The detection of nonhybrid, trisomic, and triploid offspring in sexual progeny of a mating of Phytophthora infestans.

Eighty single-oospore offspring of Phytophthora infestans from a mating of isolates, which had previously been analyzed for segregation of avirulence/virulence, were assessed for the inheritance of 20 RFLP markers. Three offspring were triploid; they inherited three alleles at all loci where this could be detected and when heterozygous, showed unequal intensities of hybridization with most probes. Twenty-four offspring were trisomic, as each had three doses of one or a few markers, evident from their inheritance of three alleles or from unequal hybridization to one probe. Coinheritance of the extra allele(s) and mitochondrial haplotype in the majority of trisomic offspring suggested that meiosis in oogonia was more aberrant than in antheridia. Linkage analysis was performed on 50 offspring, which were assumed to be euploid; six small linkage groups were detected and several avirulence loci were found to be linked. The origins of aberrant offspring are discussed.     

Non-Mendelian inheritance revealed in a genetic analysis of sexual progeny of Phytophthora cinnamomi with microsatellite markers

We report the development of four microsatellite loci into genetic markers for the diploid oomycete plant pathogen Phytophthora cinnamomi and that (AC)(n) and (AG)(n) microsatellites are significantly less frequent than in plant and mammal genomes. A minisatellite motif 14 bp long was also discovered. The four microsatellite loci were used to analyze sexual progeny from four separate crosses of P. cinnamomi. A large proportion of non-Mendelian inheritance was observed across all loci in all four crosses, including inheritance of more than two alleles at a locus and noninheritance of alleles from either parent at a locus. The aberrant inheritance is best explained by nondisjunction at meiosis in both the A1 parent and the A2 trisomic parents, resulting in aneuploid progeny. Two loci on the putative trisomic chromosome showed linkage and no loci were linked to mating type. One aneuploid offspring was shown to have lost alleles at two loci following subculture over 4 years, indicating that aneuploid progeny may not be mitotically stable.     

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Inheritance of Microsatellite DNA Markers in the Pacific Abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Microsatellite markers have been developed for a variety of abalones, and locus-specific homozygote excesses at population level have been recorded for microsatellite loci. To ascertain whether null alleles exist at microsatellite loci in the Pacific abalone, we studied the mode of inheritance of 7 microsatellite loci in 4 families with a reciprocal cross of 2 females × 2 males. All loci segregated codominantly, but only 3 loci (Hdh1321, Hdh78, and Hdd108C) conformed to Mendelian segregation and can be used for parental analysis and population genetic studies. When null alleles were considered, 2 loci (Hdh1761 and Hdh1457) confirmed Mendelian expectations in all families, while the remaining 2 loci (Hdd114B and Hdd229) showed deviation from Mendelian segregation in at least one family even though null alleles were considered. These results indicated the need to test the inheritance pattern for microsatellite markers in abalones before using them for population genetic or parentage analysis.     

Inheritance in turnip of variable-number tandem-repeat genetic markers revealed with synthetic repetitive DNA probes

Oligomers (16–26 mers) composed of short, tandemly repeated DNA sequences (3–10 bases) were used individually with their complementary oligomer in separate polymerase chain reactions (PCRs) that extended the number of repeats to make 15 different PCR synthetic tandem-repeat (STR) probes. These PCR-STR probes were used to examine the inheritance of variable-number tandem-repeat (VNTR) genetic markers from two parent plants of turnip (Brassica rapa L.) to 20 offspring. Following HinfI digestion and PCR-STR probing of Southern blots, interpretable variable parental and offspring band profiles were found with 9 of the 15 probes used. Each of these nine probes produced a unique set of fragments, and no cases of different probes revealing the same fragment were detected. Seventy-nine parental fragments were found and, of these, 65% (51) appeared to be heterozygous in one or both parents, with 52% (41) appearing to be heterozygous in one of the parents exclusively. That these fragments are transmitted as though heterozygous in the parents implies that they are derived from the nuclear complement of the genome. Chi-square analyses of the transmission of markers are, in general, consistent with Mendelian expectations, although three non-parental bands were found accounting for approximately 0.5% of these transmitted bands. For the fragments heterozygous in one of the parents exclusively, seven alleles exhibited complete linkage in three groups, 12 alleles were incompletely linked in six groups, and four allelic groups involving 11 alleles were identified. PCR-STR probes are relatively rapid to generate and apply (no cloning, clone screening, or sequencing steps are required), and have been shown to reveal VNTR genetic markers in a wide variety of plant species. These results add to the list of studies showing that VNTR genetic markers (and in this case, markers revealed by PCR-STR probes) are transmitted for the greater part in a Mendelian fashion.     

