Polymorphic microsatellite loci in Linum usitatissimum

We report the characterization of 28 polymorphic microsatellite markers in Linum usitatissimum that allow distinguishing almost all cultivars of both flax and linseed. Polymorphism was low, ranging from two to 10 alleles per locus in the 93 cultivars screened. Linkage disequilibrium was found at about a third of the pairs of loci likely due to self‐fertilization and strong selection by breeders. We tested these loci for cross‐amplification in nine additional species of Linum and found that three species amplified a majority of loci.     

Identification of quantitative trait loci contributing to Fusarium wilt resistance on an AFLP linkage map of flax (Linum usitatissimum)

 An AFLP genetic linkage map of flax (Linum usitatissimum) was used to identify two quantitative trait loci (QTLs) on independent linkage groups with a major effect on resistance to Fusarium wilt, a serious disease caused by the soil pathogen Fusarium oxysporum (lini). The linkage map was constructed using a mapping population from doubled-haploid (DH) lines. The DH lines were derived from the haploid component of F₂ haploid-diploid twin seed originating from a cross between a polyembryonic, low-linolenic-acid genotype (CRZY8/RA91) and the Australian cultivar ‘Glenelg’. The AFLP technique was employed to generate 213 marker loci covering approximately 1400 cM of the flax genome (n=15) with an average spacing of 10 cM and comprising 18 linkage groups. Sixty AFLP markers (28%) deviated significantly (P<0.05) from the expected segregation ratio. The map incorporated RFLP markers tightly linked to flax rust (Melamspora lini) resistance genes and markers detected by disease resistance gene-like sequences. The study illustrates the potential of the AFLP technique as a robust and rapid method to generate moderately saturated linkage maps, thereby allowing the molecular analysis of traits, such as resistance to Fusarium wilt, that show oligogenic patterns of inheritance. Received: 8 December 1997 / Accepted: 7 April 1998     

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.     

THE DISTRIBUTION AND ORIGIN OF GENES FOR RACE-SPECIFIC RESISTANCE TO MELAMPSORA LINI IN LINUM MARGINALE

The genetic basis of resistance in wild flax (Linum marginale) to its host-specific pathogen Melampsora lini was investigated in seven lines collected from a single population growing at Kiandra, New South Wales and in an additional ten lines collected more widely across southeastern Australia. All lines showed different phenotypic patterns of resistance and susceptibility. Genetic analyses indicated the presence of single dominant genes for race-specific resistance in all but one of these lines. That particular line appeared to carry two linked dominant genes for resistance. Intercrosses between lines in each of these groups of L. marginale detected substantially more linkage between the resistance genes in the Kiandra population sample than between those in the broader geographic collection. This result is interpreted to indicate a possible mechanism whereby resistance genes are generated in natural populations.     

Use of short microsatellites from database sequences to generate polymorphisms among Lycopersicon esculentum cultivars and accessions of other Lycopersicon species

A search of nearly 2000 sequences from Solanaceae species in the EMBL and Genbank databases yielded 220 microsatellites. Among these were 80 microsatellites from 675 Lycopersicon entries. Dinucleotide repeats, as well as (CAA)_n and (TAA)_n repeats, were over-represented in non-coding DNA. The other trinucleotide repeats were predominantly found in exonic DNA. PCR analysis of 44 of the microsatellite-containing Lycopersicon loci identified 36 primer pairs that yielded well-scorable fragments, or groups of fragments, in L. esculentum cultivars and accessions of Lycopersicon species. Twenty-nine of these amplified bands that were polymorphic among the four Lycopersicon species. Ten primer pairs generated polymorphic bands among seven tomato cultivars. Upon examining the number of microsatellites and the degree of polymorphisms in relation to the repeat type and motif, the type of DNA the microsatellite resided in, the length of the microsatellite, and the presence of imperfections in the microsatellite, only two significant correlations were found. (i) Imperfect repeats were less polymorphic among species than perfect repeats. (ii) The percentage of loci polymorphic among cultivars increased from 6% for the shortest loci (with eight or less repeat units) to 60% for the group with the longest repeats (12 repeat units or longer). Among the species, however, all length classes contained about 83% polymorphic loci. In general, 2–4 alleles were found for each locus among the samples of the test set. In a few cases, up to eight alleles were found. A combination of these microsatellite loci can therefore be useful in distinguishing cultivars of tomato, which are genetically very closely related to each other. Received: 9 August 1996 / Accepted: 23 August 1996     

