Fine mapping a locus controlling leg morphology in the domestic dog.

The domestic dog offers a remarkable opportunity to disentangle the genetics of complex phenotypes. Here, we explore a locus, previously identified in the Portuguese water dog (PWD), associated with PC2, a morphological principal component characterized as leg width versus leg length. The locus was initially mapped to a region of 26 Mb on canine chromosome 12 (CFA12) following a genome-wide scan. Subsequent and extensive genotyping of single-nucleotide polymorphisms (SNPs) and haplotype analysis in both the PWD and selected breeds representing phenotypic extremes of PC2 reduced the region from 26 Mb to 500 kb. The proximity of the critical interval to two collagen genes suggests that the phenotype may be controlled by cis-acting mechanisms.

Understanding the genetics of autoimmune disease: two loci that regulate late onset Addison's disease in Portuguese Water Dogs.

Addison's disease, an immune-mediated disorder caused by destruction of the adrenal glands, is a rare disorder of Western European populations. Studies indicate that the disorder is polygenic in nature, involving specific alleles of the CTLA-4, DRB1*04 and DQ, Cyp27B1, VDR and MIC-A and -B loci. A similar immune form of Addison's disease occurs in several breeds of domestic dog, with frequencies ranging from 1.5 to 9.0%. The high frequency of the disease in domestic dog breeds likely reflects the small number of founders associated with many breeds, subsequent inbreeding, and the frequent use of popular sires. The Portuguese Water Dog (PWD) is a significantly affected breed. An analysis of 11,384 PWDs surveyed between 1985 and 1996 suggests a breed-specific disease incidence of 1.5%. As with humans, the disease is typically of late onset. This study involves a genetic comparison of Addison's disease in the PWD to the analogous disease in humans. The study is facilitated by the existence of complete pedigrees and a relatively high degree of inbreeding among PWDs. The breed originated from 31 founders, with 10 animals responsible for 90% of the current gene pool. We describe, specifically, the identification of two disease-associated loci, on Canis familiaris (CFA) chromosomes CFA12 and 37, which are syntenic with the human DRB1 histocompatibility locus alleles HLA-DRB1*04 and DRB1*0301, and to a locus for immunosuppression syntenic with CTLA-4. Strong similarities exist therefore in the complex genetic background of Addison's disease in humans and in the PWD. With the completion of the canine and human genome sequence, the purebred dog is set to become an important comparative model for Addison's as well as other human immune disorders.

A new integrated genetic linkage map of the soybean.

A total of 391 simple sequence repeat (SSR) markers designed from genomic DNA libraries, 24 derived from existing GenBank genes or ESTs, and five derived from bacterial artificial chromosome (BAC) end sequences were developed. In contrast to SSRs derived from EST sequences, those derived from genomic libraries were a superior source of polymorphic markers, given that the mean number of tandem repeats in the former was significantly less than that of the latter ( P<0.01). The 420 newly developed SSRs were mapped in one or more of five soybean mapping populations: "Minsoy" x "Noir 1", "Minsoy" x "Archer", "Archer" x "Noir 1", "Clark" x "Harosoy", and A81-356022 x PI468916. The JoinMap software package was used to combine the five maps into an integrated genetic map spanning 2,523.6 cM of Kosambi map distance across 20 linkage groups that contained 1,849 markers, including 1,015 SSRs, 709 RFLPs, 73 RAPDs, 24 classical traits, six AFLPs, ten isozymes, and 12 others. The number of new SSR markers added to each linkage group ranged from 12 to 29. In the integrated map, the ratio of SSR marker number to linkage group map distance did not differ among 18 of the 20 linkage groups; however, the SSRs were not uniformly spaced over a linkage group, clusters of SSRs with very limited recombination were frequently present. These clusters of SSRs may be indicative of gene-rich regions of soybean, as has been suggested by a number of recent studies, indicating the significant association of genes and SSRs. Development of SSR markers from map-referenced BAC clones was a very effective means of targeting markers to marker-scarce positions in the genome.

Teaching a new dog old tricks: identifying quantitative trait loci using lessons from plants.

