Interactions of the Solanum spp. of the Etuberosa group and nine potato-infecting viruses and a viroid

All 26 accessions of Solanum brevidens, one accession of S. etuberosum and one accession of S. fernandezianum tested were all extremely resistant to potato leafroll virus (PLRV) and potato viruses Y (PVY) and A (PVA). S. brevidens and S. etuberosum were also resistant to Andean potato mottle virus (APMV) and moderately resistant to potato virus X (PVX), whereas S. fernandezianum was susceptible to these viruses. Additionally, S. brevidens was resistant to sap-inoculated potato viruses M (PVM) and S (PVS). All the Etuberosa accessions were susceptible by graft-inoculation to PVM, PVS, potato virus T (PVT) and Andean potato latent virus (APLV). Infections by the above mentioned viruses were symptomless in all of the Etuberosa spp. S. etuberosum and S. fernandezianum were infected by mechanical inoculation with potato spindle tuber viroid, S. etuberosum developing severe stunting and leaf-curl symptoms, but S. brevidens was infected only by graft-inoculation. The genes conferring resistance to PVY and PVX in S. brevidens and S. etuberosum appeared to be different from those currently utilised by plant breeders.     

Nuclear DNA content of the Solanum spp. in the series Etuberosa as determined by laser flow cytometry

Nuclear DNA content was determined in three accessions of Solanum brevidens, three accessions of S. etuberosum, and one accession of S. fernandezianum, which are diploid (2n = 2×= 24), closely related, non tuber-bearing wild potato species belonging to the series Etuberosa (Solanaceae). The plants were grown in vitro at 18°C or at 25°/22°C (day/night). S. brevidens was also grown in soil in the glasshouse at 25°/19°C (day/night), and in growth chambers at 18°C or 32°C. Leaf nuclei were isolated using a chopping method and stained with propidium iodide. Chicken red blood cells (CRBC; 2.33 pg) were added to the samples of nuclei as internal standards. The fluorescence of plant nuclei relative to CRBC was measured with an EPICS PROFILE flow cytometer. The 2C values of in vitro-grown S. brevidens and S. etuberosum were similar (1.48–1.54 pg, depending on the accession), but they were smaller than the 2C value of S. fernandezianum (1.63 pg). The 2C values of S. brevidens and S. etuberosum were generally smaller than those of the diploid species S. berthaultii (1.60–1.61 pg) and the diploid clones of S. tuberosum (1.60–1.72 pg). A similar relative difference of nuclear DNA content was found also between tetraploid S. brevidens and tetraploid S. tuberosum (2C = 3.15–3.16 pg and 3.50–3.62 pg, respectively). High (32°C) and low (18°C) growth temperatures caused abnormal changes in morphology and reduced fertility in S. brevidens in the growth chamber. The 2C values of S. brevidens grown at 25°/19°C (day/night) or at 32°C were similar, whereas the 2C values were c. 10% lower at 18°C.     

Comparison of four molecular markers in measuring relationships among the wild potato relatives Solanum section Etuberosum (subgenus Potatoe)

We evaluated chloroplast DNA (cpDNA), isozymes, single to low-copy nuclear DNA (RFLPs), and random amplified polymorphic DNAs (RAPDs) in terms of concordance for genetic distance of 15 accessions each of Solanum etuberosum and S. palustre, and 4 accessions of S. fernandezianum. These self-compatible, diploid (2n=24), and morphologically very similar taxa constitute all species in Solanum sect. Etuberosum, a group of non-tuber-bearing species closely related to Solanum sect. Petota (the potato and its wild relatives). Genetic distance and multidimentional scaling results show general concordance of isozymes, RFLPs and RAPDs between all three taxa; cpDNA shows S. etuberosum and S. palustre to be more similar to each other than to S. fernandezianum. Interspecific sampling variance shows a gradation of resolution from allozyme (low) to RAPD to RFLP (high); while intraspecific comparisons graded from RFLPs (low) to RAPDs (high; lack of sufficient allozyme variability within species precluded comparisons for allozymes). Experimental error was low in RFLPs and RAPDs.Names are necessary to report factually and available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

RFLP analysis of asymmetric somatic hybrids between Solanum tuberosum and irradiated S. brevidens

The nuclear genome composition of five asymmetric somatic hybrids, obtained by fusion of leaf protoplasts from Solanum tuberosum and gamma-irradiated leaf protoplasts from S. brevidens, have been analyzed at the molecular level. An analysis of 21 loci using linkage group-specific restriction fragment length polymorphism (RFLP) was included in the study. All five hybrids contained a complete set of the loci studied from S. tuberosum. The degree of elimination of alleles from the irradiated S. brevidens donor genome ranged from 10–65% in the five asymmetric hybrids analyzed. The detection of incomplete chromosomes, as well as non-parental bands in Southern hybridizations with RFLP markers, revealed extensive chromosome rearrangements in the asymmetric hybrids.     