Isolation and characterization of polymorphic microsatellite loci for the northern flicker (Colaptes auratus; Aves)

To investigate the mating system of northern flickers (Colaptes auratus), we developed primers for 14 microsatellite loci and screened them in 68 unrelated adults and their offspring. All markers were highly polymorphic with 9 to +36 alleles per locus. One marker was Z‐chromosome linked; one marker exceeded the size standard range and could not be analysed further. We checked the other 12 markers for Mendelian inheritance in 36 broods for which the social parents were known. Seven markers showed evidence for the presence of null alleles, and three of those showed significant deviation from Hardy–Weinberg equilibrium. Markers were generally unlinked.     

The use of a triploid hybrid for introgression in Lolium species

Summary Triploid hybrids of Lolium multiflorum (4x) x L. perenne (2x) behaved cytologically as autotriploids but the segregation of isozyme variants did not always agree with the expected trisomic ratios. The overall effect of these deviations from expectation was a greater proportion than expected of diploid progeny from the cross L. multiflorum (2x) x triploid hybrid which did not include any of the L. perenne alleles at the three marker isozyme loci. The possible mechanisms for these aberrant segregation ratios are discussed together with the advantages of the crossing scheme to accelerate the recovery of the genotype of the recurrent parent in a backcrossing programme to transfer characters from one species to another.     

Isozyme analysis of primary trisomies in beet (Beta vulgaris L.). Genetical characterization and techniques for chromosomal assignment of two enzyme-coding loci: leucine aminopeptidase and glutamate oxaloacetate transaminase

Segregating families of beet (Beta vulgaris) were used to verify the monofactorial inheritance of two enzyme-coding loci, leucine aminopeptidase (Lap1) and glutamate oxaloacetate transaminase (Got3). With a series of primary trisomies and using three methods to discriminate between the critical trisomic (the locus is situated on the triplicated chromosome) and the non-critical ones, it was possible to allocate the two loci to beet chromosomes I and II, respectively. For the locus Lap1 distorted segregation ratios were estimated, and the incorporation of three alleles into one plant was attempted. In the case of Got3 the measurement of the allele dosage effect after electrophoresis was chosen as the major strategy. The output of laser densitometric scans were subjected to the non-parametrical Wilcoxon-Mann-Whitney test.     

The inheritance of restriction fragment length polymorphisms in the flax rust Melampsora lini

Summary Random cDNA sequences synthesized from poly A⁺ RNA extracted from germinated urediospores of the flax rust fungus, Melampsora lini, were used as probes to detect restriction fragment length polymorphisms (RFLPs) in three races of M. lini originating from cultivated flax, Linum usitatissimum, and one race originating from Australian native flax, L. marginale. Fourteen out of 22 probes tested detected RFLPs in the three races from cultivated flax while 19 of the probes detected polymorphisms between these three races and the race from L. marginale. The segregation of seven RFLPs was determined in a family of 19 F₂ progeny derived from a cross between two of the rust races. With six of these the inheritance was consistent, in each case, with the segregation of alleles at a single locus. Inheritance of the seventh was unusual and an explanation involving two loci with null alleles at each was proposed. No linkage was detected between any of the RFLP loci and nine unlinked loci specifying avirulence.     

Comparative analysis of microsatellite DNA polymorphism in landraces and cultivars of rice

Genetic polymorphisms of ten microsatellite DNA loci were examined among 238 accessions of landraces and cultivars that represent a significant portion of the distribution range for both indica and japonica groups of cultivated rice. In all, 93 alleles were identified with these ten markers. The number of alleles varied from a low of 3 or 4 at each of four loci, to an intermediate value of 9–14 at five loci, and to an extra-ordinarily high 25 at one locus. The numbers of alleles per locus are much larger than those detected using other types of markers. The number of alleles detected at a locus is significantly correlated with the number of simple sequence repeats in the targeted microsatellite DNA. Indica rice has about 14% more alleles than japonica rice, and such allele number differences are more pronounced in landraces than in cultivars. The indica-japonica differentiation component accounted for about 10% of the diversity in the total sample, and twice as much differentiation was detected in cultivars as in landraces. About two-thirds as many alleles were observed in cultivars as in landraces; another two-thirds of the alleles in the cultivar group were found in modern elite cultivars or parents of hybrid rice. The majority of the simple sequence repeat (SSR) alleles that were present in high or intermediate frequencies in landraces ultimately survived into modern elite cultivars and hybrids. The greater resolving power and the efficient production of massive amounts of SSR data may be particularly useful for germplasm assessment and evolutionary studies of crop plants.     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