Locating quantitative trait loci (QTL) in mammalian systems has proven difficult due to the lack of genetic control and reproducibility, as well as the expense of maintaining sufficiently large populations for genotyping and phenotyping. In plants, populations of recombinant inbred lines (progeny bred to homozygosity from a single cross) do not have these problems. Methods developed to identify QTL in a recombinant inbred soybean population provide a basis for analysis of a suitable mammalian population, such as Portuguese water dogs in the United States. The more than 6,000 dogs have accurate pedigrees, available phenotypic data and samples for genotyping, as well as interesting quantitative trait variation. The computer program Georgie allows us to choose large subpopulations with desirable characteristics such as high degrees of consanguinity that capture some of the benefits of recombinant inbred lines in plants. Computer simulations extending methods developed for simpler plant populations indicate that QTL with realistic effects can be identified from such subpopulations. Currently we are developing markers and collecting phenotypic and genotypic data from this population to begin the process of unraveling the genetic basis of quantitative traits in dogs.

Characterization of Soymar1, a mariner element in soybean.

Mariner elements, a family of DNA-mediated transposable elements with short, inverted terminal repeats, have been reported in a wide variety of arthropods, as well as planarians, nematodes, and humans. No such element has been reported in a plant. Here we report a mariner element in the plant soybean (Glycine max (L.) Merr.). Although this sequence belongs to the mariner family, it is clearly distinct from previously reported mariner-like elements, as well as from the Tc1 transposon family. Novel aspects of its sequence could be useful as a starting point to identify mariner-like elements in new organisms, and it may prove useful in creating a transformation vector for plants.

Interactions between quantitative trait loci in soybean in which trait variation at one locus is conditional upon a specific allele at another.

A large recombinant inbred population of soybean has been characterized for 220 restriction fragment-length polymorphism (RFLP) markers. Values for agronomic traits also have been measured. Quantitative trait loci (QTL) for height, yield, and maturity were located by their linkage to RFLP markers. QTL controlling large amounts of trait variation were analyzed for the dependence of trait variation on particular alleles at a second locus by comparing cumulative distributions of the trait for each genotype (four genotypes per pair of loci). Interesting pairs of loci were analyzed statistically with maximum likelihood and Monte Carlo comparison of additive and epistatic models. For each locus affecting height, variation was conditional upon the presence of a particular allele at a second unlinked locus that itself explained little or no trait variation. The results show that interactions between QTL are frequent and control large effects. Interactions distinguished between different QTL in a single linkage group and between QTL that affect different traits closely linked to one RFLP marker--i.e., distinguished between pleiotropy and closely linked genes. The implications for the evolution of inbreeding plants and for the construction of agronomic breeding strategies are discussed.

Epistatic expression of quantitative trait loci (QTL) in soybean [Glycine max (L.) Merr.] determined by QTL association with RFLP alleles.

Quantitative trait values for seed oil and protein content or for maturity were measured in recombinant inbred lines (RIL) of soybean derived from a cross between two soybean cultivars: 'Minsoy' PI 27890 and 'Noir 1' PI 290136. Seed oil was found to be inversely correlated to protein content. By analyzing DNA from plants with extreme phenotypes, we were able to identify quantitative trait loci (QTL) for these traits as being linked to several restriction fragment length polymorphism (RFLP) loci, including R183 for oil and protein content and R79 for maturity. Cumulative distributions of trait values were graphed for those RIL with 'Minsoy' alleles and for those with 'Noir 1' alleles. As already suggested by the alleles found associated with extreme phenotypes, the distributions were consistent with an independent and additive expression of the maturity QTL linked to R79. That is, the cumulative distributions for plants with 'Minsoy' alleles and for plants with 'Noir 1' alleles were similar in shape, but the entire 'Noir 1' curve had been shifted to later maturity dates. In contrast, the trait distributions for a locus affecting oil and protein content linked to R183 were not compatible with an additive model. These results suggest that this approach can be used for rapid identification of QTLs with epistatic expression.

