Applied Genomics
Y. Kashi
Analysis of haplotypes is an important tool in population genetics, familial heredity, and gene mapping. Determination of haplotypes of multiple SNPs (single nucleotide polymorphisms) or other simple mutations is time-consuming and expensive when analyzing large populations, and often requires the help of computational and statistical procedures. Based on the procedure described by Sarkar and Sommer (1991) and Lo et al. (1991), we have developed a simple, rapid, and low-cost method for direct haplotyping of multiple SNPs and simple mutations found within specific regions or genes (micro-haplotypes). Using this method, it is possible to directly determine the physical linkage of multiple heterozygous alleles, by conducting a series of double allele-specific PCR amplification sets with simple analysis by gel electrophoresis. Application of the method requires prior information as to the sequence of the segment to be haplotyped, including the polymorphic sites. We applied the method to haplotyping of nine sites in the chicken HSP108 gene. One of the haplotypes in the population apparently arose by recombination between two existing haplotypes, and we were able to locate the point of recombination within a segment of 19 base-pairs. We anticipate rapidly growing needs for SNP haplotyping in human (medical and pharmacogenetics), animal, and plant genetics; in this context, the MD-PASA method offers a useful haplotyping tool.
Eitan, Y. Kashi, Y. Direct intragenic haplotyping by multiple double PCR amplification of specific alleles. Nucleic Acids Research, (in press 2002).
Y. Kashi
Revealing factors affecting the maintenance of genetic diversity in natural populations is of primary importance for genetic conservation, environmental protection, and recruitment of genetic resources for biotechnology. Recently, microorganisms became an important target for such studies. The budding yeast Saccharomyces cerevisiae is a unicellular eukaryote serving a favorite model in genetics and cell biology research, but remaining poorly studied in Nature. In this proposal, we focus on genetic diversity in natural populations isolated from the ’’Evolution Canyon’’ located in Nahal Oren at Mount Carmel, Israel.
Objectives: The major goal of our research program is to unravel the molecular-genetic basis of yeast population adaptation to spatially and temporarily varying environments. In particular, we plan to evaluate the genetic consequences of yeast adaptation to contrasting conditions on the opposite slopes of the ?Evolution Canyon?. The specific objectives include: (a) Evaluating variation at the ploidy level (haploid, diploid, aneu- and polyploid); (b) Assessment of mating type stability in the haploid strains, combined with DNA sequence analysis of the HO and MAT genes, and testing the fertility level of interslope vs. intraslope matings; (c) Analysis of microsatellite polymorphism and testing whether and how microsatellite loci may be a participating factor in yeast reaction to microclimatic selection; (d) Analysis of polymorphism in genes involved in resistance to radiation, heat, and drought stresses; (e) Analysis of the linkage disequilibrium between different microsatellite loci and testing whether sexual or clonal reproduction is more characteristic to the compared yeast populations. 70 Saccharomyces cerevisiae strains were isolated from opposite slopes in "Evolution Canyon". All of them were found prototrophs. The strains were tested for sporulation and for their mating type. The viability of crosses between the strains will be tested. On the DNA level, a battery of microsatellites will be checked for polymorphism and variation between the different strains. Sequences of candidate genes will be tested. DNA polymorphisms will be analyzed for linkage disequilibria. All the data will be analyzed in regards to the origin of the different populations in the canyon. The proposed study contributes to in-depth understanding of how genetic diversity arises and evolves during population adaptation to stressful environments. In particular, we expect to shed new light on questions related to: (i) role of microsatellite variation as a factor of fast fine ?tuning? of gene activity; (ii) the effect of stress on relative advantages of diploid vs. haploid stages; (iii) the evolution of the reproductive system, i.e. do more stressful conditions promote higher frequency of sex and recombination, and (iv) to what extent the ecological contrasts on the opposite slopes affect population differentiation, do yeast display interslope incipient sexual isolation, as found for other model organisms in the canyon. We expect that the revealed genetic variation in adaptively-important traits may become a source for further biotechnological improvement of industrial yeasts.
Elisabeth Nadjar- Boger et al , in preparation 2002.Submitted to the Israel Academy, 2002.
