Genomic architecture of alpha-amylase activity in mature rye grain relative to that of preharvest sprouting

Bi-directional selective genotyping (BSG) carried out on two opposite groups of F₉(541 × Ot1-3) recombinant inbred lines (RILs) with extremely low and extremely high alpha-amylase activities in mature (dry) grain of rye, followed by molecular mapping, revealed a complex system of selection-responsive loci. Three classes of loci controlling alpha-amylase activity were discerned, including four major AAD loci on chromosomes 3R (three loci) and 6RL (one locus) responding to both directions of the disruptive selection, 20 AAR loci on chromosomes 2RL (three loci), 3R (three loci), 4RS (two loci), 5RL (three loci), 6R (two loci) and 7R (seven loci) responding to selection for low alpha-amylase activity and 17 AAE loci on chromosomes 1RL (seven loci), 2RS (two loci), 3R (two loci), 5R (two loci) and 6RL (four loci) affected by selection for high alpha-amylase activity. The majority of the discerned AA loci also showed responsiveness to selection for preharvest sprouting (PHS). Two AAD loci on chromosome arm 3RL coincided with PHSD loci. The AAD locus on chromosome arm 3RS was independent from PHS, whereas that on chromosome 6RL belonged to the PHSR class. AAR-PHSR loci were found on chromosomes 4RS (one locus) and 5R (two loci) and AAE-PHSE loci were identified on chromosomes 1RL (one locus) and 5RL (one locus). Some PHSD loci represented the AAE (chromosomes 1RL, 3RS and 3RL) or AAR classes (chromosome 5RL). AAR and AAE loci not related to PHS were found on chromosomes 1RL, 2R, 3RS, 4R, 6RL and 7RL. On the other hand, several PHS loci (1RL, 3RS, 5RL, 6RS and 7RS) had no effect on alpha-amylase activity. Allele originating from the parental line 541 mapped in six AA loci on chromosomes 2R (two loci), 5R (three loci) and 7R (one locus) exerted opposite effects on PHS and alpha-amylase activity. Differences between the AA and PHS systems of loci may explain the weak correlation between these two traits observed among recombinant inbred lines. Strategies for the breeding of sprouting-resistant varieties with low alpha-amylase and high PHS resistance are discussed.     

Characterization of microsatellite loci from the solitary sweat bees Lasioglossum leucozonium and Lasioglossum oenotherae (Hymenoptera,Halictidae)

Microsatellite loci were isolated from two solitary sweat bees: the polylectic Lasioglossum leucozonium (10 loci) and the oligolectic Lasioglossum oenotherae (9 loci) (Hymenoptera, Halictidae). All loci were polymorphic with high observed heterozygosities (0.07–0.75 for L. leucozonium; 0.06–0.92 for L. oenotherae). These loci will be used to study the consequences of diet specialization on the population and conservation genetics of bees.     

Conserved genetic regions across angiosperms as tools to develop single‐copy nuclear markers in gymnosperms: an example using cycads

Several individuals of the Caribbean Zamia clade and other cycad genera were used to identify single‐copy nuclear genes for phylogeographic and phylogenetic studies in Cycadales. Two strategies were employed to select target loci: (i) a tblastX search of Arabidopsis conserved ortholog sequence (COS) set and (ii) a tblastX search of Arabidopsis‐Populus‐Vitis‐Oryza Shared Single‐Copy genes (APVO SSC) against the EST Zamia databases in GenBank. From the first strategy, 30 loci were selected, and from the second, 16 loci. In both cases, the matching GenBank accessions of Zamia were used as a query for retrieving highly similar sequences from Cycas, Picea, Pinus species or Ginkgo biloba. After retrieving and aligning all the sequences in each locus, intron predictions were completed to assist in primer design. PCR was carried out in three rounds to detect paralogous loci. A total of 29 loci were successfully amplified as a single band of which 20 were likely single‐copy loci. These loci showed different diversity and divergence levels. A preliminary screening allowed us to select 8 promising loci (40S, ATG2, BG, GroES, GTP, LiSH, PEX4 and TR) for the Zamia pumila complex and 4 loci (COS26, GroES, GTP and HTS) for all other cycad genera.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