A genetic map of soybean (Glycine max L.) using an intraspecific cross of two cultivars: 'Minosy' and 'Noir 1'.

Genetic markers were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: 'Minsoy' (PI 27.890) and 'Noir 1' (PI 290.136). A genetic linkage map was constructed (LOD [Symbol: see text] 3), consisting of 132 RFLP, isozyme, morphological, and biochemical markers. The map defined 1550cM of the soybean genome comprising 31 linkage groups. An additional 24 polymorphic markers remained unlinked. A family of RFLP markers, identified by a single probe (hybridizing to an interspersed repeated DNA sequence), extended the map, linking other markers and defining regions for which other markers were not available.

Determining the linkage of quantitative trait loci to RFLP markers using extreme phenotypes of recombinant inbreds of soybean (Glycine max L. Merr.).

An experimental test is described for linkages between RFLP markers and quantitative trait loci (QTL). Two hundred and eighty-four F7-derived recombinant inbred lines (RIL) obtained from crossing the soybean cultivars (Glycine max L. Merr.) 'Minsoy' and 'Noir 1' were evaluated for maturity, plant height, lodging, and seed yield. RIL exhibiting an extreme phenotype for each trait (earliest and latest plants for maturity, etc.) were selected, and two bulked DNA samples were prepared for each trait. A Southern transfer of the digested bulked DNA was hybridized with restriction fragement length polymorphism (RFLP) probes, and linkages with QTL were established by quantitating the amount of radioactive probe that bound to fragments defining alternative parental RFLP alleles. When an RFLP marker was linked to a QTL, one parental allele predominated in the bulked DNA from a particular phenotype; the other allele was associated with the opposite phenotype. When linkage was absent, radioactivity was associated equally with both alleles for a given phenotype (or with both phenotypes for a given allele). These results confirmed RFLP-QTL associations previously discovered by interval mapping on a smaller segregating population from the same cross. New linkages to QTL were also verified.

Interval mapping of quantitative trait loci for reproductive, morphological, and seed traits of soybean (Glycine max L.).

Quantitative trait loci (QTL) were mapped in segregating progeny from a cross between two soybean (Glycine max (L.) Merr.) cultivars: 'Minsoy' (PI 27.890) and 'Noir 1' (PI 290.136). The 15 traits analyzed included reproductive, morphological, and seed traits, seed yield and carbon isotope discrimination ratios ((13)C/(12)C). Genetic variation was detected for all of the traits, and transgressive segregation was a common phenomenon. One hundred and thirty-two linked genetic markers and 24 additional unlinked markers were used to locate QTL by interval mapping and one-way analysis of variance, respectively. Quantitative trait loci controlling 11 of the 15 traits studied were localized to intervals in 6 linkage groups. Quantitative trait loci for developmental and morphological traits (R1, R5, R8, plant height, canopy height, leaf area, etc.) tended to be clustered in three intervals, two of which were also associated with seed yield. Quantitative trait loci for seed oil were separated from all the other QTL. Major QTL for maturity and plant height were linked to RFLP markers R79 (31% variation) and G173 (53% variation). Quantitative trait loci associated with unlinked markers included possible loci for seed protein and weight. Linkage between QTL is discussed in relation to the heritability and genetic correlation of the traits.

The RecE recombination pathway mediates recombination between partially homologous DNA sequences: structural analysis of recombination products.

Escherichia coli generalized recombination, utilizing the RecA RecB recombination pathway, requires large stretches (70-200 bp) of complete DNA sequence homology. In contrast, we have found that the RecE pathway can promote recombination between DNA with only short stretches of homology. A plasmid containing 10 partially homologous direct repeats was linearized by digestion with specific restriction enzymes. After transformation, a RecE+ (sbcA) host was able to circularize the plasmid by recombination between partially homologous direct repeat sequences. Recombination occurred in regions of as little as 6 bp of perfect homology. Recombination was enhanced in the regions adjacent to restriction sites used to linearize the plasmid, consistent with a role of double-strand breaks in promoting recombination. A mechanism is proposed in which the 5' exonuclease, ExoVIII, produces 3' single-stranded ends from the linearized plasmid. These pair with other sequences of partial homology. Partial homologies in the sequences flanking the actual join serve to stabilize this recombination intermediate. Recombination is completed by a process of "copy and join." This recombination mechanism requires less homology to stabilize intermediates than the degree of homology needed for mechanisms involving strand invasion. Its role in nature may be to increase genomic diversity, for example, by enhancing recombination between bacteriophages and regions of the bacterial chromosome.