Inter-fungus interaction betweenFusarium lini bolley and some saprophytic fungi isolated fromLinum usitatissimum L. roots

Summary Necrotrophic mycoparasitic and strong competitive ability ofFusarium lini Bolley, a root pathogen of linseed (Linum usitatissimum L.), was observed against some potential fungal root colonizers. The antagonistic nature ofF. lini was studied on nutrient agar and root surface in the presence of background antagonism. The results are discussed in the light of general factors involved in antagonism during the colonization of substrate.     

Development and characterization of polymorphic microsatellite markers in Linum usitatissimum

Thirty-five microsatellite loci were isolated and characterized in Linum usitatissimum using enriched genomic libraries. These loci were screened in eight cultivars from different countries and regions and were found to be polymorphic, with the number of alleles per locus ranging from two to six, and observed and expected heterozygosities ranging from 0.125 to 0.375 (mean 0.013) and from 0.233 to 0.842 (mean 0.601), respectively. These polymorphic new microsatellite loci will be useful for genetic linkage map construction, germplasm classification and identification, gene identification and quantitative trait loci mapping, and marker-assisted selection in breeding in L. usitatissimum.     

Deletion mutation as a means of isolating avirulence genes in flax rust

Summary The interaction between flax rust,Melampsora lini, and its host, flax,Linum usitatissimum, has been extensively studied, and certain genetic features make the system an appropriate choice to utilize in isolating genes conferring avirulence in rust. A mutant that was selected for virulence on Lx plants was isolated, after treatment with gamma rays, from a strain that is genotypicallyA-L5,A-L6,A-L7,A-Lx/A-L5,A-L6,a-L7,a-Lx. These four specificities are tightly linked. Breeding tests showed that this mutant was genotypicallyA-L5,A-L6,a-L7,a-Lx/a-L5,a-L6,a-L7,a-Lx and, when made homozygous for the mutant chromosome, was virulent onL5,L6,L7, andLx. This result excludes somatic recombination as a source of the mutation and indicates deletion as a likely cause. A 250 bp genomic sequence from a strain of rust homozygous for these four linked avirulence genes (A-L5,A-L6,A-L7,A-Lx) was isolated, using a method that allows the differential cloning of the specific DNA sequences located within a deletion in the mutant genome. This clone hybridized to two EcoRI bands in genomic DNA from the strain homozygous for the four linked avirulence genes and from the strain homozygousA-L5 andA-L6 and heterozygousA-L7 andA-Lx, but showed no homology to DNA from the strain carrying the putative chromosomal deletion. The correlation between the genetically characterized deletion mutation and the isolation of a sequence from within a region of chromosome missing from this strain of rust suggests that this 250 bp tract may be part of, or closely linked to, the defined set of avirulence genes.     

Characterization of microsatellite loci in the house fly,Musca domestica L. (Diptera: Muscidae)

The house fly, Musca domestica L., is a cosmopolitan species with a capacity to transmit human pathogens. Here, we report on the development of polymorphic microsatellite loci for house flies and present preliminary results from four house fly subpopulations from Manhattan, Kansas. Twenty‐four microsatellite loci were isolated and characterized using DNA enriched for repeat sequences. Forty individuals from four locations in Kansas were assayed to identify for polymorphic loci. Eight loci were polymorphic with the number of alleles ranging from three to six.     

Polymorphic microsatellite DNA markers for Daviesia triflora (Papilionaceae)

We developed 11 polymorphic microsatellite DNA markers for an Australian native plant, Daviesia triflora. The number of alleles per locus in 40 individuals varied from four to 19, and observed and expected heterozygosities ranged from 0.450 to 0.925 and from 0.497 to 0.899, respectively. Nine loci showed no significant deviation from Hardy–Weinberg equilibrium (P > 0.05), and null alleles appear to exist at loci DT‐A102 and DT‐B103. All loci showed independent inheritance.     

