Are current ecological restoration practices capturing natural levels of genetic diversity? A New Zealand case study using AFLP and ISSR data from mahoe (Melicytus ramiflorus)

Sourcing plant species of local provenance (eco-sourcing) has become standard practice in plant community restoration projects. Along with established ecological restoration practices, knowledge of genetic variation in existing and restored forest fragments is important for ensuring the maintenance of natural levels of genetic variation and connectivity (gene flow) among populations. The application of restoration genetics often employs anonymous ‘fingerprinting’ markers in combination with limited sample sizes due to financial constraints. Here, we used two such marker systems, AFLPs and ISSRs, to estimate population-level genetic variation of a frequently used species in restoration projects in New Zealand, māhoe (Melicytus ramiflorus, Violaceae). We examined two rural and two urban forest fragments, as potential local source populations, to determine whether the māhoe population at the recently (re)constructed ecosystem at Waiwhakareke Natural Heritage Park (WNHP), Hamilton, New Zealand reflects the genetic variation observed in these four potential source populations. Both marker systems produced similar results and indicated, even with small population sizes, that levels of genetic variation at WNHP were comparable to in situ populations. However, the AFLPs did provide finer resolution of the population genetic structure than ISSRs. ISSRs, which are less expensive and technically less demanding to generate than AFLPs, may be sufficient for restoration projects where only a broad level of genotypic resolution is required. We recommend the use of AFLPs when species with a high conservation status are being used due to the greater resolution of this technique

Measuring Plant Genetic Diversity Using Inter-Simple Sequence Repeats (ISSRS)

Simple sequence repeats (SSRs) have great utility as they are conserved and present in all eukaryotic genomes. Here we report the use of a simple PCR with fluorescently-labelled primers to amplify inter-SSR markers (ISSRs) for diversity assessments. The use of ISSR markers does not rely upon specific genetic sequence information, or prolonged method development and may be measured rapidly using the automated equipment. The major restriction of the ISSR method is at the analysis stage, as the markers are dominant it is not possible to distinguish heterozygotes as loci. We obtained ISSR data from ca. 60 phenotypically characterised Capsella bursa pastoris L. Medic (shepherds purse)accessions that had been isolated from a diverse mix of arable field sites throughout the UK. We developed mathematical scripts for use with the free statistical software tool R (http://www.rproject.org/), that processed the molecular data in a binary format to estimate genetic diversity (using the Jaccard co-efficient), and that related genotype to the plant phenotypic and environmental (site specific) traits. The methodology established has the power to predict the relationship between environmental and plant morphological characteristics

An efficient protocol to perform genetic traceability of tissue and foods from Geoffroea decorticans

The quality of a DNA isolation method depends, among others, on the target tissue and the metabolites therein. Geoffroea decorticans Burkart (chanar) is a species that has nutritional and pharmacological potential. However, an effective method of DNA extraction capable of facilitating population studies and food genetic traceability has not been studied yet. The objective of the present work was to evaluate four methods of DNA extraction from leaves and chanar-based foods. The methods were evaluated based on yield, DNA purity, and molecular markers. The CCI-P (CTAB/Chloroform-Isoamylalcohol/pellet) method showed the highest yield of DNA obtained from leaves. However, the CPCI-SC (CTAB/Phenol-Chloroform-Isoamylalcohol/silica-column) method was the only one that resulted in acceptable DNA quality with both parameters (A260/A280 and A260/A230). The leaf DNA obtained with this method showed a greater amount of fragments with RAPD, and an acceptable amount of fragments with ISSR. On the other hand, the CCI-P method showed a higher yield of DNA from arrope de chanar (syrup). However, the CPCI-SC method was the only one that had relatively better DNA quality, which allowed the amplification of molecular markers. Regarding chanar flour, the CPCI-SC method showed the highest yield, DNA quality and good amplification with molecular markers. Therefore, the CPCI-SC extraction method is efficient for obtaining DNA from different matrices, and can support studies for a possible designation of origin of chanar-based foods

High Genetic Diversity and Low Differentiation of Michelia coriacea (Magnoliaceae), a Critically Endangered Endemic in Southeast Yunnan, China

Michelia coriacea, a critically endangered tree, has a restricted and fragmented distribution in Southeast Yunnan Province, China. The genetic diversity, genetic structure and gene flow in the three extant populations of this species were detected by 10 inter-simple sequence repeat (ISSR) markers and 11 simple sequence repeat (SSR) markers. Examination of genetic diversity revealed that the species maintained a relatively high level of genetic diversity at the species level (percentage of polymorphic bands) PPB = 96.36% from ISSRs; PPL (percentage of polymorphic loci) = 95.56% from SSRs, despite several fragmental populations. Low levels of genetic differentiation among the populations of M. coriacea were detected by Nei’s Gst = 0.187 for ISSR and Wright’s Fst = 0.090 for SSR markers, which is further confirmed by Bayesian model-based STRUCTURE and PCoA analysis that could not reveal a clear separation between populations, although YKP was differentiated to other two populations by ISSR markers. Meanwhile, AMOVA analysis also indicated that 22.84% and 13.90% of genetic variation existed among populations for ISSRs and SSRs, respectively. The high level of genetic diversity, low genetic differentiation, and the population, structure imply that the fragmented habitat and the isolated population of M. coriacea may be due to recent over-exploitation. Conservation and management of M. coriacea should concentrate on maintaining the high level of genetic variability through both in and ex-situ conservation actions