Eighteen novel microsatellite markers for the Chinese sea perch, Lateolabrax maculatus

Chinese sea perch (Lateolabrax maculates) is one of the most important commercial species of mariculture in China. In this study, we constructed a repeat-enriched genomic DNA library of L. maculates. Eighteen dinucleotide microsatellite markers were characterized by genotyping 32 samples. The number of alleles ranged from three to nine, and the observed and expected heterozygosities ranged from 0.4516 to 1.0000 and from 0.4045 to 0.8676, respectively. Significant deviations from Hardy–Weinberg expectations were detected at four loci and linkage disequilibrium between two loci was significant after applying Bonferroni correction. The 18 highly polymorphic microsatellite markers should provide sufficient level of genetic diversity to investigate the population structure and evaluate the breeding strategy in L. maculates.     

A genetic map of Blumeria graminis based on functional genes, avirulence genes, and molecular markers

A genetic map of the powdery mildew fungus, Blumeria graminis f. sp. hordei, an obligate biotrophic pathogen of barley, is presented. The linkage analysis was conducted on 81 segregating haploid progeny isolates from a cross between 2 isolates differing in seven avirulence genes. A total of 359 loci were mapped, comprising 182 amplified fragment length polymorphism markers, 168 restriction fragment length polymorphism markers including 42 LTR-retrotransposon loci and 99 expressed sequence tags (ESTs), all the seven avirulence genes, and a marker closely linked to the mating type gene. The markers are distributed over 34 linkage groups covering a total of 2114 cM. Five avirulence genes were found to be linked and mapped in clusters of three and two, and two were unlinked. The Avr(a6) gene was found to be closely linked to markers suitable for a map-based cloning approach. A linkage between ESTs allowed us to demonstrate examples of synteny between genes in B. graminis and Neurospora crassa.     

Isolation and characterization of polymorphic nuclear microsatellite loci in <Emphasis Type="Italic">Taxus baccata</Emphasis> L.

Seven polymorphic nuclear microsatellite markers for Taxus baccata L. (English yew) were developed using an enriched-library method. An additional polymorphic SSR was obtained by testing eight primer pairs from the congeneric species Taxus sumatrana. Mendelian inheritance for the seven Taxus baccata SSRs was proved by genotyping 17 individuals and 124 megagametophytes (conifer seed haploid tissue). A total of 96 individuals from 5 different populations (10–26 samples per population) were used to estimate genetic diversity parameters. High levels of genetic diversity, with values ranging from 0.533 to 0.929 (6–28 alleles per SSR) were found. No linkage disequilibrium between pairs of loci was detected. All loci but one showed significant departures from Hardy–Weinberg equilibrium. Excess of homozygosity was probably due to high inbreeding in English yew populations, an outcome of low effective population size and/or fragmented distribution. Highly polymorphic SSRs will be used to conduct population genetic studies at different geographical scales and to monitor gene flow.     

QTL analysis of potato tuberization

Quantitative trait loci (QTLs) affecting tuberization were detected in reciprocal backcrosses between Solanum tuberosum and S. berthaultii. Linkage analyses were performed between traits and RFLP alleles segregating from both the hybrid and the recurrent parent using a set of framework markers from the potato map. Eleven distinct loci on seven chromosomes were associated with variation in tuberization. Most of the loci had small effects, but a QTL explaining 27% of the variance was found on chromosome 5. More QTLs were detected while following alleles segregating from the recurrent S. tuberosum parent used to make the backcross than were detected by following alleles segregating from the hybrid parent. More than half of the alleles favoring tuberization were at least partly dominant. Tuberization was favored by an allele from S. berthaultii at 3 of the 5 QTLs detected by segregation from the hybrid parent. The additive effects of the QTLs for tuberization explained up to 53% of the phenotypic variance, and inclusion of epistatic effects increased this figure to 60%. The most common form of epistasis was that in which presence of an allele at each of 2 loci favoring tuberization was no more effective than the presence of a favorable allele at 1 of the 2 loci. The QTLs detected for tuberization traits are discussed in relationship to those previously detected for trichome-mediated insect resistance derived from the unadapted wild species.Paper number 54 of the Department of Fruit and Vegetable Science, Cornell University     

Development of polymorphic microsatellite markers for the zebra finch (Taeniopygia guttata)

To study the population genetics as well as the mating system of captive zebra finch (Taeniopygia guttata) populations, we developed primers for 12 microsatellite loci and screened them in 529 individuals from two successive generations of a single captive population. All markers were polymorphic with five to 14 alleles per locus. We checked all markers for Mendelian inheritance in 307 offspring whose parents were known for sure. Four markers showed evidence for the presence of null alleles. Once allowing for null alleles, we found no mismatches between offspring and parents, suggesting a very low rate of mutation. Average observed and expected heterozygosities across the eight loci showing no evidence for null‐alleles was 0.819 and 0.812, respectively.     