Isolation and characterization of microsatellite loci in the Pacific pleasure oyster,Crassostrea corteziensis,and their cross‐species amplification in four other oyster species

Microsatellite loci were isolated in Crassostrea corteziensis using (GT)_n, (CT)_n and (CTGT)_n‐enriched genomic libraries. Within each of 45 sequenced clones, an average of three microsatellite regions (156 total) were observed. Thirty‐three primers were designed, from which 11 microsatellite loci amplified. Ten of those were polymorphic, with a range of two to 30 alleles. Three loci were not in Hardy–Weinberg equilibrium, and linkage disequilibrium was found for six pairs of loci. These microsatellite loci will be further tested for segregation distortions and null alleles to establish a set for population genetic studies of the species in the Northwest coasts of Mexico, and for optimization of aquaculture development. Seven of the microsatellite loci cross‐amplified in Crassostrea palmula, a sympatric species, and will be useful in further genetic studies.     

Isolation and characterization of microsatellites in Drosophila virilis and their cross species amplification in members of the D. virilis group

We report the isolation and cross species amplification of 42 Drosophila virilis microsatellite loci. Nine loci were isolated from mapped P1 bacteriophage clones and 33 were obtained from genomic DNA or GenBank searches. Cross species amplification was tested for all members of the D. virilis group. The amplification success was high (varying from 45% to 100%) and most of the loci were polymorphic. This set of loci can be applied for several genetic studies such as mapping behavioural quantitative trait loci (QTL) and for studying population structure in a phylogeographical framework in D. virilis group species.     

Primers from the orders Osteoglossiform and Siluriform detect polymorphic microsatellite loci in sun-catfish, Horabagrus brachysoma (Teleostei: Bagridae)

Horabagrus brachysoma (sun‐catfish, Bagridae, Siluriformes) is a valuable ornamental and food fish. The stock structure of H. brachysoma, necessary to conserve its declining natural populations, is not known. Twenty‐five primers developed for four fish species belonging to the orders Siluriform (3) and Osteoglossiform (1) were tested and eight primers amplified microsatellite loci in H. brachysoma. The results demonstrate that cross‐priming between fish species belonging to different families and even to different orders can yield microsatellite loci. Five of eight primers each amplified two loci. However, the loci that had repeat motifs after sequencing were considered only for genotyping. Finally, eight loci were polymorphic with hree to seven alleles. Individual fish genotype data (n = 42; 21 each in two rivers) at each locus was analysed. Significant genetic heterogeneity was detected at six loci. The identified loci exhibited potential for use in population genetics application in H. brachysoma.     

Different endogenous viral loci in Cornish and White Plymouth rock chickens

Summary Endogenous viral (ev) loci were studied in three broiler lines. In 5 birds of each of line cw1 and line cw2 (White Plymouth Rock lines) 19 and 14, respectively, different SstI ev-junction fragments were found, while in 8 R line birds (Cornish type) 15 different Sst I junction fragments were found. Further characterization of the line R loci with a second restriction enzyme, BamHI, revealed that these junction fragments represent 25 different loci, of which at least 21 have not been reported previously. SstI RFLP analysis of progeny from crosses between chickens of the three broiler lines and White Leghorns demonstrated that within line R and cw1 approximately 90% of the ev loci were hemizygous. In line cw2 at least 50% of the ev loci were hemizygous. There was no evidence for polymorphic loci, and only two ev loci were found to be linked genetically. Intertype crosses revealed that overall differences in the RFLP patterns observed between Cornish, White Plymouth Rock and White Leghorn chicken lines were due to the presence of different ev loci in each of the lines rather than to polymorphism. The few shared ev loci always contained similar allelic fragments.     