Y. Kashi
The cyanobacterium Nostoc linckia tested here is a sessile microorganism, growing as a carpet on rock surface, permanently exposed to environmental fluctuations of solar radiation, temperature and drought. We demonstrated remarkable interslope and intraslope genetic divergence of the genome (including both coding and noncoding regions) of Nostoc linckia, by using 211 AFLP (Amplified Fragment Length Polymorphism) DNA molecular marker loci. Genetic polymorphism of N. linckia sub-populations on the ecologically harsher SFS was significantly (p<0.05) higher (P=99.53%) than was that of the sub-populations on the climatically milder NFS (P=85.78%). We suggest that the climatically stressed SFS environment is responsible for this marked increase of genetic polymorphism, which is maintained by the combined evolutionary forces of diversifying and balancing selection.
As in other tested organisms at "Evolution Canyon", but even more exceptionally because of it is completely sedentary nature, the higher genetic polymorphism of Nostoc linckia on the SFS is naturally selected as an adaptive trait to cope with the higher ecological heterogeneity (the wider niche breadth theory) and stress. This could highlight the importance of ecological stress and selection in evolution, and its remarkable effect on the genetic system across the prokaryotic genome.
Krugman T., Satish N., Vinogadova O.N., Beharav A., Kashi Y., Nevo E. Genome diversity in a cyanobacterium Nostoc linckia at ?Evolution Canyon?, Israel, revealed by inter-HIP1 size polymorphisms. Evolutionary Ecology Research,3: 899-915, 2001.
Satish N., Krugman T., Vinogadova O.N. Nevo E., and Kashi Y. Genome Evolution of the Cyanobacterium Nostoc Linckia under Sharp Microclimatic Divergence at ?Evolution Canyon?, Israel. Microbial Ecology, 42: 306-316, 2001.
Y. Kashi
Every year tens of millions of dollars are lost worldwide due to mortality and morbidity resulting from heat stress of poultry. Therefore, broiler growers in developing countries in tropical and subtropical climates are looking for a more thermotolerant broiler.
The Solution: We have identified desert chickens that are distinctly more thermotolerant than modern commercial chickens due to an evolutionary adaptation. However, classical breeding methods do not provide the means for combining the thermotolerance of the desert bird with the superior commercial qualities of the modern broiler.
In the past decade, we contributed to the development of a new biotechnological approach to genetic improvement, termed "Marker Assisted Selection" (MAS) which involves a shift from selection based on observed performance, to selection based on detailed examination of DNA-level genetic variation. The MAS approach is ideally suited to identify DNA markers that are closely linked to genes determining physiological resistance to heat stress. We are developing and identifying DNA markers that are closely linked to genes determining physiological resistance to heat stress. This is a long term project - mapping the genes will take up to 2 years and the development of the thermotolerant broiler will take three more years. Field-testing, and flock expansion and improvement will take another few years. Since a large budget is required for such a project, we have set up a privet company "Sollargene inc." with the help of the Technion R&D business office".
Kashi, Y., King, D., Soller, M. Simple sequence repeats as a source of quantitative genetic variation. Trends in Genetics 13: 74-78, 1997.
Revealed by Inter-Simple Sequence Repeats (ISSR)
Y. Kashi
Inter-simple sequence repeats (ISSR) polymorphism was used to determine genetic relationships among 75 Sesamum indicum L. accessions of Korean and exotic sesame. Fourteen reliable ISSR primers were selected for the assessment of genetic diversity, yielding 79 amplification products. Of these PCR products, 33% revealed polymorphism among the 75 accessions. Genetic distances ranged from 0.000 to 0.255 with mean genetic distance of 0.0687. The 75 accessions were divided into 7 groups on the basis of UPGMA cluster analysis. The largest group consisted of 25 Korean cultivars, 8 Korean breeding lines and 17 world wide accessions. The other groups included 25 accessions several of which contained useful traits. The dendrogram did not indicate any clear division among sesame accessions based on geographical origin. However, all Korean sesame cultivars except Namsankkae, were clustered in the same group, indicating a narrow gene pool. Some of the Korean breeding lines were spread along the dendrogram showing enlargement of the genetic diversity. The genomic diversity data, uncovered in this study, can be used in future breeding programs
Kim, D. H., Zur, G., Danin- Poleg, Y., Lee,S.W., Shim, K.B., Kang, C.W. and Kashi, Y. Genetic Relationships of Seasame (Sesamum indicum) Germplasm Collection as Revealed by Inter-Simple Sequence Repeats (ISSR), Plant Breeding, 121: 1-4, 2002.