Inheritance of RFLP loci in a loblolly pine three-generation pedigree

Summary A high-density restriction fragment length polymorphism (RFLP) linkage map is being constructed for loblolly pine (Pinus taeda L.). Loblolly pine cDNA and genomic DNA clones were used as probes in hybridizations to genomic DNAs prepared from grandparents, parents, and progeny of a three-generation outbred pedigree. Approximately 200 probes were evaluated for their ability to detect polymorphic loci between DNAs prepared from the two parent trees, 20–1010 and 11–1060, and cut with four different restriction enzymes: BamHI, DraI, EcoRI, and HindIII. More than half of the probes detecting single- or low-copy number sequences (56%) revealed polymorphisms between the two parents with at least one restriction enzyme. If necessary, an additional hybridization to DNAs prepared from the four grandparent trees was conducted to determine the zygosity of parent trees. Ten of these probes were hybridized to progeny DNAs from this cross and, as expected, the markers were inherited as simple codominant Mendelian alleles. Four of the ten probes detected segregation of three alleles at one locus, and four probes detected more than one independently segregating locus. RFLPs can be used immediately to assess genetic diversity in conifer populations and to efingerprint genotypes in tree improvement programs.     

Isolation and characterization of 13 microsatellite loci in Rhinolophus pusillus (least horseshoe bat) with cross-amplification in five related species

We used the enriched genomic library method to isolate and characterize dinucleotide microsatellite loci in the least horseshoe bat, Rhinolophus pusillus. Seventeen loci were obtained and tested on 31 individuals sampled from Guangxi Province in southern China. Thirteen of these markers were polymorphic with expected heterozygosity ranging from 0.821 to 0.909. A total of 164 alleles were detected and the number of alleles per locus ranged from 9 to 16 (mean 12.6). These polymorphic markers will be used to assess population structure in R. pusillus. In addition, successful cross-amplification in five congeneric bat species suggests most of these markers will also be useful for studying related species.     

Expression of unilateral incompatibility in pollen of Lycopersicon pennellii is determined by major loci on chromosomes 1, 6 and 10

Summary We have previously described gene introgression from the wild nightshade Solanum lycopersicoides into tomato (Lycopersicon esculentum) through the use of either diploid or sesquidiploid hybrids (the latter consisting of two genomes of L. esculentum and one genome of S. lycopersicoides). Both types of intergeneric hybrids display pollen sterility, but workable ovule fertility. Unilateral incompatibility prevents their direct hybridization with staminate L. esculentum. Pollen of a self-compattible form of the related wild species L. pennellii is compatible with pistils of L. esculentum x S. lycopersicoides hybrids. This trait was backcrossed from L. pennellii to L. esculentum in order to develop bridging lines that could be used to obtain progeny from the intergeneric hybrids and to study the inheritance of bridging ability. In progeny of L. esculentum x S. lycopersicoides hybrids pollinated with L. pennellii-derived bridging lines, preferential transmission of L. pennellii alleles was observed for certain isozyme and RFLP markers on chromosomes 1, 6 and 10. The skewed segregations suggest linkage to three major pollen-expressed compatibility loci. This was confirmed by observations of pollen tube growth, which indicated that compatibility with pistils of the diploid intergeneric hybrid occurred only in bridging lines at least heterozygous for the L. pennellii markers on chromosomes 1, 6 and 10. Compatibility with the sesquidiploid hybrid required only the chromosome 1 and 6 loci, indicating an apparent effect of gene dosage on expression of incompatibility in the pistil. In an F₂ L. esculentum x L. pennellii population, preferential transmission of L. pennellii alleles was observed for the same markers on chromosomes 1 and 10, as well as other markers on chromosomes 3, 11, and 12, but not 6. The chromosome 1 pollen compatibility locus maps to or near the S-locus, which determines S-allele specificity. The results are discussed in relation to existing genetic models for unilateral incompatibility, including the possible involvement of the S-locus.     

Isolation and characterization of microsatellite loci of Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta)

Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta) is a gardening plant with a native, fragmentary distribution in Taiwan. In this study, we described the development of eleven microsatellite loci in L. fordii for genetic studies. These new markers were tested in 16 individuals of three populations. The number of alleles ranged from 1 to 5 and the expected heterozygosity from 0.41746 to 0.72222. Four of the nine polymorphic loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. The microsatellite markers have also been proved as informative genetic markers for other 15 Lycopodium species.