Co-mapping studies of QTLs for fruit acidity and candidate genes of organic acid metabolism and proton transport in sweet melon (Cucumis melo L.).

Sweet melon cultivars contain a low level of organic acids and, therefore, the quality and flavor of sweet melon fruit is determined almost exclusively by fruit sugar content. However, genetic variability for fruit acid levels in the Cucumis melo species exists and sour fruit accessions are characterized by acidic fruit pH of <5, compared to the sweet cultivars that are generally characterized by mature fruit pH values of >6. In this paper, we report results from a mapping population based on recombinant inbred lines (RILs) derived from the cross between the non-sour 'Dulce' variety and the sour PI 414323 accession. Results show that a single major QTL for pH co-localizes with major QTLs for the two predominant organic acids in melon fruit, citric and malic, together with an additional metabolite which we identified as uridine. While the acidic recombinants were characterized by higher citric and malic acid levels, the non-acidic recombinants had a higher uridine content than did the acidic recombinants. Additional minor QTLs for pH, citric acid and malic acid were also identified and for these the increased acidity was unexpectedly contributed by the non-sour parent. To test for co-localization of these QTLs with genes encoding organic acid metabolism and transport, we mapped the genes encoding structural enzymes and proteins involved in organic acid metabolism, transport and vacuolar H+ pumps. None of these genes co-localized with the major pH QTL, indicating that the gene determining melon fruit pH is not one of the candidate genes encoding this primary metabolic pathway. Linked markers were tested in two additional inter-varietal populations and shown to be linked to the pH trait. The presence of the same QTL in such diverse segregating populations suggests that the trait is determined throughout the species by variability in the same gene and is indicative of a major role of the evolution of this gene in determining the important domestication trait of fruit acidity within the species.

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes.

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and 'Dulce' (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.

A 4,103 marker integrated physical and comparative map of the horse genome.

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

First radiation hybrid map of the river buffalo X chromosome (BBUX) and comparison with BTAX.

We report the first radiation hybrid map of the river buffalo X chromosome generated from a recently constructed river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel (BBURH(5000)). This map contains a total of 33 cattle-derived markers, including 10 genes, four ESTs and 19 microsatellites. The markers are distributed in two linkage groups: LG1 contains eight markers spanning 125.6 cR, and LG2 contains 25 markers spanning 366.3 cR. LG1 contains six markers in common with bovine sequence assembly build 3.1. With the exception of BMS2152, the order of these markers on our BBUX map is shuffled when compared to the cow X chromosome (Bos taurus; BTAX). From LG2, two markers (AMELX and BL22) map to a more distal portion of BTAX compared to BBUX. In addition, two pairs of LG2 markers exhibit inversions compared to BTAX (ILSTS017 and ATRX; XBM38 and PPEF1). Alternatively, when compared to the most recent bovine RH map (Bov-Gen 3000rads), BL1098 and BMS2227 from LG1 as well as PLS3 and BMS1820 from LG2 showed inverted positions on the BBUX map. These discrepancies in buffalo and cattle maps may reflect evolutionary divergence of the chromosomes or mapping errors in one of the two species. Although the set of mapped markers does not cover the entire X chromosome, this map is a starting point for the construction of a high-resolution map, which is necessary for characterization of small rearrangements that might have occurred between the Bubalus bubalis and Bos taurus X chromosomes.

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 1 (BBU1).

The largest chromosome in the river buffalo karyotype, BBU1, is a submetacentric chromosome with reported homology between BBU1q and bovine chromosome 1 and between BBU1p and BTA27. We present the first radiation hybrid map of this chromosome containing 69 cattle derived markers including 48 coding genes, 17 microsatellites and four ESTs distributed in two linkage groups spanning a total length of 1330.1 cR(5000). The RH map was constructed based on analysis of a recently developed river buffalo-hamster whole genome radiation hybrid (BBURH(5000)) panel. The retention frequency of individual markers across the panel ranged from 17.8 to 52.2%. With few exceptions, the order of markers within linkage groups is identical to the order established for corresponding cattle RH maps. The BBU1 map provides a starting point for comparison of gene order rearrangements between river buffalo chromosome 1 and its bovine homologs.

Preliminary radiation hybrid map for river buffalo chromosome 6 and comparison to bovine chromosome 3.

