A Genomic Resource for the Development,Improvement, and Exploitation of Sorghum for Bioenergy

With high productivity and stress tolerance, numerous grass genera of the Andropogoneae have emerged as candidates for bioenergy production. To optimize these candidates, research examining the genetic architecture of yield, carbon partitioning, and composition is required to advance breeding objectives. Significant progress has been made developing genetic and genomic resources for Andropogoneae, and advances in comparative and computational genomics have enabled research examining the genetic basis of photosynthesis, carbon partitioning, composition, and sink strength. To provide a pivotal resource aimed at developing a comparative understanding of key bioenergy traits in the Andropogoneae, we have established and characterized an association panel of 390 racially, geographically, and phenotypically diverse Sorghum bicolor accessions with 232,303 genetic markers. Sorghum bicolor was selected because of its genomic simplicity, phenotypic diversity, significant genomic tools, and its agricultural productivity and resilience. We have demonstrated the value of sorghum as a functional model for candidate gene discovery for bioenergy Andropogoneae by performing genome-wide association analysis for two contrasting phenotypes representing key components of structural and non-structural carbohydrates. We identified potential genes, including a cellulase enzyme and a vacuolar transporter, associated with increased non-structural carbohydrates that could lead to bioenergy sorghum improvement. Although our analysis identified genes with potentially clear functions, other candidates did not have assigned functions, suggesting novel molecular mechanisms for carbon partitioning traits. These results, combined with our characterization of phenotypic and genetic diversity and the public accessibility of each accession and genomic data, demonstrate the value of this resource and provide a foundation for future improvement of sorghum and related grasses for bioenergy production.     

Retrospective genomic analysis of sorghum adaptation to temperate-zone grain production

Background

Sorghum is a tropical C₄ cereal that recently adapted to temperate latitudes and mechanized grain harvest through selection for dwarfism and photoperiod-insensitivity. Quantitative trait loci for these traits have been introgressed from a dwarf temperate donor into hundreds of diverse sorghum landraces to yield the Sorghum Conversion lines. Here, we report the first comprehensive genomic analysis of the molecular changes underlying this adaptation.

Results

We apply genotyping-by-sequencing to 1,160 Sorghum Conversion lines and their exotic progenitors, and map donor introgressions in each Sorghum Conversion line. Many Sorghum Conversion lines carry unexpected haplotypes not found in either presumed parent. Genome-wide mapping of introgression frequencies reveals three genomic regions necessary for temperate adaptation across all Sorghum Conversion lines, containing the Dw1, Dw2, and Dw3 loci on chromosomes 9, 6, and 7 respectively. Association mapping of plant height and flowering time in Sorghum Conversion lines detects significant associations in the Dw1 but not the Dw2 or Dw3 regions. Subpopulation-specific introgression mapping suggests that chromosome 6 contains at least four loci required for temperate adaptation in different sorghum genetic backgrounds. The Dw1 region fractionates into separate quantitative trait loci for plant height and flowering time.

Conclusions

Generating Sorghum Conversion lines has been accompanied by substantial unintended gene flow. Sorghum adaptation to temperate-zone grain production involves a small number of genomic regions, each containing multiple linked loci for plant height and flowering time. Further characterization of these loci will accelerate the adaptation of sorghum and related grasses to new production systems for food and fuel.     

Spatio-temporal dynamics of genetic diversity in Sorghum bicolor in Niger

The dynamics of crop genetic diversity need to be assessed to draw up monitoring and conservation priorities. However, few surveys have been conducted in centres of diversity. Sub-Saharan Africa is the centre of origin of sorghum. Most Sahel countries have been faced with major human, environmental and social changes in recent decades, which are suspected to cause genetic erosion. Sorghum is the second staple cereal in Niger, a centre of diversity for this crop. Niger was submitted to recurrent drought period and to major social changes during these last decades. We report here on a spatio-temporal analysis of sorghum genetic diversity, conducted in 71 villages covering the rainfall gradient and range of agro-ecological conditions in Niger’s agricultural areas. We used 28 microsatellite markers and applied spatial and genetic clustering methods to investigate change in genetic diversity over a 26-year period (1976–2003). Global genetic differentiation between the two collections was very low (F _st = 0.0025). Most of the spatial clusters presented no major differentiation, as measured by F _st, and showed stability or an increase in allelic richness, except for two of them located in eastern Niger. The genetic clusters identified by Bayesian analysis did not show a major change between the two collections in the distribution of accessions between them or in their spatial location. These results suggest that farmers’ management has globally preserved sorghum genetic diversity in Niger.     

