Genetic diversity and differentiation in natural and reintroduced populations of Bencomia exstipulata and comparisons with B. caudata (Rosaceae) in the Canary Islands: an analysis using microsatellites

Variation at five polymorphic microsatellite loci was used to investigate genetic diversity and differentiation of two tetraploid Canarian endemics, Bencomia exstipulata and B. caudata. Data were analysed and are discussed in terms of tetrasomic (autotetraploid) and disomic (allotetraploid) inheritance. In both cases, genetic diversity values were similar to those described in other tetraploid plant species. High genetic differentiation between the only two described natural populations of B. exstipulata was detected (F_ST = 0.411). Bayesian cluster analysis revealed a geographical structure with distinct genetic groups from each island. High genetic differentiation and low genetic diversity of the B. exstipulata population from Tenerife suggest a recent population bottleneck, perhaps caused by the most recent major volcanic eruption, for this natural locality. This may be heightened by possible inbreeding depression and the monoecy of these species. Polymorphic microsatellite loci were also tested across all species in the Bencomia alliance. These reliably amplified the target sequence, suggesting a high degree of conservation of the sequences flanking the microsatellites. © 2009 The Linnean Society of London, Botanical Journal of the Linnean Society, 2009, 160 , 429–441.     

Dual influence of terrestrial and marine historical processes on the phylogeography of the Brazilian intertidal red alga Gracilaria caudata

In this study, we explored how past terrestrial and marine climate changes have interacted to shape the phylogeographic patterns of the intertidal red seaweed Gracilaria caudata, an economically important species exploited for agar production in the Brazilian north‐east. Seven sites were sampled along the north‐east tropical and south‐east sub‐tropical Brazilian coast. The genetic diversity and structure of G. caudata was inferred using a combination of mitochondrial (COI and cox2‐3), chloroplast (rbcL) and 15 nuclear microsatellite markers. A remarkable congruence between nuclear, mitochondrial and chloroplast data revealed clear separation between the north‐east (from 03° S to 08° S) and the south‐east (from 20° S to 23° S) coast of Brazil. These two clades differ in their demographic histories, with signatures of recent demographic expansions in the north‐east and divergent populations in the south‐east, suggesting the maintenance of several refugia during the last glacial maximum due to sea‐level rise and fall. The Bahia region (around 12° S) occupies an intermediate position between both clades. Microsatellites and mtDNA markers showed additional levels of genetic structure within each sampled site located south of Bahia. The separation between the two main groups in G. caudata is likely recent, probably occurring during the Quaternary glacial cycles. The genetic breaks are concordant with (i) those separating terrestrial refugia, (ii) major river outflows and (iii) frontiers between tropical and subtropical regions. Taken together with previously published eco‐physiological studies that showed differences in the physiological performance of the strains from distinct locations, these results suggest that the divergent clades in G. caudata correspond to distinct ecotypes in the process of incipient speciation and thus should be considered for the management policy of this commercially important species.     

Transferability and characterization of microsatellite markers from Byrsonima cydoniifolia A. Juss. (MALPIGHIACEAE) in seven related taxa from Cerrado biome reveal genetic relationships

Byrsonima Rich. is one of the largest genera of the Malpighiaceae family with 97 species occurrence in Brazil and multiple potentialities, including pharmaceutical and food industries. In this study, 17 microsatellite markers characterized in Byrsonima cydoniifolia were tested for seven related taxa, all species are native to Brazil and four are endemic. Genomic DNA was extracted from leaves tissues and 17 microsatellite markers were used to cross-amplification of microsatellite regions. Polymorphism and genetic diversity were evaluated for B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. umbellata, B. linearifolia. from 16 individuals and for B. viminifolia from 14 individuals. Transferred microsatellite markers panels ranged from 11 (64.8%) in B. viminifolia to 6 (35.2%) in B. umbellata. The total number of alleles per locus ranged from 5 (B. linearifolia) to 8 (B. subterranea) alleles. B. umbellata showed lower values of observed and expected heterozygosity (H_O?=?0.312; H_E?=?0.436) and B. subterranea presented the highest values (H_O?=?0.687; H_E?=?0.778). A greater number of microsatellite markers should be developed for B. umbellata. The microsatellite marker panels transferred to the species B. intermedia, B. verbascifolia, B. laxiflora, B. subterranea, B. viminifolia and B. linearifolia are very informative, with a high combined probability of exclusion of paternity (Q?≥?0.976) and the low combined probability of identity (I?≤?9.91?×?10^–6), potentially suitable for future genetic-population studies, supporting strategies for maintaining the genetic diversity and for exploration of Byrsonima species as genetic resources.