Genetic analysis of an interspecific hybrid swarm of Populus: occurrence of unidirectional introgression.

Restriction fragment length polymorphisms were used to distinguish genotypes of two species of Populus, P. fremontii ('Fremont') and P. angustifolia ('narrowleaf'). Both inter- and intraspecific polymorphisms were detected in these cottonwood trees. The interspecific variation was much greater than the intraspecific variation. This permitted identification of parental genotypes within individual trees of a hybrid swarm which exists in an overlap zone between the two species. Within this hybrid swarm, individual trees are either F1 hybrids or backcrosses with a pure 'narrowleaf' parent; no progeny were found that could be attributed to crossing between F1 hybrid trees, or to backcrossing between F1 hybrid trees and 'Fremont'.

Genetic variation in an inbred plant: variation in tissue cultures of soybean [Glycine max (L.) Merrill].

Although soybean [Glycine max (L.) Merrill] grows as an inbreeding, generally homozygous, plant, the germplasm of the species contains large amounts of genetic variation. Analysis of soybean DNA has indicated that variation of RFLP (restriction fragment length polymorphism) markers within the species usually entails only two alleles at any one locus and that mixtures of such dimorphic loci account for virtually all of the restriction fragment variation seen in soybean (G. max), and in its ancestors, G. soja and G. gracilis. We report here that tissue cultures prepared from root tissue of individual soybean plants develop RFLP allelic differences at various loci. However, these newly generated alleles are almost always the same as ones previously found and characterized in other varieties of cultivated soybean (cultivars). This repeated generation of particular alleles suggests that much of the genetic variation seen in soybean could be the consequence of specific, relatively frequently employed, recombinational events. Such a mechanism would allow inbred cultivars to generate genetic variation (in the form of alternative alleles) in a controlled manner, perhaps in response to stress.

The sequence of the 3.3-kilobase repetitive element from Dipodomys ordii suggests a mechanism for its amplification and interspersion.

DNA from the kangaroo rat, Dipodomys ordii, contains a 3.3-kb, highly repeated sequence that is interspersed throughout the genome in small tandem clusters. One 3.3-kb unit has been cloned into pBR322 and the nucleotide sequence determined. The clone used was shown to be representative of the bulk of such sequences found in the genomic DNA. The sequence contains 10 homologous subunits each ca. 260 bp in length. Comparison of these to one another yielded a 258-bp consensus sequence containing a 35-bp terminal inverted repeat. Two unique stretches also occur. One of these contains a region that could serve as a promoter for RNA polymerase III; the other contains a sequence related to the ARS sequences of yeast. It is proposed that an ancestral sequence similar to the consensus sequence was amplified to 10 or more units, and that, subsequently, two other sequences were inserted. The properties of these insertions may have led to the dispersal of the sequence throughout the genome.

Changes in methylation of tissue cultured soybean cells detected by digestion with the restriction enzymes HpaII and MspI.

Each of the tandemly arranged 5S RNA genes of soybean contain two CCGG sites which, if unmethylated, can be digested by both MspI and HpaII. Methylation of the internal cytosine (CmeCGG) prevents digestion by HpaII but allows digestions by MspI.Suspension cultures were prepared from soybean plants and the DNA from these cultures was examined for the susceptibility of 5S RNA genes to digestion by MspI and HpaII. 5S genes from DNA extracted from intact plants can be partially digested with MspI but not at all by HpaII. In contrast, shortly after cells were cultured the 5S RNA could be hydrolyzed by both HpaII and MspI. After prolonged cell culture, the 5S genes from some cell lines were found to have become partially or even completely resistant to HpaII digestion. The results suggest that lack of methylation can occur when cells are cultured and that such methylation may play a role in the heritable changes observed in cell culture.