Protoplast-fusion-derived Solanum cybrids: application and phylogenetic limitations

Summary We established interspecific Solanum cybrids in order to study the intrageneric nuclear-organelle compatibility and the introgression of advantageous plasmone-coded breeding traits into potato. Cybridization was performed by the donor-recipient protoplast-fusion procedure. We found that the plastomes of S. chacoense, S. brevidens, and S. etuberosum could be transferred into the cybrids having S. tuberosum nuclear genomes; chondriome components were likewise transferred from the former species into these cybrids. The combination with S. chacoense as organelle donor and potato as recipient resulted in green fertile plants with potato morphology. By using S. etuberosum as an organelle donor and potato as recipient, male-sterile cybrid plants, most of them having pigmentation abnormalities, were obtained. The combination of S. brevidens with potato resulted in palegreen (almost albino) regenerants. The latter albino plantlets had both the chloroplast DNA and the mitochondrial DNA of the donor (S. brevidens) and did not survive the transfer into the greenhouse. An immediately applicative result of this study is the de novo establishment of male-sterile plants in a potato cultivar. Such plants should be useful as seed parents in the production of hybrid, true-potato seeds.     

Isolation and characterization of microsatellite loci in the highland papaya Vasconcellea × heilbornii V. Badillo (Caricaceae)

Nine polymorphic microsatellite loci were identified in the tropical plant Vasconcellea ×heilbornii and used to estimate allelic diversity in two populations of southern Ecuador. Allelic richness ranged from two to five alleles, observed heterozygosity ranged from 0.150 to 0.947 and expected heterozygosity ranged from 0.186 to 0.701. Most of these markers also amplified microsatellite loci in two other Vasconcellea species (Vasconcellea stipulata and Vasconcellea cundinamarcensis). Hence, these markers will be useful for population genetic analysis and the evaluation of genetic diversity and gene flow in these species.     

Isolation and characterization of 14 polymorphic microsatellite loci in the ringneck snake <Emphasis Type="Italic">Diadophis punctatus</Emphasis> (Colubridae: Dipsadinae)

We constructed a genomic library and developed 14 polymorphic loci to assess species boundaries between previously described mtDNA lineages and to examine the genetic variability between populations of the ringneck snake Diadophis punctatus species complex. Polymorphism was investigated for these markers in 34 individuals from populations in southeastern Louisiana and southern Florida. The number of alleles ranged from 5 to 29, expected heterozygosity ranged from 0.816 to 0.962 and observed heterozygosity ranged from 0.452 to 0.889. These new loci provide the tools necessary to examine species boundaries, population genetics within species and to determine the conservation status of imperiled populations.     

Concordance between life history traits, invasion history, and allozyme diversity of the Everglades invader Melaleuca quinquenervia

During the century following its initial introduction in 1886, the Australian tree Melaleuca quinquenervia (Myrtaceae) dispersed from a few introduction points to occupy over 200,000 ha, primarily in historic Everglades wetlands of southern Florida. Cellulose acetate gel electrophoresis (CAGE) was used to investigate the allozyme diversity and population genetic structure of 208 individuals in a dozen populations resulting from this invasion. The analyses showed that these populations have a high (82%) rate of polymorphic loci and an average of 2 alleles/locus. There was substantial heterozygosity (mean H_e = 0.356), which concords well with recent studies reporting a greater number of introduction events and sources than generally recognized. The introduction history and distributional patterns within Florida have led to geographic structuring (G_ST = 0.419) in which the Gulf Coast metapopulation has a greater effective number of alleles and greater heterozygosity than the Atlantic Coast metapopulation. The gene diversity in M. quinquenervia was comparable to other tropical woody species. Its strong population divergence was reminiscent of pioneer species and consistent with its status as a plant invader in Florida.     