     

Isolation and characterization of microsatellite markers from Musa balbisiana

This is the first report of targeted development of B genome microsatellite markers in Musa. A total of 44 sequences with microsatellites were isolated from an enriched library of Musa balbisiana cv. ‘Tani’ (BB genome). Of these, 25 were polymorphic when screened on 14 diverse diploid and triploid Musa accessions. The number of alleles detected by each marker ranged between one and seven. All 25 microsatellite markers generated amplification products in all species and genome complements. These new microsatellite markers fill an important gap for diversity assessment and linkage mapping studies in plantain (AAB) and cooking banana (ABB).     

Isolation and characterization of microsatellite markers from malaria vector,Anopheles culicifacies

Anopheles culicifacies, an important vector in the Indian subcontinent is a complex of five sibling species of which four are vectors. We describe the isolation of 31 microsatellite markers from the recently recognized isomorphic species A of which 13 were characterized in sympatric populations of Anopheles culicifacies isomorphic species A and B. The allele frequencies ranges from two to 12 in species A and two to seven in species B. Species A being a vector, and that these markers can be used in closely related species, makes the isolation of these markers important to study population structure of all sibling species in this complex.     

Development and characterization of microsatellite markers in Eucommia ulmoides Oliver (Eucommiaceae)

Twenty‐three microsatellite markers were developed from an AC‐enriched genomic library of Eucommia ulmoides, an economically important tree species for both herbal medicine and organic chemical industry in China. Nineteen microsatellite loci were found polymorphic by testing 36 individuals from 10 populations, with two to 14 alleles per locus. The expected heterozygosity ranged from 0.054 to 0.874. This set of microsatellite markers has provided a useful tool for the ongoing efforts in studying population genetic structure of E. ulmoides.     

Assessment of genetic diversity and relationships of Krachaai Sayam,an endemic plant in Thailand using microsatellite markers

Nineteen microsatellite markers were developed from Boesenbergia siamensis (Gagnep.) P. Sirirugsa, an endemic plant of Kanchanaburi Province, Thailand and used to determine genetic diversity of this plant. The number of alleles of each locus varied from 4 to 16 in 25 individual samples. The average values of observed and expected heterozygosities were 0.577 and 0.804, respectively. The polymorphic information content value ranged between 0.486 and 0.912. Ten loci significantly deviated from Hardy–Weinberg equilibrium, and eight pairs of loci showed significant evidence of linkage disequilibrium. Moreover, these newly developed microsatellite markers were used to determine cross-species amplification with three samples each of B. thorelii (Gagnep.) Loes., B. rotunda (L.) Mansf. and Kaempferia parviflora Wall. Of the 19 markers developed, 13 (68.42%) primer pairs were able to be used with B. thorelii (Gagnep.) Loes. and K. parviflora Wall., while 12 (63.16%) primer pairs were able to be used with B. rotunda (L.) Mansf. These microsatellite markers will be useful for further genetic analysis of this endemic plant as well as related species.     

Microsatellite markers for the large blue butterflies Maculinea nausithous and Maculinea alcon (Lepidoptera: Lycaenidae) and their amplification in other Maculinea species

We developed microsatellite markers for Maculinea nausithous and Maculinea alcon, two of five species of endangered large blue butterflies found in Europe. Two separate microsatellite libraries were constructed. Eleven markers were developed for M. nausithous and one for M. alcon. The primers were tested on both species as well as on the three other European Maculinea species. The number of alleles per locus ranged from two to 14. These markers will be useful tools for population genetic studies of Maculinea species.     