Isopropyl-N(3-chlorophenyl) carbamate (CIPC) induced chromosomal loss in soybean: a new tool for plant somatic cell genetics.

The properties and uses of tissue culture partial haploid soybean cell lines are explored. Partial haploid lines were prepared by CIPC treatment of a genetic heterozygote, and compared to this heterozygote and to homozygous cell lines corresponding to the parental genotypes from which the heterozygote was derived. Cell lines which lack chromosomes were characterized physiologically and with respect to a variety of isozyme markers. Often the loss of chromosomes revealed a phenotype corresponding to a recessive parental genotype. In some cases, however, new phenotypes were observed indicating a complex genotype and suggests the interaction of several genes. The implications of this for plant breeding are discussed.CIPC also was used as a tool to dissect a complex phenotype which arose as the result of mutagenesis. A mutant cell line which required asparagine for growth but also had acquired the ability to grow on allantoin as a sole source of nitrogen was treated with CIPC to remove chromosomes. The requirement for asparagine could be separated from the ability to use allantoin, demonstrating that these phenotypes were the result of separate mutations.

Ribosomal RNA synthesis in soybean suspension cultures growing in different media.

The transcription of ribosomal RNA has been studied in suspension tissue cultures of soybean Glycine max L. Merr cv. Mandarin cells (SB-1). A large precursor molecule was synthesized which contains RNA homologous to the 25 and 18S cistrons. Transcription was from one strand and appeared to start adjacent to the 18S cistron and to proceed through the 18S DNA, a <0.6-kilobase transcribed spacer and the 25S cistron. A nontranscribed spacer region was identified. When cells grew rapidly in sucrose (24 hours doubling time) they contained 7 times as many ribosomes as when they grew slowly in maltose (200 hours doubling time). Upon transfer from maltose to sucrose, cells began to accumulate ribosomes at a rapid rate (80-fold more rapid synthesis than in maltose medium) within 2 hours at 33 degrees C. The 2-hour lag is due in large part to a longer processing time during which newly synthesized RNA is packaged into ribosomes. Therefore, the increase in transcription rate may occur within a few minutes of the transfer to sucrose.

The Red function of phage lambda mediates the alteration of an interspersed repeated DNA sequence from the kangaroo rat Dipodomys ordii.

The kangaroo rat D. ordii is shown to contain an interspersed repeated sequence 3.25 kb in length. This sequence undergoes alteration (deletions) when cloned into a Red+ lambda vector but is stable in a Red- vector. Using the Red- clone it is shown that deletions are the result of the activity of the lambda Red, or E. coli RecE, recombination systems. Mixed infection experiments demonstrate that this activity is acting in trans. The deletions appear to yield fragments of specific size, suggesting that the recombination system is recognizing a specific sequence. The demonstration of small homologous fragments in the animal genomic DNA suggests that a similar system of recombination may exist in D. ordii.

Use of isopropyl-N(3-chlorophenyl) carbamate (CIPC) to produce partial haploid cells from suspension cultures of soybean (Glycine max).

Suspension cultures of a heterozygous line of soybean (Glycine max) were treated with CIPC. Surviving clones had lost 10-15 chromosomes. A recessive phenotype (non-fluorescence) not expressed in the heterozygote, was expressed with high frequency (15-20%) in these clones. Nucleoside auxotrophs were isolated from the segregant clones consistent with partial haploidy. A selective screen for nucleoside auxotrophs is described.

Isolation of an auxotrophic cell line of soybean (Glycine max) which requires asparagine or glutamine for growth.

A suspension culture of haploid soybean cells was treated with ultra-violet light. From the mutagenized culture an auxotrophic cell line was isolated which grows on 1-B5 media supplemented with asparagine or glutamine. In the absence of these additives the cells cease to grow and die. Asparagine is unable to serve as a sole source of nitrogen, i.e., cannot substitute for nitrate and ammonia.