Isolation and characterization of microsatellite DNA markers for spinner dolphin (<Emphasis Type="Italic">Stenella longirostris</Emphasis>)

Practically no studies on the population genetics of the spinner dolphin (Stenella longirostris) exist. Seventeen pairs of DNA primers, cloned from an Mbo I digestion of S. longirostris liver DNA, were selected from a total of 288 sequences. Eight polymorphic microsatellite DNA markers were selected from the 17 primer pairs following amplification of DNA from skin samples of 65 spinner dolphins. Characterization of the polymorphisms revealed between three and nine alleles per loci. The observed heterozygosity ranged from 0 to 0.6032, while the expected heterozygosity ranged from 0.5834 to 0.73. Seven of the eight designed primer pairs amplified DNA from three other delphinid species. There was a marked low observed heterozygosity in the spinner dolphin suggesting a high level of inbreeding within this species in the southern Atlantic.     

Production of asymmetric hybrids between Solanum tuberosum and irradiated S. brevidens

Summary Asymmetric somatic hybrids were obtained by fusion of Solanum tuberosum (PDH40) protoplasts with 300- or 500-Gy irradiated protoplasts of S. brevidens. These radiation doses were sufficient to prevent the growth of the S. brevidens protoplasts. Putative hybrids were selected on the basis of phenotype from regenerated shoots and identified with a S. brevidens-specific probe. From these, 31 asymmetric hybrids were confirmed by morphological characteristics, isoenzyme patterns and RFLP analysis. The morphology of the asymmetric hybrids was intermediate between that of S. tuberosum and symmetric hybrids of both species (obtained without irradiation treatment). Chromosome counts from 17 asymmetric hybrids showed that the chromosome number of the hybrids ranged from 31 to 64. The asymmetric hybrids probably had one or two genome complements (i.e. either 24 or 48 chromosomes) from S. tuberosum and 7–22 chromosomes from S. brevidens. There was no clear correlation between the radiation dose and the degree of elimination of the S. brevidens genome.     

Isolation of polymorphic microsatellite loci in Hemerocallis fulva and Hemerocallis citrina (Hemerocallidaceae)

Twenty microsatellite loci were isolated from a hybrid of two daylilies, Hemerocallis fulva and Hemerocallis citrina. We characterized individuals from two H. fulva populations and two H. citrina populations in Japan and observed three to 20 alleles per locus in H. fulva and one to 19 alleles per locus in H. citrina. Mean observed heterozygosity within populations ranged from 0.35 to 0.85 in H. fulva and from 0 to 0.95 in H. citrina. In about a half of the loci, the observed heterozygosity did not deviate from Hardy–Weinberg equilibrium. These loci are proved useful in studying gene flow and qualitative trait loci mapping using the two species.     

Somatic hybrids between Solanum etuberosum and diploid,tuber bearing Solanum clones

Electrofusion was used to obtain somatic hybrids between Solanum etuberosum (2n=2x=24) and two diploid potato lines. These hybridizations were conducted to determine if haploidxwild species hybrids are better fusion partners than conventional S. tuberosumGp. Tuberosum haploids. Restriction fragment length polymerase (RFLP) analyses of the putative somatic hybrids confirmed that each parental genome was present. The somatic hybrids between S. etuberosum and a haploid S. tuberosum clone, US-W730, were stunted and had curled, purple leaves. In contrast, somatic hybrids between S. etuberosum and a haploidxwild species hybrid (US-W 730 haploidx S. berthaultii), were vigorous and generally tuberized under field conditions. These hybrids were designated as E+BT somatic hybrids. Analyses of 23 E+BT somatic hybrids revealed a statistically significant bias towards the retention of S. etuberosum chloroplasts. Stylar incompatibilities were observed when the E+BT somatic hybrids were used as pollen donors in crosses with S. tuberosum cultivars. Reciprocal crosses did not show this incompatibility. The progeny were vigorous and had improved tuber traits when compared to the maternal E+BT parent. RFLP analyses of three sexual progeny lines confirmed the presence of all 12 S. etuberosum chromosomes. In two of these lines, RFLPs that marked each of the 24 chromosome arms of S. etuberosum were present. However, RFLP markers specific for regions on chromosomes 2, 7, and 11 were missing from the third clone. Because other markers for these chromosomes were present in the progeny line, these results indicated the likelihood of pairing and recombination between S. etuberosum and S. tuberosum chromosomes.     