Microsatellite loci for Myrmica kotokui and their application in some congeneric ant species distributed in northern Japan

We developed seven polymorphic microsatellite loci for Myrmica kotokui from RAPD (rapid analysis of polymorphic DNA) fragments. These loci showed two to six alleles with expected and observed heterozygosity of 0.13–0.73 and 0.14–0.78, respectively. These loci are transferable to the congeneric species as Myrmica rubra, Myrmica luteola and Myrmica taediosa from northern Japan. These loci will allow analyses of genetic structure of Myrmica species at both the colony level and population levels.     

Microsatellite DNA loci for Western Hemlock [Tsuga heterophylla (Raf.) Sarg]

Polymorphic microsatellite loci were developed for Western Hemlock [Tsuga heterophylla (Raf.) Sarg], a prominent forest tree species in Western North America. Microsatellite‐enriched libraries were screened for (CA)_n dinucleotide repeats from which 33 positive clones were sequenced. Polymerase chain reaction (PCR) primers for 16 microsatellite loci were prepared and tested against DNA from unrelated Western Hemlock trees. The 12 most informative microsatellite loci are reported here. From four to 22 alleles per locus were observed, with an average expected heterozygousity of 0.799.     

Characterization of novel microsatellites and development of multiplex PCR for large‐scale population studies in wild cherry,Prunus avium

Primers were developed for 14 microsatellite or simple sequence repeat (SSR) loci identified from a Prunus avium‘Charger’ genomic DNA library. In a survey of 16 wild cherry accessions 10 of the loci revealed polymorphisms of between two and six alleles. The remaining loci were found to be monomorphic. Seven polymorphic loci identified in this study and four polymorphic loci previously reported in sweet cherry were mapped and found to be unlinked. Two multiplex polymerase chain reactions (PCR) were optimized to enable the characterization of all 11 unlinked, polymorphic SSR loci.     

Genetic variation and population structure of endemic yellow catfish, <Emphasis Type="Italic">Horabagrus</Emphasis><Emphasis Type="Italic">brachysoma</Emphasis> (Bagridae) among three populations of Western Ghat region using RAPD and microsatellite markers

Random amplified polymorphic DNA (RAPD) and microsatellite markers were applied to evaluate the genetic variation in endemic and endangered yellow catfish, Horabagrus brachysoma sampled from three geographic locations of Western Ghat, South India river systems. In RAPD, of 32 10-mer RAPD primers screened initially, 10 were chosen and used in a comparative analysis of H. brachysoma collected from Meenachil, Chalakkudy and Nethravathi River systems. Of the 124 total RAPD fragments amplified, 49 (39.51%) were found to be shared by individuals of all 3 populations. The remaining 75 fragments were found to be polymorphic (60.48%). In microsatellites, six polymorphic microsatellite loci were identified by using primers developed for Pangasius hypophthalmus, Clarias macrocephalus and Clarias gariepinus. The identified loci were confirmed as microsatellite by sequencing after making a clone. The nucleotide sequences of 6 loci were published in NCBI genbank. The number of alleles across the six loci ranged from 4 to 7 and heterozygosities ranged from 0.07 to 0.93. The mean number of alleles and effective number of alleles per locus were 5.00 and 3.314, respectively. The average heterozygosity across all investigated samples was 0.72, indicating a significant deficiency of heterozygotes in this species. RAPD and microsatellite methods reported a high degree of gene diversity and genetic distances depicted by UPGMA dendrograms among the populations of H. brachysoma.     

Dinucleotide repeat microsatellite markers for buck’s‐horn plantain (Plantago coronopus)

Eleven polymorphic microsatellite loci were obtained from a GA enriched genomic library, constructed from DNA of buck’s‐horn plantain (Plantago coronopus). The microsatellite loci were tested on 24 genotypes. These plants were collected from meadows along the coast, located on 11 sites ranging from the southwest to the northeast of the Netherlands. In this set of plants the isolated microsatellites were highly polymorphic with 3–24 alleles per locus and a maximum observed heterozygosity of 0.91. Some of the microsatellite loci also showed amplification in two other plantain species (P. lanceolata and P. maritima).     

