A comparison of genetic variation among wild and cultivated Morus Species (Moraceae: Morus) as revealed by ISSR and SSR markers

In this study, inter-simple sequence repeats (ISSR) ans simple sequence repeat (SSR) markers were used to investigate genetic diversity of 27 mulberry accessions including 19 cultivated accessions (six M. multicaulis, three M. alba, two M. atropurpurea, two M. bombycis, one M. australis, two M. rotundiloba, one M. alba var. pendula, one M. alba var. macrophylla, and one M. alba var. venose) and 8 wild accessions (two M. cathayana, two M. laevigata, two M. wittiorum, one M. nigra and one M. mongolica). ISSRs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 27 mulberry genotypes. SSRs presented a higher level of polymorphism and greater information content. All index values of genetic diversity both markers analyzed using Popgene 32 software indicated that within wild species had higher genetic diversity than within cultivated species. Cultivation may caused the lose of genetic diversity of mulberry compared with wild species revealed by ISSR and SSR markers. The mean genetic similarity coefficients among all mulberry genotypes ascribed by ISSR and SSR matrices were 0.7677 and 0.6131, respectively. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. Cluster analysis of ISSR and SSR using UPGMA method revealed that the wild species are genetically distant from the domesticated species studied here. The correlation coefficients of similarity were statistically significant for both marker systems used. Principal coordinates analysis (PCA) for ISSR and SSR data also supports their UPGMA clustering. These results have an important implication for mulberry germplasm characterization, improvement, molecular systematics and conservation.     

Chloroplast Genome of Native Silene latifolia subsp. alba from Fennoscandia Shows High Level of Differences from Invasive White Campion

Silene latifolia is an herbaceous plant with great invasive potential. Spread along trade routes from Europe to almost all continents, white campion became particularly widespread in North America. We sequenced the chloroplast genome of S. latifolia subsp. alba from a native range in southeast Fennoscandia. The chloroplast genome of native S. latifolia subsp. alba forms a 151,747-bp circle, has two inverted repeat regions (25,993 bp each), large single copy (82,708 bp), and small single copy (17,106 bp) regions. It contains 77 protein-coding genes, 30 tRNA genes, and four rRNA genes. SSRs and long DNA repeats were identified. Comparison of a newly sequenced plastome of S. latifolia subsp. alba with plastomes of invasive specimens of species from North America and Japan revealed a high level of single nucleotide polymorphisms (SNPs) among them. A total of 214 SNPs were found, among which 110 were identified in intergenic spacers, 74 in exons, and 30 in introns. Intraspecific shifts in inverted repeat boundaries were identified. Our research suggests that high polymorphic regions may be potential molecular markers for population studies and that high intraspecific genetic polymorphism may contribute to a species’ invasive success.

     

Nuclear cyp73 intron fragment length polymorphism supports morphological analysis of Salix species and hybrids

Abstract

The polyploid Salix alba L.–Salix fragilis L. hybrid complex still presents major difficulties in morphological identification. Most of the measured characters show a low diagnostic value for unambiguously identifying the parental species and their hybrid Salix × rubens Schrank due to continuous variation creating a large overlap in leaf and catkin morphology. Fragment length polymorphism of nuclear cyp73 intron markers was used to identify species and hybrids. This multilocus genotyping could be applied in a morphological analysis of trees from hybrid zones and allowed to demonstrate that morphological features of leaves and catkins clearly separated S. alba from S. fragilis. The hybrid individuals largely overlapped with both parental species but appeared to be morphologically more similar to S. fragilis than to S. alba. Cyp73 analysis of 11 Salix taxa revealed intermediate positions of two hybrid taxa with S. alba, namely S. × rubens and S. × sepulcralis Simonkai with their respective parental species S. fragilis and S. babylonica L. Additionally, the cyp73 intron multilocus genotypes clustered tetraploid taxa separately from diploid willows. Cyp73 introns are valuable markers for fast, reliable and straightforward genotyping in willow species and hybrids.     