Resistance to potato leaf roll virus and potato virus Y in somatic hybrids between dihaploid Solanum tuberosum and S. brevidens

Summary Many somatic fusion hybrids have been produced between a dihaploid potato Solanum tuberosum and the sexually-incompatible wild species S. brevidens using both chemical and electrical fusion techniques. S. brevidens was resistant to both potato leaf roll virus (PLRV) and potato virus Y (PVY), the viruses being either at low (PLRV) or undetectable (PVY) concentrations as determined by enzyme-linked immunosorbent assay (ELISA). The S. tuberosum parent was susceptible to both viruses. A wide range of resistance, expressed as a decrease in virus concentration to both viruses was found amongst fusion hybrids, four of which were especially resistant. The practicality of introducing virus resistance from S. brevidens into cultivated potatoes by somatic hybridisation is discussed.     

Genetic diversity of common carp from two largest Chinese lakes and the Yangtze River revealed by microsatellite markers

Six polymorphic microsatellites (eight loci) were used to study the genetic diversity and population structure of common carp from Dongting Lake (DTC), Poyang Lake (PYC), and the Yangtze River (YZC) in China. The gene diversity was high among populations with values close to 1. The number of alleles per locus ranged from 2 to 11, and the average number of alleles among 3 populations ranged from 6.5 to 7.9. The mean observed (H _O) and expected (H _E) heterozygosity ranged from 0.4888 to 0.5162 and from 0.7679 to 0.7708, respectively. Significant deviations from Hardy–Weinberg Equilibrium expectation were found at majority of the loci and in all three populations in which heterozygote deficits were apparent. The analysis of molecular variance (AMOVA) indicated that the percent of variance among populations and within populations were 3.03 and 96.97, respectively. The Fst values between populations indicated that there were significant genetic differentiations for the common carp populations from the Yangtze River and two largest Chinese freshwater lakes. The factors that may result in genetic divergence and significant reduction of the observed heterozygosity were discussed.     

Isolation and characterization of variable microsatellite loci in Lilium philadelphicum (Liliaceae)

Here we describe the characterization of six polymorphic microsatellite loci for Lilium philadelphicum (Liliaceae). Polymorphism levels ranged from 7 to 30 alleles per locus with a mean number of 15 alleles per locus. Observed heterozygosity (H_O) ranged from 0.17 to 0.82 and did not deviate from Hardy–Weinberg expectations in the three western Montana populations included in the analysis. These loci are proving useful in studying gene flow between populations of this species distributed across its range in North America.     

基于SSR标记的大明松天然群体遗传多样性分析

大明松是广西和贵州特有的高山松类,具有很高的经济价值和生态价值,其自然种群长期没有得到充分的保护和利用,不利于该树种长期稳定发展。为了合理保护和开发利用大明松天然遗传资源,该文利用12个SSR分子标记对大明松3个天然群体进行遗传多样性研究,分析其群体间的遗传分化和基因流,为该物种的保护策略提供参考。研究结果表明:(1)12对引物共检测到37 个等位基因,多态位点百分率为100%。每个位点平均观察等位基因数(Na)为3.08,平均有效等位基因数(Ne)为1.68, 不同位点间有效等位基因数差异较大; 每个位点平均观察杂合度(Ho)为0.35,平均期望杂合度(He)为0.40,平均多态信息含量(PIC)为0.31。(2)3 个群体的Shannon''s多样性指数变化范围为0.48～0.65,Nei''s基因多样度的变化范围为0.27～0.39,与其他近缘种松类相比遗传多样性偏低。群体平均观察杂合度为0.40,平均期望杂合度为0.33,平均有效等位基因数为1.58。群体间基因分化系数(Gst)为0.10,群体间的遗传分化水平较低,大部分变异均存在群体内。群体间的基因流(Nm)为2.74,说明大明松群体间的基因交流比较充分。该研究可为大明松生物多样性保护提供重要参考依据,为科学利用大明松资源打下基础。     

Patterns of selection and allele diversity of class I and class II major histocompatibility loci across the species range of sockeye salmon (Oncorhynchus nerka)

The major histocompatibility complex (MHC), an important component of the vertebrate immune system, provides an important suite of genes to examine the role of genetic diversity at non‐neutral loci for population persistence. We contrasted patterns of diversity at the two classical MHC loci in sockeye salmon (Oncorhynchus nerka), MHC class I (UBA) and MHC class II (DAB), and neutral microsatellite loci across 70 populations spanning the species range from Washington State to Japan. There was no correlation in allelic richness or heterozygosity between MHC loci or between MHC loci and microsatellites. The two unlinked MHC loci may be responding to different selective pressures; the distribution of F_ST values for the two loci was uncorrelated, and evidence for both balancing and directional selection on alleles and lineages of DAB and UBA was observed in populations throughout the species range but rarely on both loci within a population. These results suggest that fluctuating selection has resulted in the divergence of MHC loci in contemporary populations.     

