Isolation and characterization of microsatellite markers in the South African abalone (Haliotis midae)

We report the isolation and characterization of 11 polymorphic microsatellite loci in the South African abalone Haliotis midae. These loci showed a range of five to 21 alleles per locus and observed heterozygosities ranging from 0.14 to 0.93 in a wild population of 32 individuals. All loci except four conformed to Hardy–Weinberg expectations and did not show linkage disequilibrium. The polymorphism exhibited at these loci indicate that they would be useful in determining levels of genetic variability in natural and commercial Haliotis midae populations as well as in parentage and Quantitative Trait Loci (QTL) analysis in hatchery reared abalone.     

A de novo mutation in KIT causes white spotting in a subpopulation of German Shepherd dogs

Although variation in the KIT gene is a common cause of white spotting among domesticated animals, KIT has not been implicated in the diverse white spotting observed in the dog. Here, we show that a loss‐of‐function mutation in KIT recapitulates the coat color phenotypes observed in other species. A spontaneous white spotting observed in a pedigree of German Shepherd dogs was mapped by linkage analysis to a single locus on CFA13 containing KIT (pairwise LOD = 15). DNA sequence analysis identified a novel 1‐bp insertion in the second exon that co‐segregated with the phenotype. The expected frameshift and resulting premature stop codons predicted a severely truncated c‐Kit receptor with presumably abolished activity. No dogs homozygous for the mutation were recovered from multiple intercrosses (P = 0.01), suggesting the mutation is recessively embryonic lethal. These observations are consistent with the effects of null alleles of KIT in other species.     

Genetic studies on free-ranging macaques of Cay Santiago. II. 6-phosphogluconate dehydrogenase and NADH-methemoglobin reductase (NADH-diaphorase).

For the population of 395 semi-free-ranging rhesus macaques (Macaca mulatta) that inhabited Cayo Santiago in 1976, 6-phosphogluconate dehydrogenase phenotypes of 378 animals were determined. Three phenotypes, controlled by two autosomal codominant alleles,PGD^A andPGD^B, were found by electrophoretic methods. The frequencies of the alleles are 0.898 and 0.102, respectively. The population, composed of five troops and peripheral males, is in Hardy-Weinberg equilibrium at this locus. The allele frequencies at the 6-phosphogluconate dehydrogenase locus in the population in 1976 were compared with frequencies in 1973; a statistically significant difference was found in one troop. The phenotypes of NADH-methemoglobin reductase (NADH-diaphorase) were determined electrophoretically for 372 animals. These phenotypes are probably the products of two autosomal codominant alleles,Dia¹ andDia², with frequencies of 0.786 and 0.214, respectively. The population is in equilibrium at this locus also. Tests of homogeneity at the dehydrogenase and reductase loci indicate that the allele frequencies are significantly different among the five troops in the population. Observed and expected phenotypic ratios in progeny were compared at the dehydrogenase and the reductase loci. The only significant deviation from expectation occurs among offspring of mothers heterozygous at the reductase locus. The observed distributions of alleles at the 6-phosphogluconate dehydrogenase locus and the NADH-methemoglobin reductase locus are probably the results of stochastic processes.     

Genetic instability at the cut locus of Drosophila melanogaster induced by the MR-h12 chromosome

Summary Unstable mutations were generated at the cut locus by the MR-h12 factor which induces male recombination. The unstable allele ct ^MR2, containing the MR-transposon in the cut locus is a very powerful mutator producing a number of different viable and lethal mutations both in the cut locus and outside it.I describe several types of mutations: stable reversion to wild type, which were sometimes associated with the appearance of unstable mutations in other loci; of stable deficiencies at the cut locus (lethals); new unstable mutations at different loci with the ct ^MR2 allele conserved; new unstable cut alleles with a phenotype other than that of ct ^MR2. The possible mechanisms of these mutational events are discussed. The genetic system constructed in the present work affords an opportunity for molecular studies of the cut locus and the MR-transposon, as a sequence from the cut locus has recently been cloned (Tchurikov et al. 1981).     