Identification of bioconversion quantitative trait loci in the interspecific cross Sorghum bicolor × Sorghum propinquum

For lignocellulosic bioenergy to be economically viable, genetic improvements must be made in feedstock quality including both biomass total yield and conversion efficiency. Toward this goal, multiple studies have considered candidate genes and discovered quantitative trait loci (QTL) associated with total biomass accumulation and/or grain production in bioenergy grass species including maize and sorghum. However, very little research has been focused on genes associated with increased biomass conversion efficiency. In this study, Trichoderma viride fungal cellulase hydrolysis activity was measured for lignocellulosic biomass (leaf and stem tissue) obtained from individuals in a F₅ recombinant inbred Sorghum bicolor × Sorghum propinquum mapping population. A total of 49 QTLs (20 leaf, 29 stem) were associated with enzymatic conversion efficiency. Interestingly, six high-density QTL regions were identified in which four or more QTLs overlapped. In addition to enzymatic conversion efficiency QTLs, two QTLs were identified for biomass crystallinity index, a trait which has been shown to be inversely correlated with conversion efficiency in bioenergy grasses. The identification of these QTLs provides an important step toward identifying specific genes relevant to increasing conversion efficiency of bioenergy feedstocks. DNA markers linked to these QTLs could be useful in marker-assisted breeding programs aimed at increasing overall bioenergy yields concomitant with selection of high total biomass genotypes.     

A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor (L.) Moench] Identifies SbCAD2 as the Brown midrib6 Gene

The content and composition of the plant cell wall polymer lignin affect plant fitness, carbon sequestration potential, and agro-industrial processing. These characteristics, are heavily influenced by the supply of hydroxycinnamyl alcohol precursors synthesized by the enzyme cinnamyl alcohol dehydrogenase (CAD). In angiosperms, CAD is encoded by a multigene family consisting of members thought to have distinct roles in different stages of plant development. Due to the high sequence similarity among CAD genes, it has been challenging to identify and study the role of the individual genes without a genome sequence. Analysis of the recently released sorghum genome revealed the existence of 14 CAD-like genes at seven genomic locations. Comparisons with maize and rice revealed subtle differences in gene number, arrangement, and expression patterns. Sorghum CAD2 is the predominant CAD involved in lignification based on the phylogenetic relationship with CADs from other species and genetic evidence showing that a set of three allelic brown midrib (bmr) lignin mutants contained mutations in this gene. The impact of the mutations on the structure of the protein was assessed using molecular modeling based on X-ray crystallography data of the closely related Arabidopsis CAD5. The modeling revealed unique changes in structure consistent with the observed phenotypes of the mutants.     

Agricultural Biodiversity in Northwest Somalia – an Assessment Among Selected Somali Sorghum(<Emphasis Type="Italic">Sorghum bicolor</Emphasis> (L.) Moench) Germplasm

The seed sector situation in Northwest Somalia is critical. The availability of food has decreased and many people are at risk of hunger. Food security can be restored by enhancing the local genetic resources and creating an efficient seed sector. Sorghum is important as a food and fodder crop in this region. It is close to Ethiopia, which is considered as the probable origin and domestication of Sorghum. Twelve morphological and productive characteristics were chosen to assess the phenotypic variability of 16 accessions of sorghum from Northwest Somalia. Univariate (analysis of variance and G test) and multivariate (discriminant and cluster analysis) methods were used to assess the morphological variation within the accession and to group the 16 accessions into clusters based upon quantitative and qualitative characters. Elmi Jama Cas, Masego Cas, Masego Cad and Carabi clearly represent distinct landraces with specific features suitable for different purpose, such as grain and/or forage production. Each landrace tested is able to grow under harsh environmental conditions, thus ensuring a low, but stable production for small poor resources farmers. Knowledge and conservation of local landraces will provide a broad base of genetic variability from which improved sorghum varieties can be developed, thus aiding in the stabilisation of a secure and sustainable food supply for farmers of Northwest Somalia.     