Characterization of the expression and inheritance of potato leafroll virus (PLRV) and potato virus Y (PVY) resistance in three generations of germplasm derived from Solanum etuberosum

Potato virus Y (PVY) and potato leafroll virus (PLRV) are two of the most important viral pathogens of potato. Infection of potato by these viruses results in losses of yield and quality in commercial production and in the rejection of seed in certification programs. Host plant resistance to these two viruses was identified in the backcross progeny of a Solanum etuberosum Lindl. somatic hybrid. Multiple years of field evaluations with high-virus inoculum and aphid populations have shown the PVY and PLRV resistances of S. etuberosum to be stably expressed in two generations of progeny. However, while PLRV resistance was transmitted and expressed in the third generation of backcrossing to cultivated potato (Solanum tuberosum L. subsp. tuberosum), PVY resistance was lost. PLRV resistance appears to be monogenic based on the inheritance of resistance in a BC₃ population. Data from a previous evaluation of the BC₂ progeny used in this study provides evidence that PLRV resistance was partly conferred by reduced PLRV accumulation in foliage. The field and grafting data presented in this study suggests that resistance to the systemic spread of PLRV from infected foliage to tubers also contributes to the observed resistance from S. etuberosum. The PLRV resistance contributed by S. etuberosum is stably transmitted and expressed through sexual generations and therefore would be useful to potato breeders for the development of PLRV resistant potato cultivars.     

Characterization of chromosome instability in interspecific somatic hybrids obtained by X-ray fusion between potato (Solanum tuberosum L.) and S. brevidens Phil

Summary Asymmetric somatic hybrids between Solanum tuberosum L. and S. brevidens Phil. have been obtained via the fusion of protoplasts from potato leaves and from cell suspension culture of S. brevidens. The wild Solanum species served as donor after irradiation of its protoplasts with a lethal X-ray dose (200 Gy). Selection of the putative hybrids was based on the kanamycin-resistance marker gene previously introduced into the genome of Solanum brevidens by Agrobacterium-mediated gene transfer. Thirteen out of the 45 selected clones exhibited reduced morphogenic potential. The morphological abnormalities of the regenerated plantlets were gradually eliminated during the extended in vitro culture period. Cytological investigations revealed that the number of chromosomes in the cultured S. brevidens cells used as protoplast source ranged between 28–40 instead of the basic 2n=24 value. There was a high degree of aneuploidy in all of the investigated hybrid clones, and at least 12 extra chromosomes were observed in addition to the potato chromosomes (2n=48). Interand intraclonal variation and segregation during vegetative propagation indicated the genetic instability of the hybrids, which can be ascribed to the pre-existing and X-ray irradiation-induced chromosomal abnormalities in the donor S. brevidens cells. The detection of centromeric chromosome fragments and long, poly-constrictional chromosomes in cytological preparations as well as non-parental bands in Southern hybridizations with restriction fragment length polymorphism (RFLP) markers revealed extensive chromosome rearrangements in most of the regenerated clones. On the basis of the limited number of RFLP probes used, preferential loss of S. brevidens specific markers with a non-random elimination pattern could be detected in hybrid regenerants.     

Development of polymorphic simple sequence repeat markers for Pisolithus microcarpus

Six polymorphic simple sequence repeat (SSR) markers were developed for the ectomycorrhizal fungus Pisolithus microcarpus. A polymerase chain reaction (PCR)‐based technique was used in which random amplified polymorphic DNA (RAPD) fingerprints were probed with labelled SSR oligonucleotides by southern hybridization. The number of alleles per locus ranged from two to nine with expected heterozygosity values from 0.33 to 0.76. These loci will be potentially useful for genetic structure and gene flow studies of P. microcarpus populations. Cross‐species amplification with Pisolithus albus isolates at all loci was also observed.