We present the first radiation hybrid (RH) map of river buffalo (Bubalus bubalis) chromosome 6 (BBU6) developed with a recently constructed river buffalo whole-genome RH panel (BBURH(5000)). The preliminary map contains 33 cattle-derived markers, including 12 microsatellites, 19 coding genes and two ESTs, distributed across two linkage groups. Retention frequencies for markers ranged from 14.4% to 40.0%. Most of the marker orders within the linkage groups on BBU6 were consistent with the cattle genome sequence and RH maps. This preliminary RH map is the starting point for comparing gene order between river buffalo and cattle, presenting an opportunity for the examination of micro-rearrangements of these chromosomes. Also, resources for positional candidate cloning in river buffalo are enhanced.

Construction of a river buffalo (Bubalus bubalis) whole-genome radiation hybrid panel and preliminary RH mapping of chromosomes 3 and 10.

The buffalo (Bubalus bubalis) is a source of milk and meat, and also serves as a draft animal. In this study, a 5000-rad whole-genome radiation hybrid (RH) panel for river buffalo was constructed and used to build preliminary RH maps for BBU3 and BBU10 chromosomes. The preliminary maps contain 66 markers, including coding genes, cattle expressed sequence tags (ESTs) and microsatellite loci. The RH maps presented here are the starting point for mapping additional loci that will allow detailed comparative maps between buffalo, cattle and other species whose genomes may be mapped in the future. A large quantity of DNA has been prepared from the cell lines forming the river buffalo RH panel and will be made publicly available to the international community both for the study of chromosome evolution and for the improvement of traits important to the role of buffalo in animal agriculture.

A radiation hybrid map of river buffalo (Bubalus bubalis) chromosome 7 and comparative mapping to the cattle and human genomes.

A preliminary radiation hybrid (RH) map containing 50 loci on chromosome 7 of the domestic river buffalo Bubalus bubalis (BBU; 2n = 50) was constructed based on a comparative mapping approach. The RH map of BBU7 includes thirty-seven gene markers and thirteen microsatellites. All loci have been previously assigned to Bos taurus (BTA) chromosome BTA6, which is known for its association with several economically important milk production traits in cattle. The map consists of two linkage groups spanning a total length of 627.9 cR(5,000). Comparative analysis of the BBU7 RH(5,000) map with BTA6 in cattle gave new evidence for strong similarity between the two chromosomes over their entire length and exposed minor differences in locus order. Comparison of the BBU7 RH(5,000) map with the Homo sapiens (HSA) genome revealed similarity with a large chromosome segment of HSA4. Comparative analysis of loci in both species revealed more variability than previously known in gene order and several chromosome rearrangements including centromere relocation. The data obtained in our study define the evolutionarily conserved segment on BBU7 and HSA4 to be between 3.5 megabases (Mb) and 115.8 Mb in the HSA4 (genome build 36) DNA sequence.

A 1.3-Mb interval map of equine homologs of HSA2.

A comparative approach that utilizes information from more densely mapped or sequenced genomes is a proven and efficient means to increase our knowledge of the structure of the horse genome. Human chromosome 2 (HSA2), the second largest human chromosome, comprising 243 Mb, and containing 1246 known genes, corresponds to all or parts of three equine chromosomes. This report describes the assignment of 140 new markers (78 genes and 62 microsatellites) to the equine radiation hybrid (RH) map, and the anchoring of 24 of these markers to horse chromosomes by FISH. The updated equine RH maps for ECA6p, ECA15, and ECA18 resulting from this work have one, two, and three RH linkage groups, respectively, per chromosome/chromosome-arm. These maps have a three-fold increase in the number of mapped markers compared to previous maps of these chromosomes, and an increase in the average marker density to one marker per 1.3 Mb. Comparative maps of ECA6p, ECA15, and ECA18 with human, chimpanzee, dog, mouse, rat, and chicken genomes reveal blocks of conserved synteny across mammals and vertebrates.

Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.