Y. Kashi
Most traits in biological populations appear to be under stabilizing selection, which acts to eliminate quantitative genetic variation. Yet, virtually all measured traits in biological populations continue to show significant quantitative genetic variation. The paradox can be resolved by postulating the existence of an abundant, though unspecified, source of mutations that has quantitative effects on phenotype, but does not reduce fitness. Does such a source actually exist? We suggest that it does, in the form of repeat-number variation in SSRs (simple sequence repeats, of which the triplet repeats of human neurodegenerative diseases are a special case). Viewing SSRs as a major source of quantitative mutation has broad implications for understanding molecular processes of evolutionary adaptation, including the evolutionary control of the mutation process itself.
Kashi, Y., King, D., Soller, M. Simple sequence repeats as a source of quantitative genetic variation. Trends in Genetics 13: 74-78, 1997.
King, D., Soller, M., Kashi, Y. Evolutionary tuning knobs. Endeavour, 21: 37-40, 1997.
Kashi, Y. & Soller, M. Function Roles of Microsatellites and Minisatellites. In Microsatellite evolution and application / David D. Goldstein & Christian Schlotterer. Oxford University Press. Chapter 2: 10-23, 1999.
Simple sequence repeats in E. coli: abundance, distribution, composition, and polymorphism
Y. Kashi
Computer-based genome-wide screening (http://www.technion.ac.il/~anne/ choice2.html) of the DNA sequence of E. coli strain K12 revealed tens of thousands of tandem simple sequence repeat (SSR) tracts, with motifs ranging from 1-6 nucleotides. SSRs were well distributed throughout the genome. Mononucleotide SSRs were over-represented in non-coding regions and under-represented in open reading frames (ORFs). Nucleotide composition of mono- and dinucleotide SSRs, both in ORFs and in non-coding regions, differed from that of the genomic region in which they occurred, with 93% of all mononucleotide SSRs proving to be of A or T. Computer-based analysis of the fine position of every SSR locus in the non-coding portion of the genome relative to downstream ORFs showed SSRs located in areas that could affect gene regulation. DNA sequences at 14 arbitrarily chosen SSR tracts were compared among E. coli strains. Polymorphisms of SSR copy number were observed at four of seven mononucleotide SSR tracts screened, with all polymorphisms occurring in non-coding regions. SSR polymorphism could prove important as a genome-wide source of variation, both for practical applications (including rapid detection, strain identification, and detection of loci affecting key phenotypes) and for evolutionary adaptation of microbes.
Gur-Arie, R. Cohen, J. C., Eitan, Y., Shelef, L., Hallerman, E.M., Kashi,Y. Simple Sequence Repeats in the E.coli: Abundant, Distribution, Composition, and Polymorphism Genome Research 10: 61-70, 2000.
Y. Kashi.
Analysis of the complete genomic DNA sequences of six diverse prokaryotes (mycobacterium tuberculosis, bacillus subtilis, haemophilius influenzae, archaeoglobus fulgidus, helicobacter pylori, and synechocystis sp. PCC6803) and portions of the genomes of three eukaryotes (saccharomyces cerevisiae [chromosome VII], arabidopsis thaliana [chromosome II], and homo sapiens [chromosome 22]) showed the existence of tens of thousands of well distributed simple sequence repeat (SSR) tracts. Genomic content of SSR tracts at least 6 bp in length varied from 2.2% in E. coli sp. to 4.7 % in helicobacter pylori. Although eukaryotes showed SSR content similar to prokaryotes, their SSR arrays exhibited higher repeat numbers than those of prokaryotes, where there is a 9 bp size limit for mononucleotide SSRs and a 12 bp size limit for SSRs with longer core sequences. Nucleotide compositions and fine distributions of SSRs were examined in detail in five microbial genomes. In mycobacterium tuberculosis and archaeoglobus fulgidus, SSRs were represented proportionately in open reading frames (ORFs) and non-coding regions, but in bacillus subtilis, haemophilius influenzae, and saccharomyces cerevisiae, SSRs were underrepresented in ORFs. Our findings show a tendency for high or low G+C content genome-wide to be even more extreme in SSRs, especially in non-coding regions. We found that many SSRs occurred in non-coding sequences, many proximal to gene regulatory elements, supporting hypotheses that SSR polymorphisms there could alter gene expression.
Cohen C.J., Hallerman E.M., and Kashi Y. Simple Sequence Repeats in Prokaryotic and Eukaryotic Genomes Submitted, 2001 (Genome Biology).