Identification of a mutant fatty acid elongase allele from zero-percent erucic acid Sinapis alba

The fae1 gene codes for KCS (β-keto-acyl-CoA synthase), the candidate enzyme for elongation of oleic acid to eicosenoic acid and erucic acid (C22:1) in various oilseed species. Degenerate primers for the fae1 gene were used to amplify and clone fae1 gene homologs in high and zero C22:1 Sinapis alba. Under stringent PCR conditions, a polymorphism was revealed between the two genotypes and was mapped as a fae1 marker in an F₂ population derived from a cross between high and zero C22:1 S. alba. The fae1 marker co-segregated with C22:1 content and the C22:1 phenotypic locus. In addition, a set of 11 RAPD markers for C22:1 in S. alba was identified. Cloning and sequencing of the fae1 alleles in high and zero C22:1 S. alba revealed two amino-acid substitutions specific to zero C22:1 S. alba. The underlying nucleotide substitution for one of the amino-acid substitutions and an adjacent silent nucleotide substitution were used to design primers for allele-specific amplicons for both the wild-type and zero C22:1 alleles. The two diagnostic PCR tests are reliable selection tools to identify S. alba carrying one or both of the wild-type and mutant C22:1 alleles of the KCS gene. Received: 27 July 2000 / Accepted: 1 February 2001     

Genetic linkage maps of <Emphasis Type="Italic">Populus alba</Emphasis> L. and comparative mapping analysis of sex determination across <Emphasis Type="Italic">Populus</Emphasis> species

White poplar (Populus alba L.) is native to Eurasia and is unexploited for its growth potential and stress-adaptive mechanisms. A better knowledge of its genome will allow for more effective protection and use of critical genetic resources. The main objective of this study was the construction of highly informative P. alba genetic maps. Two genotypes were selected from contrasting natural Italian populations and crossed to generate an F₁ mapping pedigree. Amplified fragment length polymorphism and simple sequence repeat markers were used to genotype 141 F₁ individuals. The pseudo-testcross strategy was applied for linkage analysis. The generated maps showed good overall colinearity to each other and allowed for a complete alignment with the 19 haploid chromosomes of the Populus genome sequence. The locus that determines sex as a morphological trait was positioned on a non-terminal position of LG XIX of the female parent map. Comparison among Populus species revealed differences in the location of the sex locus on LG XIX as well as inconsistencies in the heterogametic sex. The genetic analysis of the sex locus in P. alba provides insights into sex determination in the genus and is useful for the identification of sex-linked markers and the early assessment of plant gender. Furthermore, these genetic maps will greatly facilitate the study of the genomics of Populus and how it can be exploited in applied breeding programs.     

Conserved simple sequence repeats for the Limnanthaceae (Brassicales)

The Limnanthaceae (Order Brassicales) is a family of 18 taxa of Limnanthes (meadowfoam) native to California, Oregon, and British Columbia. Cultivated meadowfoam (L. alba Benth.), a recently domesticated plant, has been the focus of research and development as an industrial oilseed for three decades. The goal of the present research was to develop several hundred simple sequence repeat (SSR) markers for genetic mapping, molecular breeding, and genomics research in wild and cultivated meadowfoam taxa. We developed 389 SSR markers for cultivated meadowfoam by isolating and sequencing 1,596 clones from L. alba genomic DNA libraries enriched for AG _n or AC _n repeats, identifying one or more unique SSRs in 696 clone sequences, and designing and testing primers for 624 unique SSRs. The SSR markers were screened for cross- taxa utility and polymorphisms among ten of 17 taxa in the Limnanthaceae; 373 of these markers were polymorphic and 106 amplified loci from every taxon. Cross-taxa amplification percentages ranged from 37.3% in L. douglasii ssp. rosea (145/389) to 85.6% in L. montana (333/389). The SSR markers amplified 4,160 unique bands from 14 genotypes sampled from ten taxa (10.7 unique bands per SSR marker), of which 972 were genotype-specific. Mean and maximum haplotype heterozygosities were 0.71 and 0.90, respectively, among six L. alba genotypes and 0.63 and 0.93, respectively, among 14 genotypes (ten taxa). The SSR markers supply a critical mass of high-throughput DNA markers for biological and agricultural research across the Limnanthaceae and open the way to the development of a genetic linkage map for meadowfoam (x = 5).Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by O. Savolainen     

Evolution, phylogeography, and taxonomy within the Viola alba complex (Violaceae)

 Taxa of the Viola alba complex were investigated using allozymes and morphometry. A taxonomic revision is presented. A wide delimitation of V. alba with only three morphological and geographical subspecies is suggested: (1) ssp. dehnhardtii distributed in the Mediterranean eastwards to Turkey; (2) ssp. alba flanking ssp. dehnhardtii in the north and east; and (3) ssp. cretica endemic to Crete. Ssp. cretica, up to now treated as a separate species, is particularly close to ssp. dehnhardtii. Viola cadevallii (NW Mediterranean) is included in the synonymy of ssp. dehnhardtii. Ssp. scotophylla (S Europe), ssp. thessala (Balkan), V. armena (Turkey), and V. besseri (Caucasus) are reduced to synonyms of V. alba ssp. alba. Viola pentelica (Greece) might represent transitional forms between ssp. alba and ssp. dehnhardtii. Glacial refugia for ssp. alba are suggested from the eastern Mediterranean via Turkey to the Caucasus, for ssp. dehnhardtii in the Mediterranean area in general, and for ssp. cretica in Crete. A key to the subspecies is provided. Taxonomic recombination: Viola alba Bess. ssp. cretica (Boiss. & Heldr.) Marcussen, comb. nov. Received June 17, 2002; accepted November 27, 2002 Published online: March 20, 2003     

An assessment of below-ground ectomycorrhizal diversity of Abies alba miller in central Italy

ABSTRACT

In the framework of an ongoing study on the mycorrhizal associations of silver fir (Abies alba Mill., Pinaceae), we investigated the below-ground diversity of ectomycorrhizal communities in ten field sites located in five distinct natural A. alba woods, situated in the central part of the Apennine chain (Abruzzo region, Italy). Based on macro- and microscopic features, a total of 48 morphologically distinct ectomycorrhizal types have been classified on mature trees of A. alba, 37 of which are reported here for the first time. Ectomycorrhizal morphotypes were partially characterized, and their main features described; in many cases, the relevant fungal symbiont was identified at the level of species or genus. The number of distinguishable morphotypes per site was, with few exceptions, rather homogeneous, ranging from (5) 8 to 13 (20). Comparison of morphotype occurrence revealed that only few types were encountered in five or more sampled sites, whereas the vast majority of types was less frequent. The present study revealed a considerably high species diversity of the ectomycorrhizal symbionts of A. alba in a quite restricted area, thus raising interesting questions as to the ectomycorrhizal potential of this host tree over its entire, large natural range.     

Cytogenetic characterization of <Emphasis Type="Italic">Lippia alba</Emphasis> and <Emphasis Type="Italic">Lantana camara</Emphasis> (Verbenaceae) from Brazil

The aim of this work was to determine the cytogenetic characteristics of Brazilian Lippia alba (Mill) N. E. Brown and Lantana camara Plum. that could be useful for future characterization of these genera. Our analyses revealed that Li. alba has 2n=30 chromosomes consisting of ten metacentric and five submetacentric pairs, while La. camara has 44 metacentric chromosomes. The large blocks of heterochromatin seen in both species suggest an apomorphic condition. Six 45S rDNA sites were detected in both species by fluorescence in situ hybridization (FISH). Two and four 5S rDNA sites were observed in Li. alba and La. camara, respectively. Meiotic analysis revealed a normal chromosomal behaviour. The number of chromosomes and the presence of 45S rDNA and 5S rDNA sites do not exclude a possible polyploid origin. The cytogenetic differences between La. camara and Li. alba may be useful markers for differentiating these species.     

Isozyme analysis as a tool for introgression of Sinapis alba germ plasm into Brassica napus

Summary Isozyme analysis of Brassica napus cv Topas and CGRC5006 as well as of Sinapis alba cv Emergo revealed significant polymorphism between the two species for the isozymes, aconitate hydratase, glucose phosphate isomerase, and diaphorase. F₁ hybrids between B. napus 5006 and S. alba cv Emergo were backcrossed to B. napus cv Topas, and the S₁ progeny of the first two backcrosses were studied isozymically. At the backcross one level the frequency of S. alba or S. alba plus B. napus patterns observed ranged from 18% to 87% across the four lines studied. There were differences between lines for the frequency of S. alba patterns, which could have an impact on the efficiency of selection for subsequent backcrossing. By the backcross two generation in one of the two lines studied, GR86-24, the S. alba patterns for GPI and DIA had been lost, while in the other line, GR86-28, the S. alba pattern for ACO had been lost, resulting in lost opportunity for S. alba gene transfer. In a wide cross such as S. alba x B. napus, which requires an intensive effort to accomplish, the isozymes ACO, GPI, and DIA may serve as useful markers to ensure gene transfer between the two species has occurred. In addition, the identification of lines with divergent isozyme patterns from B. napus will provide the basis for establishing linkages between S. alba traits of interest and isozyme markers.     

Characterisation and inheritance of nuclear microsatellite loci for use in population studies of the allotetraploid <Emphasis Type="Italic">Salix alba</Emphasis>–<Emphasis Type="Italic">Salix fragilis</Emphasis> complex

We present nine polymorphic di- and tri-nucleotide repeat nuclear microsatellite markers selected specifically for their use in high throughput studies concerning the dioecious allotetraploid Salix alba–Salix fragilis willow complex. These taxa and their hybrids are difficult to discriminate using morphological characters. Thus, multiplex reactions were developed for these microsatellite loci and their effectiveness to distinguish individuals, especially hybrids, and their inheritance patterns in controlled crosses were determined. All loci displayed disomic–monogenic inheritance which allowed for the genotype data to be analysed as for a diploid organism. The nine loci produced a total of 67 alleles (mean, 7.4 alleles per locus; range, 3–11 alleles) in a reference panel of 57 individuals from two germplasm collections and natural populations. Gene diversity values (as measured by the expected heterozygosity) ranged from 0.000–0.820. A total of 53 distinct multilocus genotypes were observed, and ordination analysis revealed three separate clusters corresponding to S. alba, S. fragilis and hybrids. The microsatellite loci described here will be used in population genetic studies to investigate genetic variation, gene flow, levels of hybridisation and the extent of introgression in natural populations of the S. alba–S. fragilis complex. They are also useful for clonal identification, conservation and sustainable management of germplasm collections, genetic mapping and the selection of individuals and/or certification of controlled crosses for breeding programmes.     

Identification and characterization of nuclear microsatellite loci in Abies alba Mill.

Eleven polymorphic nuclear microsatellite markers for Abies alba Mill. were developed from an enriched genomic library. An average of 5.2 alleles per locus and a mean expected heterozygosity of 0.532 were found in a sample of 24 Abies alba individuals from different populations within Europe. These loci can be used in studies of genetic diversity for parentage analysis and for estimation of gene flow in silver fir populations. Moreover, successful amplifications were obtained for eight other Mediterranean Abies species, suggesting that these loci may be useful for similar applications in other fir species.     

Gap formation and regeneration of tropical mangrove forests in Ranong,Thailand

Tropical mangrove forests are characterized by clear zonation along a tidal gradient, and it has been supposed that the zonation is primarily controlled by soil factors. However, effects of disturbance on mangrove forests are still not well understood and may play an important role on the vegetation patterns and forest dynamics in some forest formations. In this study, the pattern of disturbance regime and its effects on regeneration of tropical mangrove forests along a tidal gradient were investigated in Ranong, Thailand. We established one or two 0.5 ha plots in four vegetation zones, i.e. Sonneratia alba–Avicennia alba zone, Rhizophora apiculata zone, Ra – Bruguiera gymnorrhiza zone, Ceriops tagal–Xylocarpus spp. zone. Gap size (percentage gap area to total study area and individual gap size) was the largest in Sa–Aa zone which is located on the most seaward fringe, and it declined from seaward to inland. Canopy trees of S. alba and A. alba had stunted trunks and showed low tree density. On the contrary, canopy dominants in the other three inland zones, e.g. R. apiculata, B. gymnorrhiza, and Xylocarpus spp., had slender trunks and showed high tree density. Accordingly, differences in disturbance regime among the four zones were resulted from the forest structural features of each zone. Disturbance regime matched with regeneration strategies of canopy dominants. Seedlings and saplings of S. alba and A. alba, which need sunny condition for their growth, were abundant in gaps than in understorey. By contrast, R. apiculata, B. gymnorrhiza, and Xylocarpus spp., which can tolerate less light than S. alba and A. alba, had greater seedling and sapling density under closed canopy than gaps. Many large gaps may enhance the abundance of S. alba and A. alba in Sa–Aa zone, and a few small gaps may prevent the light demanding species to establish and grow in the other inland zones. Correspondence of disturbance regime and regeneration strategies (e.g. light requirement) of canopy dominants may contribute to the maintenance of the present species composition in each of the vegetation zones.     

The development of a genetic map for meadowfoam comprised of amplified fragment length polymorphisms

Limnanthes alba Benth. (meadowfoam), a diploid (x=5) winter annual, produces novel very long-chain seed oils (C₂₀ and C₂₂) with less than 2% saturated fatty acids. The first genetic map of meadowfoam, a recently domesticated species, is described herein. Two phenotypically diverse inbred lines, OMF40–11 (L. alba ssp. alba) and OMF64 (L. alba ssp. versicolor), were screened for amplified fragment length polymorphisms (AFLPs) using 16 primer combinations. Twenty three percent of the AFLP bands (415 out of 1,801) were polymorphic between OMF40–11 and OMF64. One hundred (OMF40–11×OMF64)×OMF64 BC₁ progeny were genotyped for 107 polymorphic AFLP markers produced by nine AFLP primer combinations. One hundred and three AFLP loci amalgamated into five linkage groups with 14 to 28 loci per linkage group (four loci segregated independently). The map was 698.5-cM long with a mean interlocus spacing of 6.7 cM and no dense clustering of loci. The segregation ratios for 25 loci (23.2%) were significantly distorted. Twenty one of the distorted loci (84%) had an excess of L. alba ssp. versicolor (recurrent parent) alleles. The distorted loci, apart from one locus on linkage group 4, were distally clustered on both ends of linkage groups 1, 4 and 5. The development of the map was facilitated by the small chromosome number, an abundance of restriction site polymorphisms between the two subspecies (23%), and a high multiplex ratio of the AFLP markers (112 per primer combination). Received: 16 October 2000 / Accepted: 18 April 2001     

Microsatellite markers for the invasive plant species white sweetclover (Melilotus alba) and yellow sweetclover (Melilotus officinalis)

We describe specific primers that amplify nine microsatellite DNA loci from Melilotus alba and Melilotus officinalis, both invasive plant species (Fabaceae) throughout North America. Allelic diversity was slightly lower for M. alba than for M. officinalis, as was expected heterozygosity. For both species, heterozygote deficit was observed at several loci. Genotypic diversity was very high for both species; the 29 plant samples of each species all had different multilocus genotypes. These markers will be used to determine the origins of the sweetclover invasion in Alaska and to compare patterns of diversity between subarctic and lower latitude populations.     

Relationships among Abies nebrodensis,A. alba and A. cephalonica in the morphological and anatomical needle characteristics

We used 39 morphological and anatomical needle traits in the biometric comparisons Abies nebrodensis with A. alba and A. cephalonica. The multivariate analyses were utilised and a closer relationship of A. nebrodensis to A. cephalonica than to A. alba was detected, in contrast to what has been shown for cone characteristics.     

Carbon dioxide assimilation of hardwood seedlings in relation to community dynamics in central illinois

Summary Field measurements of net assimilation and respiration for seedlings of four hardwood species were made periodically over a growing season with soil moisture tension maintained between 0 and 0.75 bar. Total net assimilation per day was significantly greater for Acer saccharum than either Quercus rubra or Quercus alba and for Quercus macrocarpa as compared with Q. rubra, when measurements were made under natural shade conditions and light intensity varied from 80 to 120 ft-c. Mean light compensation points determined under canopy shade were 50.3, 53.5, 87.2, and 102.5 ft-c., respectively, for Acer saccharum, Quercus macrocarpa, Q. rubra, and Q. alba. In a 0.04-hectare canopy opening, total net assimilation per day was not significantly different between Q. rubra, Q. alba, and A. saccharum but was significantly greater for Q. macrocarpa than Q. alba and A. saccharum. Relationships between photosynthetic efficiency and successional characteristics of these species are inferred from the data.     

Molecular and cytological characterization of introgression lines in yellow seed derived from somatic hybrids between Brassica napus and Sinapis alba

Polymerase chain reaction and genomic in situ hybridization techniques were conducted to demonstrate the genomic introgressions in somatic hybrid progenies between Brassica napus and Sinapis alba. With minisatellite core sequence 33.6 as primer, an S. alba-specific band (288 bp) was amplified in yellow seed progenies. No hybridization signals were found using the genomic DNA of S. alba as probe. In addition, degenerate primers were used for the detection of related genes based on the genes related to flavonoid biosynthesis of the model plant Arabidopsis thaliana. Sequencing results show that flavonoid pathway genes are highly conserved in A. thaliana, S. alba and B. napus, and present subtle differences because of genetic evolution. A specific band (1,672 bp) consistent with S. alba was characterized in the yellow seed lines. This study demonstrates that the new yellow seed germplasms, derived from backcrossed and self-crossed progenies of B. napus–S. alba hybrids, are stable and homozygous introgression lines with yellow seed color, and differ from existing yellow seed materials.     

Microsatellite loci for great white herons and great blue herons (Aves,Ardeidae, Ardea herodias)

We used an enrichment technique to isolate 60 microsatellite loci in Ardea herodias. We developed primers for 17 loci, screened for variation in A. herodias and attempted to amplify these loci in three closely related species (A. alba, A. cinerea and A. cocoi). Fifteen loci were polymorphic in A. herodias. Observed heterozygosities ranged from 0.10 to 0.81. Two loci appeared to be monomorphic in A. herodias, but exhibited variation in product size among species within the genus. Our ability to amplify polymorphic products in closely related species suggests that these markers may be useful in other herons.     

Genetic diversity of Artemisia populations in central and north Saudi Arabia based on morphological variation and RAPD polymorphism

The analysis of morphological variation and RAPD polymorphism distinguished populations of A. herba alba from populations of A. judaica and A. monosperma. Higher morphological diversity is found in A. herba alba compared to the other two species, but molecular data derived from RAPD polymorphism also indicated that A. herba alba is more polymorphic than the other two species. However, RAPD fingerprinting also indicated sharp polymorphism among populations of both A. judaica and A. monosperma. Geographic and local ecological variations related to elevation of the sites of the examined populations may be regarded to have played a role in the genetic diversity of the examined populations of Artemisia species in the study area. The results are important for future plans for sustainable conservation of medicinal plants in Saudi Arabia. However, extensive sampling of the examined Artemisia species populations is required, and more regional data should be obtained from other distribution areas.