A phylogenetic network of wild Ussurian pears (Pyrus ussuriensis Maxim.) in China revealed by hypervariable regions of chloroplast DNA

In order to understand the genetic diversity of wild Ussurian pears in China, chloroplast DNA (cpDNA) of 186 wild accessions from 12 populations in Inner Mongolia, Heilongjiang and Jilin Provinces and 51 Chinese and European pear cultivars including Pyrus ussuriensis, Pyrus pyrifolia, Pyrus bretschneideri, Pyrus sinkiangensis and Pyrus communis were investigated. Each accession was classified into one of three types (types A, B and C) based on two large deletions in the hypervariable regions between the accD–psaI and rps16–trnQ genes. Thirty haplotypes were identified by 32 mutations including 17 gaps (in/dels) and 15 base changes. Haplotype network analysis revealed that wild Chinese Ussurian pears could be grouped into subgroup I of type A. A haplotype, Hcp3, in subgroup I detected in Heilongjiang and Jilin Provinces was considered to be a divergent centre in Chinese Ussurian pears. However, the genetic diversity of wild accessions revealed by the two hypervariable regions was quite low. In particular, 98 % of wild Ussurian accessions in Inner Mongolia shared an identical haplotype Hcp1 and are, therefore, monomorphic. In comparison, Chinese pear cultivars were more divergent. These results suggest that the cpDNAs from wild Ussurian pears in Inner Mongolia have specifically differentiated compared to those from pears of other areas. The number of wild Ussurian pears has been decreasing because of desertification and land development, therefore conservation is needed.     

Identification of SSR markers associated with height using pool-based genome-wide association mapping in sorghum

Sorghum has been proposed as a potential energy crop. However, it has been traditionally bred for grain yield and forage quality, not traits related to bioenergy production. To develop tools for genetic improvement of bioenergy-related traits such as height, genetic markers associated with these traits have first to be identified. Association mapping has been extensively used in humans and in some crop plants for this purpose. However, genome-wide association mapping using the whole association panel is costly and time-consuming. A variation of this method called pool-based genome-wide association mapping has been extensively used in humans. In this variation, pools of individuals with contrasting phenotypes, instead of the whole panel, are screened with genetic markers and polymorphic markers are confirmed by screening the individuals in the pools. Here, we identified several new simple sequence repeats (SSR) markers associated with height using this pool-based genome-wide association mapping in sorghum. After screening the tall and short pools of sorghum accessions from the sorghum Mini Core collection developed at the International Crops Research Institute for the Semi-Arid Tropics with 703 SSR markers, we have identified four markers that are closely associated with sorghum height on chromosomes 2, 6, and 9. Comparison with published maps indicates that all four markers are clustered with markers previously mapped to height or height-related traits and with candidate genes involved in regulating plant height such as FtsZ, Ugt, and GA 2-oxidase. The mapping method can be applied to other crop plants for which a high-throughput genome-wide association mapping platform is not yet available.     

Conservation of genetic resources for five traditional crops from Jazan,SW Saudi Arabia,at the KACST Gene-Bank

The conservation of five traditional crops is an important aspect of achieving national food security. In the present study, we aimed to collect and conserve germplasm of five local crops from the Jazan region of southwestern Saudi Arabia: Sorghum: Sorghum bicolor (L.) Moench); Barley (Hordeum vulgare L.) Millet (Pennisetum glaucum (L.) R. Br.); Sesame (Sesamum indicum L.) and Guar (Cyamopsis tetragonoloba (L.) Taub). Forty-one seed accessions of these five crops were collected and tested to determine seed moisture content (MC%) and quality as indicators of their potential to survive during long-term dry storage at ?18 °C (i.e. ex-situ conservation of genetic resources). Seed viability was assessed using germination tests, the tetrazolium chloride (TZ) test and X-ray imaging. Seeds of the five crops had very low MC% and high viability (fully developed embryos and germination >91%), indicating that they were of good quality and had high potential for long-term survival in gene banks. The genetic resources of these crops (seeds) have now been preserved at the gene-bank of King Abdulaziz City for Science and Technology (KACST-BGB), Riyadh, Saudi Arabia.     