Molecular Genetic Diversity Study of Forest Coffee Tree (Coffea arabica L.) Populations in Ethiopia: Implications for Conservation and Breeding

Coffee provides one of the most widely drunk beverages in the world, and is a very important source of foreign exchange income for many countries. Coffea arabica, which contributes over 70 percent of the world's coffee productions, is characterized by a low genetic diversity, attributed to its allopolyploidy origin, reproductive biology and evolution. C. arabica has originated in the southwest rain forests of Ethiopia, where it is grown under four different systems, namely forest coffee, small holders coffee, semi plantation coffee and plantation coffee. Genetic diversity of the forest coffee (C. arabica) gene pool in Ethiopia is being lost at an alarming rate because of habitat destruction (deforestation), competition from other cash crops and replacement by invariable disease resistant coffee cultivars. This study focused on molecular genetic diversity study of forest coffee populations in Ethiopia using PCR based DNA markers such as random amplified polymorphic DNA (RAPD), inverse sequence-tagged repeat (ISTR), inter-simple sequence repeats (ISSR) and simple sequence repeat (SSR) or microsatellites. The objectives of the study are to estimate the extent and distribution of molecular genetic diversity of forest coffee and to design conservation strategies for it’s sustainable use in future coffee breeding. In this study, considerable samples of forest coffee collected from four coffee growing regions (provinces) of Ethiopia were analysed. The results indicate that moderate genetic diversity exists within and among few forest coffee populations, which need due attention from a conservation and breeding point of view. The cluster analysis revealed that most of the samples from the same region (province) were grouped together which could be attributed to presence of substantial gene flow between adjacent populations in each region in the form of young coffee plants through transplantation by man. In addition wild animals such as monkeys also play a significant role in coffee trees gene flow between adjacent populations. The overall variation of the forest coffee is found to reside in few populations from each region. Therefore, considering few populations from each region for either in situ or ex situ conservation may preserve most of the variation within the species. For instance, Welega-2, Ilubabor-2, Jima-2 and Bench Maji-2 populations should be given higher priority. In addition, some populations or genotypes have displayed unique amplification profiles particularly for RAPD and ISTR markers. Whether these unique bands are linked to any of the important agronomic traits and serve in marker assisted selections in future coffee breeding requires further investigations

The impact of one-decade ecological disturbance on genetic changes : a study on the brine shrimp Artemia urmiana from Urmia Lake, Iran

Urmia Lake, the largest natural habitat of the brine shrimp Artemia urmiana, has progressively desiccated over the last two decades, resulting in a loss of 80% of its surface area and producing thousands of hectares of arid salty land. This ecological crisis has seriously affected the lake's native biodiversity. Artemia urmiana has lost more than 90% of its population during the decade from 1994 (rainy period) to 2004 (drought period) due to salinity increasing to saturation levels (similar to 300 g/l). We studied the influence of this ecological crisis on the genetic diversity of A. urmiana in Urmia Lake, based on one cyst collections in 1994 and 2004. AMOVA analysis on ISSR data demonstrated a 21% genetic variation and there was a 5.5% reduction of polymorphic loci between samples. PCoA showed that 77.42% and 68.75% of specimens clustered separately in 1994 and 2004, respectively. Our analyses of four marker genes revealed different genetic diversity patterns with a decrease of diversity at ITS1 and an increase for Na+/K+ ATPase. There was no notable difference in genetic variation detected for CO/ and 16S genes between the two periods. However, they represented distinctly different haplotypes. ITS1 and COI followed a population expansion model, whereas Na+/K+ ATPase and 16S were under demographic equilibrium without selective pressure in the 1994 samples. Neutrality tests confirmed the excess of rare historical and recent mutations present in COI and ITS1 in both samples. It is evident that a short-term ecological disturbance has impacted the genetic diversity and structure of A. urmiana

Multilocus haplotyping by parallel sequencing to decipher the interspecific mosaic genome structure of cultivated citrus

The most important economic Citrus species originated from natural interspecific hybridization between four ancestral taxa (C. reticulata, C. maxima, C. medica and C. micrantha) with limited further interspecific recombination due to apomixis and vegetative propagation. Such reticulate evolution coupled with vegetative propagation results in genomes that are mosaics of large chromosome fragments of the basic taxa, in frequent interspecific heterozygosity. Breeding of these species is hampered by their complex heterozygous genomic structures. Haplotyping of multiple gene fragments along the genome should be a powerful approach to resolve the evolutionary history of the gene pools, to reveal the admixture genomic structure of current species and to develop innovative breeding schemes. We have analysed the efficiency of parallel sequencing with 454 methodology to decipher the hybrid structure of modern citrus species and cultivars along chromosome 2. Four hundred fifty four amplicon libraries were established with the fluidigm array system for 48 genotypes and 16 gene fragments of chromosome 2. Haplotypes were established from the reads of each accession and phylogenetic analyses were performed from the haplotypic data of each gene fragment. The length of 454 reads and the level of differentiation between the ancestral taxa of modern citrus allowed efficient haplotype phylogenetic assignations for 12 of the 16 gene fragments. The analysis of the mixed genomic structure of modern species and cultivars (i) revealed C. maxima introgressions in modern mandarins; (ii) was consistent with previous hypothesis regarding the origin of secondary species; and (iii) provided a new picture of the evolution of chromosome 2. Perspectives to rebuild the main secondary species from the basic taxa are discussed. (Résumé d'auteur

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding.

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids