Molecular characterisation and origin of the Coffea arabica L. genome

Restriction fragment length polymorphism (RFLP) markers were used in combination with genomic in situ hybridisation (GISH) to investigate the origin of the allotetraploid species Coffea arabica (2n = 44). By comparing the RFLP patterns of potential diploid progenitor species with those of C. arabica, the sources of the two sets of chromosomes, or genomes, combined in C. arabica were identified. The genome organisation of C. arabica was confirmed by GISH using simultaneously labelled total genomic DNA from the two putative genome donor species as probes. These results clearly suggest that C. arabica is an amphidiploid formed by hybridisation between C. eugenioides and C. canephora, or ecotypes related to these diploid species. Our results also indicate low divergence between the two constituent genomes of C. arabica and those of its progenitor species, suggesting that the speciation of C. arabica took place relatively recently. Precise localisation in Central Africa of the site of the speciation of C. arabica, based on the present distribution of the coffee species, appears difficult, since the constitution and extent of tropical forest has varied considerably during the late Quaternary period. Received: 6 June 1998 / Accepted: 10 November 1998     

Evidence of Intergenomic Relationships in Triploid Hybrids of Coffee (<Emphasis Type="Italic">Coffea sp.)</Emphasis> as Revealed by Meiotic Behavior and Genomic in Situ Hybridization

Interspecific hybrids involving the cultivated C. arabica (2n = 4x = 44, E^aE^aC^aC^a) and two related diploid species (2n = 2x = 22), C. eugenioides (EE) and C. liberica (LL), were produced and analyzed for their relative genome affinity using different complementary approaches, including chromosome association analysis, genomic in situ hybridization (GISH) and pollen fertility. The mean arm pairing frequency (c) and the relative affinity index (x) of triploid hybrids with known genome combinations were used as a measure of chromosome homology. Triploid hybrids were highly sterile as a result of meiotic abnormalities (fertility ranged from 1 to 15 %). Nevertheless, all hybrids exhibited a significant occurrence of genome affinities (x = 0.96 for E^aC^aE and 0.81 for E^aC^aL). Further analysis using the GISH approach revealed that C. eugenioides was more closely related to C. arabica than to C. liberica, which was in agreement with the ancestral history of the allotetraploid C. arabica. The absence of incompatibility barriers at the stylar level in the flowers of the triploid hybrids indicates the possibility of desirable gene transfer through breeding strategies.     

In situ hybridization identifies the diploid progenitor species of Coffea arabica (Rubiaceae)

 The most important commercial coffee species, Coffea arabica, which is cultivated in about 70% of the plantations world-wide, is the only tetraploid (2n=4x=44) species known in the genus. Genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) were used to study the genome organization and evolution of this species. Labelled total genomic DNA from diploid species (C. eugenioides, C. congensis, C. canephora, C. liberica) closely related to C. arabica was separately used as a probe in combination with or without blocking DNA to the chromosome spreads of C. arabica. GISH discriminated between chromosomes of C. arabica only in the presence of an excess of unlabelled block DNA from the species not used as a probe. Among the range of different species combinations used, DNA from C. eugenioides strongly and preferentially labelled 22 chromosomes of the tetraploid C. arabica, while the remaining 22 chromosomes were labelled with C. congensis DNA. The similarity of observations between C. arabica and the two diploid species using two ribosomal genes with FISH with respect to metaphase chromosomes provided additional support to the GISH results. These results confirm the allopolyploid nature of C. arabica and show that C. congensis and C. eugenioides are the diploid progenitors of C. arabica. Received: 2 February 1998 / Accepted: 12 May 1998     

Comparative and evolutionary analysis in natural diploid and tetraploid weather loach Misgurnus anguillicaudatus based on cytochrome b sequence data in central China

To obtain the phylogenetic relationship between diploid and tetraploid Misgurnus anguillicaudatus, the mitochondrial cyt b gene in the diploid and tetraploid weather loach were isolated and sequenced. The DNA sequences were analyzed using MEGA 3.0 software to determine the phylogenetic relationship. Forty-five variable sites among cyt b gene sequences and 18 amino acid substitutions occurred within the diploid and tetraploid loaches as deduced from the nucleotide sequences analysis of the cyt b gene. The nucleotide pairwise distance between diploid and tetraploid loach ranged from 0.001 to 0.025. Phylogenetic analysis revealed evolutionary relationships between diploid and tetraploid loach. Our results indicated a significant difference between diploid and tetraploid loach about the cyt b gene. AMOVA analysis indicated that there were no significant genetic variations within diploid loaches (Fst = 0.2529, P > 0.05) and within tetraploid loaches (Fst = 0.0564, P > 0.05), neither. However, significant genetic differences were found between diploid and tetraploid loaches (Fst = 0.7634, P < 0.05). Thus, it is concluded that no reproductive isolation was found within the same cytotypes of different localities, but there was reproductive isolation between these two cytotypes. The diploid loach existed before the tetraploid loach in nature. The present study is the first to describe the phylogenetic relationships of natural polyploidy weather loach using mtDNA cyt b gene.     

Chromosomal regions associated with segregation distortion of molecular markers in F2?, backcross, doubled haploid, and recombinant inbred populations in rice ( Oryza sativa L.)

Chromosomal regions associated with marker segregation distortion in rice were compared based on six molecular linkage maps. Mapping populations were derived from one interspecific backcross and five intersubspecific (indica / japonica) crosses, including two F₂ populations, two doubled haploid (DH) populations, and one recombinant inbred (RI) population. Mapping data for each population consisted of 129–629 markers. Segregation distortion was determined based on chi-square analysis (P < 0.01) and was observed at 6.8–31.8% of the mapped marker loci. Marker loci associated with skewed allele frequencies were distributed on all 12 chromosomes. Distortion in eight chromosomal regions bracketed previously identified gametophyte (ga) or sterility genes (S). Distortion in three other chromosomal regions was found only in DH populations, where japonica alleles were over-represented, suggesting that loci in these regions may be associated with preferential regeneration of japonica genotypes during anther culture. Three additional clusters of skewed markers were observed in more than one population in regions where no gametophytic or sterility loci have previously been reported. A total of 17 segregation distortion loci may be postulated based on this study and their locations in the rice genome were estimated. Received: 31 May 1996 / Accepted: 30 September 1996     

A new karyotype for <Emphasis Type="Italic">Cavia magna</Emphasis> (Rodentia: Caviidae) from an estuarine island and <Emphasis Type="Italic">C. aperea</Emphasis> from adjacent mainland

We describe a new karyotype for Cavia magna Ximenez, 1980 from an estuarine island and the karyotype of Cavia aperea Erxleben, 1777 from an adjacent mainland. The species have differences in diploid number (2n), autosomal fundamental number, quantity, and distribution of heterochromatin as dissimilar distributions of the nucleolus-organizing regions (Ag-NORs). The C. aperea karyotype has a diploid number of 64 as previously reported for C. aperea and most other Cavia species. In contrast, this new C. magna karyotype exhibits a variant diploid number of 2n = 62, considering that previous work reported a karyotype of 2n = 64 for C. magna. The discovery of a distinct diploid number within C. magna represents the first record of intra-specific chromosomal variation in a species of Caviidae. The diploid number of 2n = 62, heterochromatin quantity, Ag-NOR distributions, and inversed X chromosome from this population of C. magna are as seen in the geographically proximate (Cavia intermedia Cherem Olimpio and Ximenez; intermediate Cavy). These data provide further evidence supporting C. magna as the sister species of C. intermedia.     

Allozyme variation and divergence in the phyllotine rodent Calomys hummelincki (Husson, 1960)

The aim of the present study was to assess the degree of genetic variation and divergence among six populations of Calomys hummelincki, a phyllotine rodent distributed in northern South America. With this information we will try to evaluate the two hypotheses of possible colonization and differentiation of this group of rodents postulated by Baskin and Reig. We studied 34 loci by electrophoretic analysis: 21 were monomorphic for all populations and 13 were polymorphic in at least one population, being P _1% = 21.6% the mean value for all populations. The mean value of heterozygosity per locus was H = 0.075. Low values of genetic distance were observed among populations of the Llanos region (0.001 < D < 0.006). There was a larger genetic distance (D = 0.024) between the population from Isiro, in the northwestern semiarid region, and those from the Llanos region. The insular population of Aruba displayed the lowest value of genetic distance with the population from Isiro (D = 0.014). The specimens from Sipao, on the right side of the Orinoco river, displayed the highest values of genetic distances in comparison with other populations of C. hummelincki (0.070 < D < 0.095). The relatively high differentiation was due to the fixation of new alleles, not found in other populations of C. hummelincki, at loci Idh-1 and Est-2. F-statistics and Nm values indicated reduced gene flow among the populations sampled. Despite the limited data, the results seem to support Reig's hypothesis about south to north colonization of genus Calomys in South America. This revised version was published online in July 2006 with corrections to the Cover Date.     

Use of allele specificity of comigrating AFLP markers to align genetic maps from different potato genotypes

The allele specificity of AFLP markers was assessed in five relatively unrelated potato genotypes. To this end, two diploid mapping populations of potato, F₁SH × RH and F₁AM × RH, were analysed using four and six AFLP primer combinations, respectively, recently applied to the analysis of the genetically well characterized backcross population BC_C × E. The AFLP profiles of the five parents revealed 733 AFLP markers and, when identical primer combinations were used, 131 comigrating AFLP markers were identified. After construction of five parental maps, the genomic positions of these comigrating AFLP markers were compared and 117 markers (89%) which targeted the same genomic region were assumed to be homologous. Of these putative homologues, 20 markers, each cloned from at least two genotypes, were sequenced and 19 sets of amplification products were shown to be nearly identical. The number of AFLP markers previously mapped in population BC_C × E ranged from three to eleven per chromosome, which allowed a reliable assessment of chromosome numbers from individual linkage groups obtained in populations F₁SH × RH and F₁AM × RH. The high incidence of corresponding AFLP alleles was confirmed by using an additional set of five primer combinations. The 733 AFLP markers localized provide a valuable reference collection for future mapping studies in potato. As a consequence AFLP analysis may replace more laborious locus-specific marker techniques. Received: 26 July 1996 / Accepted: 30 January 1997     

In vitro induction of a trisomic of Agave tequilana Weber var. Azul (Agavaceae) by para-fluorophenylalanine treatment

The effect of para-fluorophenylalanine (PFP) on the production of trisomic plants of Agave tequilana Weber var. Azul produced through somatic embryogenesis was investigated. Normal diploid plants with 2n = 2x = 60 were obtained in the control treatment and with 4 mg L⁻¹ PFP exposure, while use of 8 and 12 mg L⁻¹ PFP led to production of trisomics with 2n = 2x = 61. Normal diploid plants showed a bimodal karyotype with five pairs of large chromosomes and 25 pairs of small chromosomes. Trisomic plants also had a bimodal karyotype with a group of three chromosomes in position five of the chromosome set. More than 13 homologous chromosome pairs exhibited structural changes. Differences in chromosome arm ratio (long arm/short arm) were also found in eight chromosome pairs; all these aberrations in the chromosome complement of trisomic plants were probably caused by inversions, deletions, and/or duplications produced by high concentrations of PFP. The gross chromosome structural changes and the presence of a single extra chromosome could have been induced by the effect of PFP on the mitotic spindle by inducing nondisjunction of sister chromatids, resulting in hyperploids (2n + x) and hypoploids (2n − x). Flow cytometric analysis of nuclear DNA content was performed using nuclei isolated from young leaves of normal and trisomic plants. The 2C DNA content of 8.635 pg (1Cx = 4,223 Mbp of trisomic plants was different (p < 0.001) than that of normal plants (2C DNA = 8.389 pg (1Cx = 4,102 Mbp). The difference in genome size was correlated with the large structural changes in the trisomic plant genomes.     

Short-distance pollen dispersal and high self-pollination in a bat-pollinated neotropical tree

We investigated pollen dispersal and breeding structure in the tropical tree species Caryocar brasiliense Camb. (Caryocaraceae), using genetic data from ten microsatellite loci. All adult trees (101) within a patch of 8.3 ha were sampled, and from these adults 18 open-pollinated maternal progeny arrays were analyzed (280 seeds from 265 fruits). Most fruits presented only one seed (median equal to 1.000) and mean number of ripened seeds per fruit was 1.053 (SD = 0.828). Our results showed that C. brasiliense presents a mixed-mating system, with 11.4% of self-pollination, multilocus outcrossing rate of t _m = 0.891 ± 0.025, and high probability of full-sibship within progeny arrays (r _p = 0.135 ± 0.032). Outcrossing rate and self-pollination varied significantly among mother trees. We could detect a maximum pollen dispersal distance of ∼500 m and a mean pollen dispersal distance of ∼132 m. However, most pollination events (80%) occurred at distances less than 200 m. Our results also indicated that pollen dispersal is restricted to a neighborhood of 5.4 ha with rare events of immigration (∼1% N _e m = 0.35). C. brasiliense also presents a significant but weak spatial genetic structure (Sp = 0.0116), and extension of pollen dispersal distance was greater than seed dispersal (N _b = 86.20 m). These results are most likely due to the foraging behavior of pollinators that may have limited flight range. The highly within-population synchronous flowering, high population density, and clumped distribution reinforce pollinator behavior and affect residence time leading to a short-distance pollen dispersal.     

Brewing trouble: coffee invasion in relation to edges and forest structure in tropical rainforest fragments of the Western Ghats, India

While the conservation impacts of invasive plant species on tropical biodiversity is widely recognised, little is known of the potential for cultivated crops turning invasive in tropical forest regions. In the Western Ghats biodiversity hotspot, India, fragmented rainforests often adjoin coffee plantations. This study in the Anamalai hills assessed the effects of distance from edges and forest structure on the occurrence and abundance of shade-tolerant coffee (Arabica Coffea arabica and Robusta C. canephora) in four fragments (32–200 ha) using replicate line transects laid from the edges into the interiors. The coffee species cultivated in adjoining plantations was more abundant than the other coffee species inside study fragments, showing a clear decline in stem density from edge (0 m) to interior (250 m), suggesting the influence of propagule pressure of adjoining plantations, coupled with edge effects and seed dispersal by animals. Significant positive correlations of coffee density with canopy cover indicate the potential threat of coffee invasion even in closed canopy rainforests. Stem density of Coffea arabica (150–1,825 stems/ha) was higher in more disturbed fragments, whereas Coffea canephora had spread in disturbed and undisturbed sites achieving much higher densities (6.3–11,486 stems/ha). In addition, a negative relationship between C. canephora and native shrub density indicates its potential detrimental effects on native plants.     

Zinc and Iron Concentration and SOD Activity in Human Semen and Seminal Plasma

The aim of the present study was to measure zinc (Zn) and iron (Fe) concentration in human semen and superoxide dismutase (SOD) activity in seminal plasma and correlate the results with sperm quality. Semen samples were obtained from men (N = 168) undergoing routine infertility evaluation. The study design included two groups based on the ejaculate parameters. Group I (n = 39) consisted of males with normal ejaculate (normozoospermia), and group II (n = 129) consisted of males with pathological spermiogram. Seminal Zn and Fe were measured in 162 samples (group I, n = 38; group II, n = 124) and SOD activity in 149 samples (group I, n = 37; group II, n = 112). Correlations were found between SOD activity and Fe and Zn concentration, and between Fe and Zn concentration. SOD activity was negatively associated with volume of semen and positively associated with rapid progressive motility, nonprogressive motility, and concentration. Negative correlation was stated between Fe concentration and normal morphology. Mean SOD activity in seminal plasma of semen from men of group I was higher than in seminal plasma of semen from men of group II. Fe concentration was higher in teratozoospermic males than in males with normal morphology of spermatozoa in group II. Our results suggest that Fe may influence spermatozoa morphology.     

Analysis of alien introgression in coffee tree (<Emphasis Type="Italic">Coffea</Emphasis><Emphasis Type="Italic">arabica</Emphasis> L.)

The transfer of desired traits from related wild diploid Coffea species into the cultivated allotetraploid C. arabica is essential in coffee breeding to develop pest/disease-resistant cultivars. The present work is an attempt to gain insights into alien introgression in C. arabica. An F2 population derived from a cross between T5296 and Et6 was analysed with simple sequence repeat (SSR; microsatellite) and amplified fragment length polymorphism (AFLP) molecular markers. The T5296 is a derivative of an interspecific hybrid introgressed by the diploid C. canephora species and Et6 is a wild Ethiopian accession of C. arabica. The origin of the revealed polymorphism was determined by comparisons using representative accessions from C. arabica and its two diploid parental species, C. eugenioides and C. canephora. The number and mode of inheritance of canephora-introgressed segments were investigated, as well as their sub-genome localisation and rate of recombination. The results suggested that the transfer of desirable genes into C. arabica from C. canephora is not limited by the ploidy level differences or the suppression of recombination between the different genomes.     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996     

Estimation of population structure in coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] using allozyme and microsatellite markers

Characterizing population structure using neutral markers is an important first step in association genetic studies in order to avoid false associations between phenotypes and genotypes that may arise from non-selective demographic factors. Population structure was studied in a wide sample of ∼1,300 coastal Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] trees from Washington and Oregon. This sample is being used for association mapping between cold hardiness and phenology phenotypes and single-nucleotide polymorphisms in adaptive-trait candidate genes. All trees were genotyped for 25 allozyme and six simple sequence repeat (SSR) markers using individual megagametophytes. Population structure analysis was done separately for allozyme and SSR markers, as well as for both datasets combined. The parameter of genetic differentiation (θ or F _ST) was standardized to take into account high within-population variation in the SSR loci and to allow comparison with allozyme loci. Genetic distance between populations was positively and significantly correlated with geographic distance, and weak but significant clinal variation was found for a few alleles. Although the STRUCTURE simulation analysis inferred the same number of populations as used in this study and as based on previous analysis of quantitative adaptive trait variation, clustering among populations was not significant. In general, results indicated weak differentiation among populations for both allozyme and SSR loci (θ _s = 0.006–0.059). The lack of pronounced population subdivision in the studied area should facilitate association mapping in this experimental population, but we recommend taking the STRUCTURE analysis and population assignments for individual trees (Q-matrix) into account in association mapping.     

Prevalence of <Emphasis Type="Italic">Chlamydophila psittaci</Emphasis> in wild birds—potential risk for domestic poultry,pet birds,and public health?

To determine the prevalence of Chlamydophila psittaci in wild birds, cloacal swabs from 527 songbirds, 442 waterfowl, 84 feral pigeons, and 38 cormorants were examined by Chlamydiaceae-specific real-time polymerase chain reaction (PCR) and ArrayTube microarray assays for chlamydial species determination and genotyping of C. psittaci. Inconclusive cases were further characterized by conventional PCR methods targeting the chlamydial outer membrane protein A, 16S, 23S, and intergenic spacer genes followed by sequencing of the PCR product. Swabs of 19 water birds (tufted ducks and pochards), 12 pigeons, and one songbird were tested positive by the Chlamydiaceae-specific real-time PCR. While C. psittaci genotypes B (n = 5) and E (n = 1) were identified in feral pigeons (n = 9), the genotype could not be identified in the remaining three cases. Sequence data of Chlamydiaceae-positive cases (n = 23; 19 waterfowl, three pigeons, one songbird) indicated the presence of nonclassified chlamydial agents (n = 20) and C. psittaci (n = 3) by 16S rRNA PCR and sequencing. In conclusion, C. psittaci was not detected in waterfowl and songbirds, but C. psittaci proved prevalent in urban feral pigeons, where it poses a significant risk for humans.     

Transgenic coffee fruits from <Emphasis Type="Italic">Coffea arabica</Emphasis> genetically modified by bombardment

The genetic modification of Coffea arabica fruits is an important tool for the investigation of physiological characteristics and functional validation of genes related to coffee bean quality traits. In this work, plants of C. arabica cultivar Catuaí Vermelho were successfully genetically modified by bombardment of embryogenic calli. Calli were obtained from 90% of the leaf explants cultivated in a callogenesis-inducing medium modified with 20 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The resulting calli were bombarded with the pBI426 vector containing a uidA and nptII gene fusion that was driven by the double CaMV35s promoter. Kanamycin-selected embryos were positive for β-glucuronidase (GUS) activity in histochemical assays and for target gene amplification by polymerase chain reaction. Integration of the nptII gene was confirmed by Southern blot and showed a low copy number (one to three) of insertions. Transformed plants showed normal development and settled fruits. GUS expression was assessed in the flower and fruit organs demonstrating the capacity of the double CaMV35s promoter to drive long-term stable expression of uidA in C. arabica fruit tissues. Moreover, we obtained a T₁ progeny presenting 3:1 Mendelian segregation of the uidA gene. This investigation is the first to report exogenous gene expression in coffee fruits and transgenic inheritance in C. arabica plants.     

Genetic structure analysis of natural <Emphasis Type="Italic">Sargassum muticum</Emphasis> (Fucales,Phaeophyta) populations using RAPD and ISSR markers

Sargassum muticum is important in maintaining the structure and function of littoral ecosystems, and is used in aquaculture and alginate production, however, little is known about its population genetic attributes. In this study, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to investigate the genetic structure of four populations of S. muticum and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China. The selected 24 RAPD primers and 19 ISSR primers amplified 164 loci and 122 loci, respectively. Estimates of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index) revealed low or moderate level of genetic variations within each S. muticum population, and a high level of genetic differentiations were determined with pairwise unbiased genetic distance (D) and fixation index (F _ST ) among the populations. The Mantel test showed that two types of matrices of D and F _ST were highly correlated whether from RAPD (r = 0.9706, P = 0.009) or ISSR data (r = 0.9161, P = 0.009). Analysis of molecular variance (AMOVA) was conducted to apportion the variations among and within the S. muticum populations. It indicated that variations among populations were higher than those within populations, being 55.82% verse 44.18% by RAPD and 55.21% verse 44.79% by ISSR, respectively. Furthermore, the Mantel test suggested that genetic differentiations among populations were related to the geographical distances (r > 0.6), namely, conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. On the whole, the high genetic structuring among the four S. muticum populations along the distant locations was clearly indicated in RAPD and ISSR analyses (r > 0.9, P < 0.05) in our study.