The functional interaction of BAFF and APRIL with TNF receptor superfamily members BAFFR, TACI and BCMA is crucial for development and maintenance of humoral immunity in mice and humans. Using a candidate gene approach, we identified homozygous and heterozygous mutations in TNFRSF13B, encoding TACI, in 13 individuals with common variable immunodeficiency. Homozygosity with respect to mutations causing the amino acid substitutions S144X and C104R abrogated APRIL binding and resulted in loss of TACI function, as evidenced by impaired proliferative response to IgM-APRIL costimulation and defective class switch recombination induced by IL-10 and APRIL or BAFF. Family members heterozygous with respect to the C104R mutation and individuals with sporadic common variable immunodeficiency who were heterozygous with respect to the amino acid substitutions A181E, S194X and R202H had humoral immunodeficiency. Although signs of autoimmunity and lymphoproliferation are evident, the human phenotype differs from that of the Tnfrsf13b-/- mouse model.

Second-generation integrated genetic linkage/radiation hybrid maps of the domestic cat (Felis catus).

We report construction of second-generation integrated genetic linkage and radiation hybrid (RH) maps in the domestic cat (Felis catus) that exhibit a high level of marker concordance and provide near-full genome coverage. A total of 864 markers, including 585 coding loci (type I markers) and 279 polymorphic microsatellite loci (type II markers), are now mapped in the cat genome. We generated the genetic linkage map utilizing a multigeneration interspecies backcross pedigree between the domestic cat and the Asian leopard cat (Prionailurus bengalensis). Eighty-one type I markers were integrated with 247 type II markers from a first-generation map to generate a map of 328 loci (320 autosomal and 8 X-linked) distributed in 47 linkage groups, with an average intermarker spacing of 8 cM. Genome coverage spans approximately 2,650 cM, allowing an estimate for the genetic length of the sex-averaged map as 3,300 cM. The 834-locus second-generation domestic cat RH map was generated from the incorporation of 579 type I and 255 type II loci. Type I markers were added using targeted selection to cover either genomic regions underrepresented in the first-generation map or to refine breakpoints in human/feline synteny. The integrated linkage and RH maps reveal approximately 110 conserved segments ordered between the human and feline genomes, and provide extensive anchored reference marker homologues that connect to the more gene dense human and mouse sequence maps, suitable for positional cloning applications.

Linkage of autosomal dominant common variable immunodeficiency to chromosome 5p and evidence for locus heterogeneity.

Common variable immunodeficiency (CVID, OMIM 240500) and selective immunoglobulin A deficiency (IgAD) are the most frequent primary immunodeficiencies in humans. Of the cases with CVID/IgAD, 20%-25% are familial, but the only previous claims of linkage or association are to the HLA region on chromosome 6p. We report the results of a genome-wide scan in three multiplex families with CVID, IgAD, and dysgammaglobulinemia, where affection is inherited in an autosomal dominant pattern. Two of the families are consistent with linkage to the telomeric region of chromosome 5p, whereas the third is consistent with linkage to the HLA region. Using a locus heterogeneity model and a conservative penetrance model, we obtained a LOD score of 3.35 for the 5p region. We sequenced the exons of one promising candidate gene within this region (PDCD6, also known as ALG-2) but found no causative mutation.

Radiation hybrid mapping of 304 novel microsatellites in the domestic cat genome.

Effective utilization of the domestic cat as an animal model for hereditary and infectious disease requires the development and implementation of high quality gene maps incorporating microsatellites and conserved coding gene markers. Previous feline linkage and radiation hybrid maps have lacked sufficient microsatellite coverage on all chromosomes to make effective use of full genome scans. Here we report the isolation and genomic mapping of 304 novel polymorphic repeat loci in the feline genome. The new loci were mapped in the domestic cat radiation hybrid panel using an automated fluorescent TAQ-Man based assay. The addition of these 304 microsatellites brings the total number of microsatellites mapped in the feline genome to 580, and the total number of loci placed onto the RH map to 1,126. Microsatellites now span every autosome with an average spacing of roughly one polymorphic STR every five centimorgans, and full genome coverage of one marker every 2.7 megabases. These loci now provide a useful tool for undertaking full-genome scans to identify genes associated with phenotypes of interest, such as those relating to hereditary disease, coat color, patterning and morphology. These resources can also be extended to the remaining 36 species of the cat family for population genetic and evolutionary genomic analyses.

Genomic imbalances in the progression of endocrine pancreatic tumors.