Comprehensive population viability study of a rare endemic shrub from the high mountain zone of the Canary Islands and its conservation implications

Oceanic island ecosystems harbour many endemic plant and animal species, which are often threatened because they have only a few small populations. Many factors contribute to the biological viability of such populations, such as demography and population dynamics, breeding system and pollination ecology, seed dispersal and genetic variation. In a collaborative project, all these factors were studied in the rare endemic, predominantly monoecious shrub Bencomia exstipulata Svent. (Rosaceae), which grows exclusively in the national parks of El Teide (Tenerife) and La Caldera de Taburiente (La Palma). Demography was monitored through annual censuses of individual plants in a natural and an augmented population on Tenerife. The breeding system and reproductive success were studied through bagging and pollination experiments, and insect visitation censuses. Seed dispersal by animals was assessed using cafetaria experiments. With matrix projection models and stochastic simulations, we show that the Tenerife population was demographically stable. This was largely explainable by the high survival of adult individuals. Despite frequent germination, successful seedling recruitment was very rare. Male and female flowers occurred in separate inflorescences within individuals, although some inflorescences were mixed and some shrubs were entirely male or female. Despite frequent visits by honeybees, the species is predominantly wind pollinated. Insect-proof bags reduced seed set by 12.5%, and pollen-proof bags by 44%. Large quantities of airborne pollen were detected on unbagged sticky microscope slides, this was 56% reduced by insect-proof and 96% by pollen-proof bags. Hence, some self-pollination also seems to occur. Cafetaria experiments showed that the local lizards (Gallotia galloti Oudart) readily eat the fruits and that the seeds pass through their intestines unharmed and germinable. Since other dispersal vectors are unknown, saurochory seems the most likely mode of dispersal. Our study strongly suggests that the population of B. exstipulata on Tenerife is viable, and that there are no significant threats associated with its breeding system, pollination or seed dispersal. To alleviate the natural extinction risk typical of narrow endemics, five main conservation measures are proposed.     

Comparative effectiveness of sugar beet microsatellite markers isolated from genomic libraries and GenBank ESTs to map the sugar beet genome

Sugar beet (Beta vulgaris) is an important root crop for sucrose production. A study was conducted to find a new abundant source of microsatellite (SSR) markers in order to develop marker assistance for breeding. Different sources of existing microsatellites were used and new ones were developed to compare their efficiency to reveal diversity in mapping population and mapping coverage. Forty-one microsatellite markers were isolated from a B. vulgaris ssp maritima genomic library and 201 SSRs were extracted from a B. vulgaris ssp vulgaris library. Data mining was applied on GenBank B. vulgaris expressed sequence tags (ESTs), 803 EST-SSRs were identified over 19,709 ESTs. Characteristics, polymorphism and cross-species transferability of these microsatellites were compared. Based on these markers, a high density genetic map was constructed using 92 F₂ individuals from a cross between a sugar and a table beet. The map contains 284 markers, spans over 555 cM and covers the nine chromosomes of the species with an average markers density of one marker every 2.2 cM. A set of markers for assignation to the nine chromosomes of sugar beet is provided.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Development and characterization of microsatellite loci for Rosa odorata var. gigantea Rehder & E. H. Wilson (Rosaceae)

Eighteen microsatellite markers were developed from Rosa odorata var. gigantea (Rosaceae), including 11 new microsatellite markers and 7 modified microsatellite loci having been developed from other Rosa species. About 27 wild individuals from 3 populations were used to screen polymorphism of these 18 microsatellite makers. The average allele number of these markers was 3.9 per locus, ranging from 2 to 9. The expected and observed heterozygosities varied from 0.2711 to 0.8043 and from 0.0370 to 0.5556, respectively. Cross-species amplification in other eight Rosa species showed their potential use for congeneric species. These microsatellite primers will be used for population genetics studies, constructing genetic linkage maps or locating quantitative trait locus (QTL) of R. odorata var. gigantea and related species.     