Genotype Dependent Somatic Embryogenesis and Regeneration from Leaf Base Cultures of Sorghum bicolor

A simple and reproducible protocol for callus induction and regeneration of plantlets from leaf base cultures of agronomically important Indian cultivars of Sorghum bicolor(L.) Moench (296 Band RS 585) has been developed. A strong genotype dependent response was observed and the genotype 296 B was found to be the most responsive as compared to the other genotypes tested. Cultures were raised from the III, IV and V leaf bases, excised from 12-day-old in vitro raised plantlets and cultured on Callus Induction Medium (CM). Callus initiation took place after 10-14 days of culture. The explants were maintained on this medium for 30- 35 days, after which they were transferred to Regeneration Medium (RM). Histological examination indicated that somatic embryogenesis was prevalent in the leaf base cultures and the embryos started to germinate after 15-20 days of transfer to RM. Plantlets with complete shoot and root system have been raised with as many as 30 plantlets regenerating from a single explant. These plantlets could be easily separated from one another and transferred to culture tubes for faster growth and development. Later, individual plants numbering more than 50 were transferred to pots containing soil: soilrite (1:1) for hardening. A high regeneration frequency of up to 40 % could be obtained in the genotype 296 B followed by 10.8 % in the genotype RS 585 and 7.8 % in C 43.     

Molecular genetic analysis of the soriz genome (<Emphasis Type="Italic">Sorghum oryzoidum</Emphasis>)

A molecular genetic analysis of soriz genotypes (Sorghum oryzoidum), its parental form Sorghum bicolor (L.) Moench (grain sorghum), possible parents (Sorghum sudanense (Piper.) Stapf. (Sudan grass) and Oryza sativa L. (Rice planting), as well as its closest relatives, has been carried out with the use of microsatellite loci of sorghum and rice. Based on the obtained data, the genetic distances were calculated and the examined species were clustered. It was shown that soriz did not carry rice DNA fragments, but its genome contained DNA fragments, which belonged to Sudan grass. This confirms that the origin of soriz is associated with representatives of Sorghum sudanense.     

Genetic studies of the lac repressor. III. Additional correlation of mutational sites with specific amino acid residues

The complete results of the analysis of over 5300 independently derived nonsense mutations in the lacI gene are presented. These have been mapped and divided into specific sites. A total of 105 nonsense mutations derived from 90 different codons can be distinguished, of which several are the result of tandem double base changes induced by ultraviolet light. With the aid of results determined in a preceding paper (Miller et al., 1977), the majority of these mutations have been assigned to points in the gene coding for specific residues in the lac repressor. This allows a detailed correlation of the physical and genetic map.Recombination studies have been carried out using mutations at known sites. For crosses involving mutations separated by less than 30 nucleotides (the main object of this study), a significant lack of agreement between distance and recombination frequency has been found.     

Systematic exploration of a class of hydrophobic unnatural base pairs yields multiple new candidates for the expansion of the genetic alphabet

We have developed a family of unnatural base pairs (UBPs), which rely on hydrophobic and packing interactions for pairing and which are well replicated and transcribed. While the pair formed between d5SICS and dNaM (d5SICS-dNaM) has received the most attention, and has been used to expand the genetic alphabet of a living organism, recent efforts have identified dTPT3-dNaM, which is replicated with even higher fidelity. These efforts also resulted in more UBPs than could be independently analyzed, and thus we now report a PCR-based screen to identify the most promising. While we found that dTPT3-dNaM is generally the most promising UBP, we identified several others that are replicated nearly as well and significantly better than d5SICS-dNaM, and are thus viable candidates for the expansion of the genetic alphabet of a living organism. Moreover, the results suggest that continued optimization should be possible, and that the putatively essential hydrogen-bond acceptor at the position ortho to the glycosidic linkage may not be required. These results clearly demonstrate the generality of hydrophobic forces for the control of base pairing within DNA, provide a wealth of new structure–activity relationship data and importantly identify multiple new candidates for in vivo evaluation and further optimization.     