Endocrine pancreatic tumors (EPTs) are neoplasms with malignant potential. To explore the molecular basis of metastatic progression in human EPTs, we analyzed 17 paired specimens of primary EPTs and their metastases and 28 nonmetastatic EPTs using comparative genomic hybridization (CGH). Genomic alterations were detected in all of the matched primary/metastatic tumors and 19 (58%) nonmetastatic EPTs. The mean number of genomic changes was 17.3 in metastases, 12.5 in their primary tumors, and 4.5 in nonmetastatic EPTs. Statistical analysis of shared genomic changes in matched pairs of primary tumors and metastases showed a high probability (>95%) of a clonal relationship in 15 of the 17 cases. A closely related genetic pattern was also demonstrated on the basis of concordance analysis of the two groups. The most striking genomic changes which were enriched in metastases included gains of chromosomes 4 and 7 and losses of 21q. Other common regions of frequent losses (>40%) identified in metastases and/or their primary tumors involved 2p, 2q, 3p, 3q, 6q, 10p, and 11p, whereas frequently detected gains (>40%) in the paired tumors involved 5p, 5q, 12q, 14q, 17q, 18q, and 20q. These chromosomal aberrations were found in significantly fewer nonmetastatic EPTs. Some of these chromosomal loci may harbor genes contributing to the progression of EPT.

Characterization of native and yeast-expressed tomato fruit fructokinase enzymes.

Three fructokinase isozymes (FKI, FKII, FKIII) were separated from both immature and ripe tomato fruit pericarp. All three isozymes were specific for fructose with undetectable activity towards glucose or mannose. The three isozymes could be distinguished from one another with respect to response to fructose, Mg and nucleotide donor concentrations and this allowed the comparison of the fruit enzymes with the gene products of the two known cloned tomato fructokinase genes, LeFRK1 and LeFRK2. FKI was characterized by both substrate (fructose), as well as Mg, inhibition; FKII was inhibited by neither fructose nor Mg; and FKIII was inhibited by fructose but not by Mg. ATP was the preferred nucleotide donor for all three FKs and FKI showed inhibition by CTP and GTP above 1 mM. All three FKs showed competitive inhibition by ADP. During the maturation of the tomato fruit total FK activity decreased dramatically. There were decreases in activity of all three FKs, nevertheless, all were still observed in the ripe fruit. The two tomato LeFRK genes were expressed in yeast and the gene products were characterized with respect to the distinguishing characteristics of fructose, Mg and nucleotide inhibition. Our results indicate that FKI is the gene product of LeFRK2 and FKII is probably the gene product of LeFRK1.

Graph models of oncogenesis with an application to melanoma.

We describe several analytical techniques for use in developing genetic models of oncogenesis including: methods for the selection of important genetic events, construction of graph models (including distance-based trees, branching trees, contingency trees and directed acyclic graph models) from these events and methods for interpretation of the resulting models. The models can be used to make predictions about: which genetic events tend to occur early, which events tend to occur together and the likely order of events. Unlike simple path models of oncogenesis, our models allow dependencies to exist between specific genetic changes and allow for multiple, divergent paths in tumor progression. A variety of genetic events can be used with the graph models including chromosome breaks, losses or gains of large DNA regions, small mutations and changes in methylation. As an application of the techniques, we use a recently published cytogenetic analysis of 206 melanoma cases [Nelson et al. (2000), Cancer Genet. Cytogenet.122, 101-109] to derive graph models for chromosome breaks in melanoma. Among our predictions are: (1) breaks in 6q1 and 1q1 are early events, with 6q1 preferentially occurring first and increasing the probability of a break in 1q1 and (2) breaks in the two sets [1p1, 1p2, 9q1] and [1q1, 7p2, 9p2] tend to occur together. This study illustrates that the application of graph models to genetic data from tumor sets provide new information on the interrelationships among genetic changes during tumor progression.

An immune defect causing dominant chronic mucocutaneous candidiasis and thyroid disease maps to chromosome 2p in a single family.