Development and characterization of ten new microsatellite markers in a mangrove tree species Bruguiera gymnorrhiza (L.)

Bruguiera gymnorrhiza is an ecologically and somewhat economically important mangrove tree species. We isolated 10 polymorphic microsatellite loci from B. gymnorrhiza using a dual‐suppression polymerase chain reaction (PCR) technique. These loci provided microsatellite markers with polymorphism of two to five alleles per locus within 216 individuals from nine natural populations of B. gymnorrhiza on Iriomote Island, the Sakishima Islands, Japan. The expected and observed heterozygosities ranged from 0.220 to 0.720 and from 0.104 to 0.447, respectively.     

Identification and characterization of 10 microsatellite primers for the calanoid freshwater copepods Eudiaptomus gracilis and E. graciloides using enriched genomic libraries

The calanoid copepods Eudiaptomus gracilis and Eudiaptomus graciloides are widespread among European lakes. We constructed enriched genomic libraries for both species in order to isolate and characterise microsatellite markers. The obtained 7 polymorphic microsatellite‐loci for E. gracilis and 3 for E. graciloides are the first for any freshwater copepod. They display an expected heterozygosity ranging from 0.60 to 0.96 and 0.63 to 0.94 and observed heterozygosity ranging from 0.53 to 0.87 and 0.57 to 0.87, respectively. We were not able to cross‐amplify the isolated loci across species, indicating long divergence among both congeneric species despite morphological similarity.     

Isolation and characterization of simple sequence repeat loci in Rubus hochstetterorum and their use in other species from the Rosaceae family

The identification of microsatellite loci in Rubus hochstetterorum provides an important tool for the characterization and conservation of wild populations of this species. Cross‐species amplification of markers may be of particular interest for the study of other Rubus species. In this study, 41 simple sequence repeat markers were identified in a genomic library of R. hochstetterorum. Fifteen of the identified microsatellite loci were characterized in a set of 30 samples and revealed to be polymorphic with three to 19 alleles per locus. All the identified markers allowed cross‐species amplification in at least one of the other three tested species from the Rosaceae family.     

A comparative study of interspecies mating of Phratora vulgatissima and P. vitellinae using behavioural tests and molecular markers

The leaf beetle genus Phratora L. (Coleoptera: Chrysomelidae) has been used to study the ecology of host plant chemicals in herbivore preference, and the evolution of host use in chemical defence. Phratora vulgatissima and P. vitellinae are sympatric species distributed widely across Europe. Their trophic niches are largely separate due to strong differences in their host feeding preference, but they have occasionally been recorded together, feeding on Salix burjatica‘Germany’ and, only in early spring, on Populus trichocarpa (Torr & A. Gray) ‘Trichobel’. Using behavioural tests and recently developed species‐specific microsatellite markers, the intra‐ and interspecific mating of both beetle species were investigated. The microsatellite markers provided evidence that interspecific mating occurred under field conditions. Interspecific mating also took place under laboratory conditions, but less frequently than mating within species. Females of both species laid fewer eggs, and fewer eggs per clutch, when isolated with an interspecific male than with a conspecific male. Female P. vulgatissima were polyandrous, as microsatellite markers showed that their larvae were the progeny of both P. vulgatissima males that had been isolated with a single female. While only 0.55% of eggs laid in interspecific pair combinations hatched, microsatellite markers provided evidence of hybridisation between beetle species; however, these larvae died within a week when reared in a Petri dish containing ‘Germany’ and P. trichocarpa leaves. It can therefore be inferred that reproductive isolation is complete. The results are discussed in relation to species integrity and the implications for diverse mixtures of short‐rotation coppice willow plantations.     