Isolation and characterization of microsatellites loci in the lemon (Citrus limon)

This study reports the isolation and characterization of seven polymorphic microsatellite loci in Citrus. The loci were isolated from two libraries constructed from genomic DNA nonenriched for TC and AC repeats and enriched for AC repeats. These markers yielded four to nine alleles per locus (mean 6.14) in a survey of 32 Citrus cultivars. Average observed heterozygosity ranged from 0.43 to 0.72. The levels of polymorphism found in this study suggest that these microsatellite loci can become an important tool for genetic studies in Citrus.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Characterization of microsatellite DNA markers for a mahseer species,Tor tambroides (Cyprinidae) and cross‐amplification in four congeners

This study reports the isolation and characterization of microsatellite DNA markers in a mahseer species, Tor tambroides (Pisces, Cyprinidae). Of a total of 14 loci evaluated, 10 were polymorphic in T. tambroides samples, with an average of 2.86 alleles per locus. Deviations from Hardy–Weinberg equilibrium were observed at one locus and there was no indication of linkage disequilibrium among loci. A high level of cross‐amplification among four congeners was achieved, with 12 loci successfully amplifying and 11 loci showing polymorphism in at least one other species. These markers will be a useful resource for population genetic studies and broodstock management of closely related mahseer species.     

Microsatellite DNA markers for the Stable Fly,Stomoxys calcitrans (Diptera: Muscidae)

Eight polymorphic microsatellite markers have been isolated from a microsatellite‐enriched DNA library from the Stable Fly, Stomoxys calcitrans. These loci exhibited four to 15 alleles per locus and an expected heterozygosity ranging from 0.19 to 0.84 in the three populations studied from La Réunion island (Indian Ocean). They should therefore be valuable for studying genetic diversity and population structure. Cross‐species amplification of these eight loci in the closely allied species Stomoxys niger niger was successful for four of these loci, one of which was monomorphic and one of which strongly departed from Hardy–Weinberg expectations.     

Instability of (GATA)<Subscript> n</Subscript> microsatellite loci in the parthenogenetic Caucasian rock lizard<Emphasis Type="Italic"> Darevskia unisexualis</Emphasis> (Lacertidae)

Mini- and microsatellites, comprising tandemly repeated short nucleotide sequences, are abundant dispersed repetitive elements that are ubiquitous in eukaryotic genomes. In humans and other bisexual species hypervariable mini- and microsatellite loci provide highly informative systems for monitoring of germline and somatic instability. However, little is known about the mechanisms by which these loci mutate in species that lack effective genetic recombination. Here, multilocus DNA fingerprinting was used to study M13 minisatellite and (GATA) _n microsatellite instability in the parthenogenetic Caucasian rock lizard Darevskia unisexualis (Lacertidae). DNA fingerprinting of 25 parthenogenetic families, from six isolated populations in Armenia (comprising a total of 84 siblings), using the oligonucleotide (GATA)₄ as a hybridization probe, revealed mutant fingerprinting phenotypes in 13 siblings that differed from their mothers in several restriction DNA fragments. In three families (8 siblings), the mutations were present in the germline. Moreover, the mutant fingerprint phenotypes detected in siblings were also present in population DNA samples. No intrafamily variations in DNA fingerprint patterns were observed with the M13 minisatellite probe. Estimates of the mutation rate for (GATA) _n microsatellite loci in D. unisexualis showed that it was as high as that seen in some bisexual species, reaching 15% per sibling or 0.95% per microsatellite band. Furthermore, in one case, a somatic (GATA) _n microsatellite mutation was observed in an adult lizard. These findings directly demonstrate that mutations in (GATA) _n microsatellite loci comprise an important source of genetic variation in parthenogenetic populations of D. unisexualis.Communicated by G. P. Georgiev