We describe a large family in which a combination of chronic mucocutaneous candidiasis (fungal infections of the skin, nails, and mucous membranes) and thyroid disease segregate as an autosomal dominant trait with reduced penetrance. The family includes (a) four members with both candidiasis and thyroid disease, (b) five members, including one pair of phenotype-concordant MZ twins, with candidiasis only, and (c) three members with thyroid disease only. A whole-genome scan using DNA samples from 20 members of the family identified a candidate linkage region on chromosome 2p. By sampling additional individuals and genotyping supplementary markers, we established linkage to a region of approximately 15 cM bounded by D2S367 and D2S2240 and including seven adjacent markers consistent with linkage. With a penetrance estimate of.8, which was based on pedigree and affected status, the peak two-point LOD score was 3.70 with marker D2S2328, and the peak three-point LOD score was 3.82. This is the first linkage assignment of a dominant locus for mucocutaneous candidiasis.

Towards a complete North American Anabaptist Genealogy II: analysis of inbreeding.

We describe a large genealogy data base, which can be searched by computer, of 295,095 Amish and Mennonite individuals. The data base was constructed by merging our existing Anabaptist Genealogy Database 2.0 containing approximately 85,000 individuals with a genealogy file containing approximately 242,000 individuals, kindly provided by Mr. James Hostetler. The merging process corrected thousands of inconsistencies and eliminated hundreds of duplicate individuals. Geneticists have long been interested in Anabaptist populations because they are closed and have detailed written genealogies. The creation of an enlarged and unified data base affords the opportunity to examine inbreeding trends and correlates in these populations. We show the following results. The frequency of consanguineous marriages shows steady increase over time and reached approximately 85% for individuals born in 1940-1959. Among consanguineous marriages, the median kinship coefficient stayed stable in the 19th century, but rose from 0.0115 to 0.0151 in the 20th century. There are statistically significant associations (p < 0.0001) between inbreeding and family size and interbirth intervals in the 20th century. There is an association (p < 0.0005) between inbreeding and early death for individuals born in 1920-1959. However, this association reverses dramatically (p < 0.0005 in the opposite direction) for individuals born in 1960-1979. We tested for an association between inbreeding and being the mother of twins, but found none.

Heritability of life span in the Old Order Amish.

Although a familial contribution to human longevity is recognized, the nature of this contribution is largely unknown. We have examined the familial contribution to life span in the Old Order Amish (OOA) population of Lancaster County, Pennsylvania. Analyses were conducted on 1,655 individuals, representing all those born prior to 1890 and appearing in the most widely available genealogy, surviving until at least age 30 years, and with known date of death. Mean age at death (+/-SD) in this population was 70.7 +/- 15.6 years, and this did not change appreciably over time. Parental and offspring ages at death were significantly correlated, as were ages of death among siblings. Offspring longevity was correlated with longevity of both parents, and in more or less additive fashion. For example, mean offspring age at death was 69.4 +/- 15.3 years in individuals for whom both parents died before the age of 75 years (n = 280) and increased to 73.5 +/- 16.0 years in individuals for whom neither parent died before the age of 75 years (n = 311). These differences were highly significant (P = 0.006). We estimated heritability of life span to be 25% +/- 5%, suggesting that the additive effects of genes account for one quarter of the total variability in life span in the OOA. We conclude that longevity is moderately heritable in the OOA, that the genetic effects are additive, and that genetic influences on longevity are likely to be expressed across a broad range of ages. Published 2001 Wiley-Liss, Inc.

Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements.

PSI-BLAST is an iterative program to search a database for proteins with distant similarity to a query sequence. We investigated over a dozen modifications to the methods used in PSI-BLAST, with the goal of improving accuracy in finding true positive matches. To evaluate performance we used a set of 103 queries for which the true positives in yeast had been annotated by human experts, and a popular measure of retrieval accuracy (ROC) that can be normalized to take on values between 0 (worst) and 1 (best). The modifications we consider novel improve the ROC score from 0.758 +/- 0.005 to 0.895 +/- 0.003. This does not include the benefits from four modifications we included in the 'baseline' version, even though they were not implemented in PSI-BLAST version 2.0. The improvement in accuracy was confirmed on a small second test set. This test involved analyzing three protein families with curated lists of true positives from the non-redundant protein database. The modification that accounts for the majority of the improvement is the use, for each database sequence, of a position-specific scoring system tuned to that sequence's amino acid composition. The use of composition-based statistics is particularly beneficial for large-scale automated applications of PSI-BLAST.