Characterization of tetranucleotide microsatellite loci and development and validation of multiplex reactions for the study of right whale species (genus Eubalaena)

A North Atlantic right whale (Eubalaena glacialis) genomic library was developed and screened with a (GATA)₈ probe to identify tetranucleotide microsatellite loci. Sixteen characterized loci were polymorphic in North Atlantic and/or South Atlantic (Eubalaena australis) right whales, 12 being polymorphic in E. glacialis, and 15 in E. australis. Fourteen of these were combined with 21 other previously identified loci for a suite of 35 loci which can be used to increase resolution of genetic analyses of these species. Multiplex reactions were developed for genotyping samples at these loci, providing a method that is rapid, reliable and cost‐effective.     

Genetic Mapping of Laminaria japonica and L. Iongissima Using Amplified Fragment Length Polymorphism Markers in a ＂Two-Way Pseudo- Testcross＂ Strategy

With a ＂two-way pseudo-testcross＂ mapping strategy, we applied the amplified fragment length polymorphism （AFLP） markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F1 cross family （Laminaria iongissima Aresch. × L. Japonica Miyabe） with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1：1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3：1 Mendelian segregation ratio. Among the loci with 1：1 segregating ratios, 79 loci were ordered in 14 linkage groups （648.6 cM） of the paternal map, and 72 loci were ordered in 14 linkage groups （601.9 cM） of the maternal map. The average density of loci was approximately 1 per 8 cM. To Investigate the homologies between two parental maps, we used 58 loci segregated 3：1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.     

Microsatellite markers for the fungal banana pathogens Mycosphaerella fijiensis, Mycosphaerella musicola and Mycosphaerella eumusae

We developed a total of 50 microsatellite markers for the three fungal pathogens causing the most important leaf spot diseases of banana: 32 loci for Mycosphaerella fijiensis are presented, and nine loci each for Mycosphaerella musicola and Mycosphaerella eumusae. All these loci were polymorphic within each species on a sample of isolates collected from various locations around the world. Within M. fijiensis and M. musicola, most of the loci tested (> 80%) in a sample of isolates from a single location in Cameroon were also polymorphic. Multiplex polymerase chain reaction systems were developed with 15 loci for M. fijiensis.     

Analysis of microsatellite locus polymorphism in potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis>) cultivars of Russian breeding

Polymorphism of microsatellite loci of the nuclear genome was examined in 29 cultivars and accessions of wild potato (S. tuberosum, S. stoloniferum, S. demissum, and S. phureja). Nine SSR markers, most informative (PIC = 0.61–0.92) for genotyping of the cultivars of Russian breeding were selected. Polymorphism of the selected SSR loci was characterized, and prevailing, as well as unique SSR allele phenotypes were described. A total of 87 allele phenotypes were identified. The highest number of allele phenotypes was detected for the SSR1 (17), ST83/84 (12), and STRBCS1b (12) loci. The least numbers of allele phenotypes were typical of the ST47/48 (5) and STWIN12G (6) loci. Based on the microsatellite loci analyzed, for each of the cultivars examined, its allele formula was established. The latter can be uses as the cultivar molecular genetic passport. Diagnostic sets of most informative loci (SSR markers), enabling identification of the genotypes of all potato cultivars of Russian breeding examined, were determined     

Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Inheritance and linkage relationships of isozyme loci in cucumber (Cucumis sativus L.)

Summary Genetic analyses were conducted among 18 provisionary isozyme loci in Cucumis sativus L. Fourteen loci demonstrated simple Mendelian inheritance while observed variation at four loci (Gpi2, Gr2, Pgm3, Skdh2) was determined not to have a predictable genetic basis. Joint segregation analyses among the 14 genetically predictable polymorphic loci resulted in the assignment of 12 loci to four linkage groups. Linkage groups contain the following loci: (1) Gr1, Pgm1, Idh, Pgd1; (2) Pep-pap, Mdh2, Mdh3, Gpi1; (3) Pep-la, Per4; (4) Pgd2, G2dh. Mpi2 and Mdh1 segregated independently. Recombination fractions for linked loci ranged between 0.051 (Pgm1-Idh) to 0.385 (Pep-la-Per4). Some practical applications of isozyme marker loci for cucumer improvement are discussed.     

