogura- and Polima-type male sterility and the original sterile form were investigated. Molecular weight markers 1 Kb ("SibEnzim", Russia) were used. Gels were documented using a Nikon D50 camera. Amplicon numbers and sizes were determined using a demo version of Totallab100. Frequencies of locus alleles were calculated in Excel. Amplification profiles were evaluated by fingerprinting using informative parameters, such as the index of primer informativeness (IPI). The Botstein’s classification and Shannon’s indices were used. Polymorphism of ISSR markers was assessed using the Nei index of genetic diversity (He). To evaluate the divergence between accessions by ISSR markers, the PHYLIP program was used. Dendrograms were constructed by the nearest neighbor algorythm.Results and discussion. Amplification with five ISSR primers identified 49 DNA fragments, 31 of which were polymorphic, in the accessions under investigation. The amplicon number varied from 5 to 14 fragments, on average 9.8 per primer. The maximum number of loci was identified using primers UBC810 and UBC 834.Amplification with five ISSR primers found 12 allelic variants (UBC810 [587 bp], UBC834 [988, 774, 709 bp], UBC842 [1127, 475 bp], UBC847 [1349, 969, 496 bp] and UBC857 [791, 520, 446 bp]). The Polima accession had no marker loci. The results proved that there were no identical genotypes in the test sample.The absolute and normalized values of theShannonindex showed that UBC 810 was the most polymorphic ISSR marker was (H '= 0.495, H'norm = 0.715). The ISSR marker UBC 857 had the lowest level of polymorphism (H '= 0.364, H'norm = 0.525). The total level of variability in ISSR markers was H'= 0.445 and H'norm = 0.642.The dendrogram distinguished three opposite clusters of rape accessions. Cluster 1 included Bekr (1) D 21, Bekr (1) D 2, Bekr (2) D 16/2 (descendants of the original sterile form); cluster 2 - Polima and MON accessions (descendants with Polima sterility); cluster 3 - Inra and D8 (in vivo) (descendants with Ogura sterility).Conclusions. A high range of intraspecies variability among the rape accessions under investigation was detected by ISSR markers. We established the general patterns of grouping rape accessions, using classification, which was formulated by cluster analysis using the nearest neighbor (NJ) algorithm. The study proved that descendants of the original sterile form had one root and showed that there were significant genetic differences from accessions with the Ogura- and Polima-type male sterility

AFLP, RAPD, and ISSR analysis of intraspecific polymorphism and interspecific differences of allotetraploid species Aegilops kotschyi Boiss. and Aegilops variabilis Eig

To evaluate genetic variation, 27 accessions of allotetraploid species Aegilops kotschyi and Ae. variabilis with the US genome were analyzed using the AFLP, RAPD, and ISSR methods. A total of 316 polymorphic RAPD fragments, 750 polymorphic AFLP fragments, and 234 polymorphic ISSR fragments were obtained. It was demonstrated that the analyzed species were characterized by a considerable level of nuclear genome variation. According to the data of ISSR and RAPD analysis, the average value of the Jaccard similarity coefficient for the accessions of Ae. variabilis from different geographical regions was slightly lower than that for the accessions of Ae. kotschyi. At the same time, AFLP analysis showed no considerable differences in the levels of intraspecific variation of the studied species. Analysis of the summarized RAPD, ISSR, and AFLP marking data in the Structure software program showed that most of the analyzed accessions with high degree of probability could be assigned to one of two groups, the first of which corresponded to Ae. kotschyi and the second corresponded to Ae. variabilis, thereby confirming the species independence of Ae. kotschyi and Ae. variabilis. Accessions k900, k907, k908, and v90 could not with a sufficiently high degree of probability be assigned to one of the species, which possibly was the result of interspecific hybridization. Analysis of the species diversity using different molecular markers made it possible to identify the accessions that were notably different from other accessions of its species.