Identification of quantitative trait loci controlling heading date in rice using a high-density linkage map

 Quantitative trait locus (QTL) analysis has been carried out to identify genes conferring heading date in rice. One hundred and eighty six F₂ plants derived from a cross between a japonica variety, Nipponbare, and an indica variety, Kasalath, were used as a segregating population for QTL mapping and more than 850 markers were employed to identify QTLs. Scan-analysis revealed the existence of two QTLs with large effects, Hd-1 and Hd-2, one in the middle of chromosome 6 and one at the end of chromosome 7, respectively. For both loci, the Kasalath alleles reduced days-to-heading. In addition, three QTLs with minor effects, Hd-3, Hd-4 and Hd-5, were found to be located on chromosomes 6, 7 and 8 based on a secondary scan analysis which was carried out by removing the phenotypic effects of Hd-1 and Hd-2. For the three secondary loci, the Nipponbare alleles reduced days-to-heading. The five QTLs explained 84% of the total phenotypic variation in the F₂ population based on a multiple-QTL model. The presence of a digenic interaction between Hd-1 and Hd-2 was clearly suggested. Received: 18 March 1997 / Accepted: 24 June 1997     

Isolation and characterization of microsatellite markers from Brown Eared-pheasant (Crossoptilon mantchuricum)

The Brown Eared-pheasant (Crossoptilon mantchuricum) is an endangered species endemic to China. Here we developed eight microsatellite loci using a modified biotin-capture method. In the analyses of 28 individuals sampled, the number of alleles per locus ranged from four to nine. The expected (H _E) and observed (H _O) heterozygosities were 0.466–0.825 and 0.619–0.847, respectively. Results that eight microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as genetic diversity and population genetic structure of C. mantchuricum.     

Quantitative trait loci affecting growth in high growth (hg) mice

A genome-wide scan was performed in order to identify Quantitative Trait Loci (QTL) associated with growth in a population segregating high growth (hg), a partially recessive mutation that enhances growth rate and body size in the mouse. A sample of 262 hg/hg mice was selected from a C57BL/6J-hg/hg× CAST/EiJ F₂ cross and typed with 79 SSLP markers distributed across the genome. Eight significant loci were identified through interval mapping. Loci on Chromosomes (Chrs) 2 and 8 affected the growth rate of F₂ mice. Loci on Chr 2 and 11 affected growth rate and carcass lean mass (protein and ash). A locus on Chr 9 modified femur length and another one in Chr 17 affected both carcass lean mass and femur length, but none of these had significant effects on growth rate. Loci on Chrs 5 and 9 modified carcass fat content. Additive effects were positive for C57BL/6J alleles, except for the two loci affecting carcass fatness. Typing of selected markers in 274 +/+ F₂ mice revealed significant interactions between hg and other growth QTL, which were detected as changes in gene action (additive or dominant) and in allele substitution effects. Knowledge about interactions between loci, especially when major genes are involved, will help in the identification of positional candidate genes and in the understanding of the complex genetic regulation of growth rate and body size in mammals. Received: 29 June 2000 / Accepted: 22 November 2000     

A Genotypic-Oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macrocategories of Cystic Fibrosis

Cystic fibrosis (CF) is a monogenic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The genotype–phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, there were 125 different mutated alleles (11 with novel mutations and 10 with complex mutations) and 225 genotypes. A strong correlation between mutational patterns at the genotypic level and phenotypic macrocategories emerged. This specificity appears to largely depend on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macrocategories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype–phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype–phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway.     

Isolation and characterization of microsatellite loci in wild and domestic turkeys (Meleagris gallopavo)

We describe the isolation, development and application of seven microsatellite loci in the eastern wild turkey, Meleagris gallopavo silvestris, as well as their amplification and levels of polymorphism in the domestic turkey. The number of alleles per locus ranged from 5 to 15 and average heterozygosity was high for almost all loci. Domestic turkeys showed significantly reduced numbers of alleles per locus and overall heterozygosities when compared to eastern wild turkeys. The high variability in these markers should provide the level of resolution required to continue studies of wild turkey population genetics.     

QTL analysis of flowering time inArabidopsis thaliana

Quantitative trait loci (QTL) analyses based on restriction fragment length polymorphism maps have been used to resolve the genetic control of flowering time in a cross between twoArabidopsis thaliana ecotypes H51 and Landsbergerecta, differing widely in flowering time. Five quantitative trait loci affecting flowering time were identified in this cross (RLN1-5), four of which are located in regions containing mutations or loci previously identified as conferring a late-flowering phenotype. One of these loci is coincident with theFRI locus identified as the major determinant for late flowering and vernalization responsiveness in theArabidopsis ecotype Stockholm.RLN5, which maps to the lower half of chromosome five (between markers mi69 and m233), only affected flowering time significantly under short day conditions following a vernalization period. The late-flowering phenotype of H51 compared to Landsbergerecta was due to alleles conferring late flowering at only two of the five loci. At the three other loci, H51 possessed alleles conferring early flowering in comparison to those of Landsbergerecta. Combinations of alleles conferring early and late flowering from both parents accounted for the transgressive segregation of flowering time observed within the F₂ population. Three QTL,RLN1,RLN2 andRLN3 displayed significant genotype-by-environment interactions for flowering time. A significant interaction between alleles atRLN3 andRLN4 was detected.     

253 Novel polymorphic microsatellites for the saltwater crocodile (Crocodylus porosus)

Genomic elucidation and mapping of novel organisms requires the generation of large genetic resources. In this study, 253 novel and polymorphic microsatellite loci were isolated and characterized for the saltwater crocodile (Crocodylus porosus) by constructing libraries enriched for microsatellite DNA. All markers were evaluated on animals obtained from Darwin Crocodile Farm in the Northern Territory, Australia, and are intended for future use in the construction of a genetic-linkage map for the saltwater crocodile. The 253 loci yielded an average of 4.12 alleles per locus, and those selected for mapping had an average polymorphic information content (PIC) of 0.425.     

A method for rapid mapping of mutations by plasmid rescue strategy inSaccharomyces cerevisiae

The products ofPRP17 andPRP18 genes are required for the second step of pre-mRNA splicing reactions inSaccharomyces cerevisiae. Temperature-sensitive mutants at either of these loci accumulate products of the first splicing reaction at nonpermissive temperature. To characterize functional regions in these proteins the mutations in three temperature-sensitive alleles ofPRP17 and two temperature-sensitive alleles ofPRP18 were mapped by the plasmid rescue strategy, One of the procedures adopted in the past is plasmid rescue of the mutant allele followed by sequencing of the entire gene. In this work we describe an adaptation of the above procedure that allows, first, rapid mapping of chromosomal segments bearing the mutations, followed by sequence characterization of the minimal segment. The strategy adopted was to integrate a wild-type copy of the gene at the homologous mutant chromosomal locus, followed by recovery of the chromosomal fragments from these integrants as plasmids inE. coli. The recovered plasmids were screened by a complementation assay for those that contained in them the chromosomal mutation. The mutations in all the three alleles ofPRP17 map to a small region in the N-terminal half of the protein, whereas the temperature-sensitive mutations in the two alleles ofPRP18 map to different regions of the PRP18 protein. The recovered mutant plasmids from all five alleles at the two loci were sequenced and the nucleotide changes were found to result in missense mutations in each case. Our strategy is therefore a rapid method to map chromosomal mutations and is of general use in structure-function analysis of cloned genes.     

Impact of white-spotting alleles,including W20, on phenotype in the American Paint Horse

The American Paint Horse Association (APHA) records pedigree and performance information for their breed, a stock-type horse valued as a working farm or ranch horse and as a pleasure horse. As the name implies, the breed is also valued for its attractive white-spotting patterns on the coat. The APHA utilizes visual inspections of photographs to determine if coat spotting exceeds threshold anatomical landmarks considered characteristic of desirable patterns. Horses with sufficient white patterning enter the ‘Regular’ registry, rather than the ‘Solid Paint-Bred’ division, providing a threshold modeled phenotype. Genetic studies previously defined sequence variants corresponding to 35 alleles for white spotting in the horse. Here, we calculate the allele frequencies for nine common white-spotting alleles in the American Paint Horse using a sample of 1054 registered animals. The APHA spotting phenotype is altered by additive interactions among spotting loci, and epistatically by the MC1R and ASIP genes controlling pigment production. The W20 allele within the KIT gene, independent of other known spotting alleles, was strongly associated with the APHA-defined white-spotting phenotype (P = 1.86 × 10⁻¹⁸), refuting reports that W20 acts only as a modifier of other underlying white-spotting patterns. The parentage of an individual horse, either American Paint or American Quarter Horse, did not alter the likelihood of its entering the APHA Regular Registry. An empirical definition of the action of these genetic loci on the APHA-defined white-spotting phenotype will allow more accurate application of genome-assisted selection for improving color production and the marketability of APHA horses.     

Isolation and characterization of polymorphic microsatellite markers in the gray,short‐tailed opossum (Monodelphis domestica)

Twenty‐six polymorphic microsatellite markers were isolated from (AC)_n and (AG)_n microsatellite‐enhanced genomic libraries of the gray, short‐tailed opossum Monodelphis domestica. All 26 loci showed high allelic diversity, with allele numbers ranging from five to 11 in a subset of 35 animals. Normal Mendelian inheritance was confirmed for 24 loci by analysing allelic segregation in 10, two‐generation, families. Non‐amplifying (null) alleles were detected at two loci, which we recommend be used only if pedigree data are available. We conclude that all of these microsatellite markers would be useful for quantitative trait locus mapping and population genetic studies.     