Isolation and characterization of microsatellites in Drosophila montana and their cross‐species amplification in D. virilis

We report the isolation of 20 microsatellite loci from Drosophila montana and their cross amplification in the relative D. virilis. All microsatellite loci were polymorphic in the focal species D. montana, with gene diversities ranging from 0.23 to 0.93. In D. virilis only eight loci (40%) amplified and two loci were polymorphic (10%). These markers represent the first report of microsatellites isolated in D. montana. They could be applied for studying population structure and phylogeography. The largest benefit, however, will be their use in studies of quantitative trait loci, such as the mapping of behavioural quantitative trait loci.     

Characterization of microsatellite loci isolated from the blacktip shark and their utility in requiem and hammerhead sharks

We isolated and characterized 16 microsatellite loci from the blacktip shark, Carcharhinus limbatus, and tested cross‐species amplification in 11 Carcharhinus species and five additional shark genera. Thirty‐six (1.6%) and 180 (48%) colonies were positive for dinucleotide repeat motifs from unenriched and enriched libraries, respectively. Heterozygosities of polymorphic loci ranged from 0.04 to 0.96 with two to 22 alleles per locus. Amplification products were observed at nine to 13 loci (five to 11 of which where polymorphic) in 10 Carcharhinus species. Several loci were also polymorphic in each of the additional genera examined.     

Characterization of microsatellite loci isolated in trumpeter swan (Cygnus buccinator)

Primers for 16 microsatellite loci were developed for the trumpeter swan (Cygnus buccinator), a species recovering from a recent population bottleneck. In a screen of 158 individuals, the 16 loci were found to have levels of variability ranging from two to seven alleles. No loci were found to be linked, although two loci repeatedly revealed significant departures from Hardy–Weinberg equilibrium. Amplification in the closely related tundra swan (Cygnus columbianus) was successful for all except one locus. These microsatellite loci will be applicable for population genetic analyses and ultimately aid in management efforts.     

Characterization of microsatellite loci for five members of the minnow family Cyprinidae found in the Sacramento–San Joaquin Delta and its tributaries

Two microsatellite‐enriched libraries [(CAGA)_n, (TAGA)_n] were constructed using pooled DNA from three cyprinid species native to the Sacramento–San Joaquin Delta of California: Sacramento splittail (Pogonichthys macrolepidotus); Sacramento pikeminnow (Ptychocheilus grandis); and tui chub (Siphateles bicolor). Primers were designed for 105 loci and tested for levels of polymorphism in five cyprinid species found in the Delta: Sacramento splittail, Sacramento pikeminnow, tui chub, hitch (Lavinia exilicauda), and Sacramento blackfish (Orthodon microlepidotus). Fifty‐one loci were polymorphic for at least one species and 31 loci were polymorphic for multiple species. The number of polymorphic loci per species ranged from 16 to 26.     

Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization

The 17s-5.8s-25s ribosomal RNA gene (rDNA) loci in Oryza spp. were identified by the fluorescence in-situ hybridization (FISH) method. The rDNA loci were located on one-to-three chromosomes (two-to-six sites) within the eight diploid Oryza spp. One of the rDNA loci gave the weakest hybridization signal. This locus is reported for the first time in the genus Oryza. The chromosomes containing the rDNA loci were determined to be numbers 9, 10 and 11 in descending order of the copy number of rDNA. The application of image analysis methods, after slide preparation treatments (post-treatments), and the use of a thermal cycler, greatly improved the reproducibility of the results. The evolutionary significance of the variability of rDNA loci among the Oryza spp. is discussed.     

Polymorphic microsatellite repeats are not conserved between Leishmania donovani and Leishmania major

Thirteen sets of polymerase chain reaction (PCR) primers were designed to amplify microsatellite loci identified in the genome sequence of Leishmania major. Polymorphisms were detected in L. major at all loci. In Leishmania donovani only two of these loci were informative for classification purposes with this data set. The PCR products of all loci from one L. donovani strain were sequenced and it was found that the number of repeats in the microsatellite loci were either substantially reduced with respect to L. major or absent altogether. Consequently it is unlikely to be possible to use the genome sequence of L. major to identify polymorphic microsatellite loci in other Leishmania species.