Phenotyping, Genotyping, and Selections within Italian Local Landraces of Romanesco Globe Artichoke

Ten Italian globe artichoke clones belonging to the Romanesco typology were characterized in the western coastal area of Italy (Cerveteri, Rome), using a combination of morphological (UPOV descriptors), biochemical (HPLC analysis), and molecular (AFLP, ISSR, and SSR markers) traits. Significant differences among clones were found for many of the quantitative and qualitative morphological traits. Multivariate analyses (Principal Component Analysis) showed that, of the 47 morphological descriptors assessed, four (i.e., plant height, central flower-head weight, earliness, and total flower-head weight) presented a clear grouping of the clones. Biochemical analyses showed that the clones significantly differed in the polyphenolic profiles of the flower-head, with the suggestion that some of these, such as S2, S3, S5, and S18, are more suitable for the fresh market. The clones, clustered by a UPGMA dendrogram based on 393 polymorphic AFLP and ISSR loci, showed that the clones were genetically separated from each other. This highlights the importance of characterizing, evaluating, and conserving autochthonous germplasm for future plant breeding activities. Overall, these studies resulted in the identification of two new clones, selected on the basis of flower-head morphology and earliness. These clones, named Michelangelo and Raffaello, are registered on the Italian National Register of Varieties (DM n. 6135, 3/29/2013 G.U. 91, 18 April 2013).

Largely invariant trnL-F/nrITS sequences and ISSRs do not resolve morphospecies of Warburgia (Canellaceae)

The aim of this study was to provide a molecular perspective on the taxonomy of Warburgia across most of its range, including South Africa, Zimbabwe, Mozambique, Tanzania, Kenya and Uganda. An accurate taxonomic treatment of Warburgia is important for the construction of sound conservation strategy, as a number of the proposed species and subspecies are regarded as endangered or critically endangered. Analyses based on 702 nucleotides of the plastid trnL-F region, 532 nucleotides of the nuclear ribosomal (nr) ITS region and eight polymorphic ISSR primer pairs yielded congruent results. Molecular clock analyses yielded a crown age of 45.5 (31.2–64.9) MY for Canellaceae. The crown age of Warburgia was dated to the early Miocene, 19.6 (11.4–29.1) MYA, with diversification into moderately supported lineages in the later Miocene, 11.6 (6.0–19.0) and 7.2 (3.2–12.7) MYA. Warburgia samples formed three major clades: a widespread clade covering most of the range, a northeast clade from parts of Kenya and Tanzania, and a stuhlmannii clade formed by GenBank samples accessioned as W. stuhlmannii. There was some ambiguity in the association between these clades and morphologically defined Warburgia species. We found no evidence to support W. elongata as a genetically distinct species. Genetic diversity in Warburgia was low in both DNA markers. We propose inclusion of these haplotypes in conservation strategies. Further molecular analyses based on a greater number of more variable DNA regions and an expanded sample set with more extensive geographical coverage are required to clarify whether the above groupings warrant status as genetic or phylogenetic species and to refine conservation strategies so as to include greater genetic variability.

Genetic diversity ofAchillea fragrantissimain Egypt inferred from phenotypic variations and ISSR markers associated with traits of plant size and seed yield

Substantial variation in phenotypic traits and ISSR fingerprinting was observed among twenty populations ofAchillea fragrantissima(Forssk.) Sch. Bip. in Egypt. Such variation was reflected in the clustering of the examined populations into two major groups: one representing populations in the mountainous area of South Sinai and the other comprising populations growing at low elevations in the middle of Sinai and the desert west of the Suez Canal from Suez in the east to Cairo in the west. Five populations in the eastern part of Sinai near Nuwieba and Taba on the Gulf of Aqaba were loosely assigned to the first group. The populations growing at high elevations in South Sinai, under lower temperature and higher humidity, were characterized by a higher number of total and polymorphic ISSR markers compared with other populations. Unique ISSR markers were more often observed in the fingerprinting of seven populations including five populations growing in the high mountains of Saint Catherine in South Sinai and two populations growing at low elevations but at Wadi Hof south-east of Cairo. Interestingly, unique bands were found in the populations that possessed traits associated with larger plant size and seed yield as well as better vigour. These are important criteria for the selection ofA. fragrantissimapopulations for conservation and sustainable commercial use.