Evaluation of allelic diversity at chloroplast microsatellite loci among common wheat and its ancestral species

Twenty four chloroplast microsatellite loci having more than ten mononucleotide repeats were identified from the entire chloroplast DNA sequence of common wheat, Triticum aestivum cv Chinese Spring. For each microsatellite, a pair of primers were designed to produce specific PCR products in the range of 100– 200 bp. The allelic diversity at the microsatellite loci was evaluated using 43 accessions from 11 Triticum and Aegilops species involved in wheat polyploid evolution. Polymorphic banding patterns were obtained at 21 out of 24 chloroplast microsatellite loci. The three monomorphic microsatellites were found to be located in coding regions. For the polymorphic microsatellites, the number of alleles per microsatellite ranged from 2 to 7 with an average of 4.33, and the diversity values (H) ranged from 0.05 to 0.72 with an average of 0.47. Significant correlations (P<0.01) were observed between the number of repeats and the number of alleles, and between the number of repeats and diversity value, respectively. The genetic diversity explained by chloroplast microsatellites and nuclear RFLP markers were compared using 22 tetraploid accessions. Although the number of alleles for nuclear RFLP markers was found to be higher than that for chloroplast microsatellites, similar diversity values were observed for both types of markers. Among common wheat and its ancestral species, the percentages of common chloroplast microsatellite alleles were calculated to examine their phylogenetic relationships. As a result, Timopheevi wheat species were clearly distinguished from other species, and Emmer and common wheat species were divided into two main groups, each consisting of a series of wild and cultivated species from tetraploid to hexaploid. This indicates that the two types of chloroplast genomes of common wheat might have independently originated from the corresponding types of wild and cultivated Emmer wheat species. Received: 6 October 2000 / Accepted: 13 March 2001     

Development, inheritance and cross-species amplification of microsatellite markers from Acacia mangium

Microsatellite markers were developed in Acacia mangium Willd. to provide highly variable co-dominant markers for linkage mapping and studies of the breeding system. After an enrichment procedure 40% of colonies contained microsatellites in contrast with less than 1% from a non-enriched library. The majority of microsatellite sequences were AC repeats. Co-dominant segregation of alleles in two full-sib crosses of A. mangium was demonstrated at 33 microsatellite loci. The markers were highly variable relative to restriction fragment lengths polymorphisms (RFLPs). In the two pedigrees 53% of microsatellite loci were fully informative compared with 15% of RFLPs. Based on alleles detected among four parental genotypes, the microsatellites consisting of dinucleotide repeats were more polymorphic than those with tri- and tetra-nucleotide repeats. The microsatellite markers were not as transferable across species in the genus Acacia as RFLPs. Two thirds of the primers developed in A. mangium (subgenus Phyllodineae, section Juliflorae) amplified DNA from other species within the same section but failed to amplify in species from the subgenus Acacia. The availability of multiallelic, PCR-based, co-dominant microsatellite loci makes possible efficient studies of gene flow and breeding systems in A. mangium, a species with low allozyme variation. Received: 30 December 1999 / Accepted: 10 May 2000     

Cross-species amplification of microsatellite loci within the dioecious, polyploid genus Actinidia (Actinidiaceae)

Microsatellite marker transfer across species in the dioecious genus Actinidia (kiwifruit) could offer an efficient and time-effective technique for use during trait transfer for vine and fruit improvement in breeding programmes. We evaluated the cross-species amplification of 20 EST-derived microsatellite markers that were fully informative in an Actinidia chinensis mapping family. We tested all 20 markers on 120 genotypes belonging to 21 species, 5 with varieties and/or chromosome races. These 26 taxa included 16 diploids, 7 tetraploids, 2 hexaploids and 1 octaploid, and represented all four taxonomic sections in the genus. All 20 markers showed some level of cross-species amplification. The most successful marker amplified in all genotypes from all species from all sections of the genus, the least successful amplified fragments only in A. chinensis and A. deliciosa. One species, A. glaucophylla, failed to amplify with all but 2 markers. PIC (Polymorphism information content) values were high, with 14 of 17 markers recording values of 0.90 and above. Sequence data demonstrated the presence of the microsatellite in all the amplified products. Sequence homology was less 5′ of the microsatellite and increased toward the start codon of the translated region of the EST from which the marker was derived. The data confirm that EST-derived microsatellite markers from Actinidia species show cross-species amplification with high levels of polymorphism which could make them useful markers in breeding programmes.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.