Eliminating expression of erucic acid-encoding loci allows the identification of “hidden” QTL contributing to oil quality fractions and oil content in <Emphasis Type="Italic">Brassica juncea</Emphasis> (Indian mustard)

Oil content and oil quality fractions (viz., oleic, linoleic and linolenic acid) are strongly influenced by the erucic acid pathway in oilseed Brassicas. Low levels of erucic acid in seed oil increases oleic acid content to nutritionally desirable levels, but also increases the linoleic and linolenic acid fractions and reduces oil content in Indian mustard (Brassica juncea). Analysis of phenotypic variability for oil quality fractions among a high-erucic Indian variety (Varuna), a low-erucic east-European variety (Heera) and a zero-erucic Indian variety (ZE-Varuna) developed by backcross breeding in this study indicated that lower levels of linoleic and linolenic acid in Varuna are due to substrate limitation caused by an active erucic acid pathway and not due to weaker alleles or enzyme limitation. To identify compensatory loci that could be used to increase oil content and maintain desirable levels of oil quality fractions under zero-erucic conditions, we performed Quantitative Trait Loci (QTL) mapping for the above traits on two independent F1 doubled haploid (F1DH) mapping populations developed from a cross between Varuna and Heera. One of the populations comprised plants segregating for erucic acid content (SE) and was used earlier for construction of a linkage map and QTL mapping of several yield-influencing traits in B. juncea. The second population consisted of zero-erucic acid individuals (ZE) for which, an Amplified Fragment Length Polymorphism (AFLP)-based framework linkage map was constructed in the present study. By QTL mapping for oil quality fractions and oil content in the ZE population, we detected novel loci contributing to the above traits. These loci did not co-localize with mapped locations of the fatty acid desaturase 2 (FAD2), fatty acid desaturase 3 (FAD3) or fatty acid elongase (FAE) genes unlike those of the SE population wherein major QTL were found to coincide with mapped locations of the FAE genes. Some of the new loci identified in the ZE population could be detected as ‘weak’ contributors (with LOD < 2.5) in the SE population in which their contribution to the traits was “masked” due to pleiotropic effects of erucic acid genes. The novel loci identified in this study could now be used to improve oil quality parameters and oil content in B. juncea under zero-erucic conditions.     

Using a limited mapping strategy to identify major QTLs for resistance to grapevine powdery mildew (<Emphasis Type="Italic">Erysiphe necator</Emphasis>) and their use in marker-assisted breeding

A limited genetic mapping strategy based on simple sequence repeat (SSR) marker data was used with five grape populations segregating for powdery mildew (Erysiphe necator) resistance in an effort to develop genetic markers from multiple sources and enable the pyramiding of resistance loci. Three populations derived their resistance from Muscadinia rotundifolia ‘Magnolia’. The first population (06708) had 97 progeny and was screened with 137 SSR markers from seven chromosomes (4, 7, 9, 12, 13, 15, and 18) that have been reported to be associated with powdery or downy mildew resistance. A genetic map was constructed using the pseudo-testcross strategy and QTL analysis was carried out. Only markers from chromosome 13 and 18 were mapped in the second (04327) and third (06712) populations, which had 47 and 80 progeny, respectively. Significant QTLs for powdery mildew resistance with overlapping genomic regions were identified for different tissue types (leaf, stem, rachis, and berry) on chromosome 18, which distinguishes the resistance in ‘Magnolia’ from that present in other accessions of M. rotundifolia and controlled by the Run1 gene on chromosome 12. The ‘Magnolia’ resistance locus was termed as Run2.1. Powdery mildew resistance was also mapped in a fourth population (08391), which had 255 progeny and resistance from M. rotundifolia ‘Trayshed’. A locus accounting for 50% of the phenotypic variation mapped to chromosome 18 and was named Run2.2. This locus overlapped the region found in the ‘Magnolia’-based populations, but the allele sizes of the flanking markers were different. ‘Trayshed’ and ‘Magnolia’ shared at least one allele for 68% of the tested markers, but alleles of the other 32% of the markers were not shared indicating that the two M. rotundifolia selections were very different. The last population, 08306 with 42 progeny, derived its resistance from a selection Vitis romanetii C166-043. Genetic mapping discovered a major powdery mildew resistance locus termed Ren4 on chromosome 18, which explained 70% of the phenotypic variation in the same region of chromosome 18 found in the two M. rotundifolia resistant accessions. The mapping results indicate that powdery mildew resistance genes from different backgrounds reside on chromosome 18, and that genetic markers can be used as a powerful tool to pyramid these loci and other powdery mildew resistance loci into a single line.     

Isolation and characterization of microsatellites in the Asian walking catfish Clarias macrocephalus

Microsatellite loci were characterized in the walking catfish, Clarias macrocephalus, random clones from a small genomic library using a (GT)₁₅ probe. Primers for DNA amplifications using polymerase chain reaction (PCR) were designed and synthesized for 23 loci. Twelve loci were polymorphic, with the number of alleles ranging from two to 13 alleles per locus. Developed microsatellite primers should prove useful for population studies and genetic mapping of the walking catfish.