Genomic analyses reveal distinct genetic architectures and selective pressures in Chinese donkeys

Donkey (Equus asinus) is an important livestock animal in China because of its draft and medicinal value. After a long period of natural and artificial selection, the variety and phenotype of donkeys have become abundant. We clarified the genetic and demographic characteristics of Chinese domestic donkeys and the selection pressures by analyzing 78 whole genomes from 12 breeds. According to population structure, most Chinese domestic donkeys showed a dominant ancestral type. However, the Chinese donkeys still represented a significant geographical distribution trend. In the selective sweep, gene annotation, functional enrichment, and differential expression analyses between large and small donkey groups, we identified selective signals, including NCAPG and LCORL, which are related to rapid growth and large body size. Our findings elucidate the evolutionary history and formation of different donkey breeds and provide theoretical insights into the genetic mechanism underlying breed characteristics and molecular breeding programs of donkey clades.     

Correlation analyses between molecular perspective and phytochemical variations in Coptis chinensis Franch

Coptis chinensis Franch. (Weilian in Chinese) is an important medicinal plant used in traditional Chinese medicines. The identification of habitats associated with good quality plant material is a challenge. In this study, we determined 59 samples from 12 different habits. Other than the samples from Zhenping, the content of six selected alkaloids in C. chinensis did not differ significantly among the habits. Furthermore, the results of the genetic analysis showed that the genetic diversity and the genetic distance among the samples were low, suggesting that the C. chinensis samples from different habits had the same genetic characteristics. These results would suggest that the quality of the drugs are not influenced by the habitats the plant is growing in.     

Phylogenetic Analysis of Different Ploidy Saccharum spontaneum Based on rDNA-ITS Sequences

Saccharum spontaneum L. is a crucial wild parent of modern sugarcane cultivars whose ploidy clones have been utilized successfully in improving the stress resistance and yield related traits of sugarcane cultivars. To establish knowledge regarding the genetic variances and evolutional relationships of ploidy clones of Saccharum spontaneum collected in China, the rDNA-ITS sequences of 62 ploidy clones including octaploid clones (2n = 64), nonaploid clones (2n = 72), decaploid clones (2n = 80), and dodecaploid clones (2n = 96), were obtained and analyzed. The rDNA-ITS sequences of four species from Saccharum and Sorghum bicolor selected as controls. The results showed that decaploid clones (2n = 80) possess the most abundant variances with 58 variable sites and 20 parsim-informative sites in ITS sequences, which were then followed by octaploid clones with 43 variable sites and 17 parsim-informative sites. In haplotype diversity, all four population exhibited high diversity, especially nonaploid and decaploid populations. By comparing the genetic distances among four ploidy populations, the dodecaploid population exhibited the closest relationship with the nonaploid population, and then the relationship strength decreased successively for the decaploid population and then for the octaploid population. Population differentiation analysis showed that the phenomena of population differentiation were not found among different ploidy populations, and low coefficient of gene differentiation(Gst) and high gene flow(Nm) occur among these populations possessing close genetic relationship. These results mentioned above will contribute to the understanding of the evolution of different ploidy populations of Saccharum spontaneum and provide vital knowledge for their utilization in sugarcane breeding and innovation.     

Genetic structure of East Asian cultivated pears (Pyrus spp.) and their reclassification in accordance with the nomenclature of cultivated plants

By use of Bayesian statistical inference and allelic data for 18 microsatellite loci, we analyzed the genetic structure of Chinese, Korean, and Japanese pear cultivars and of native populations of Pyrus ussuriensis. Although Japanese pear cultivars had a simple genetic structure, Chinese and Korean pear cultivars were admixures of Japanese pear and native P. ussuriensis from the Asian continent. Genetic differentiation between groups of native populations and those of cultivars was high, but cultivars were not well differentiated from each other. Chinese and Korean cultivars, which have traditionally been classified as either P. ussuriensis, P. bretschneideri, or P. pyrifolia, were much closer to Japanese cultivars, which have traditionally been classified as P. pyrifolia, than to native P. ussuriensis. We propose a new classification of cultivars by using the Group concept in accordance with the International Nomenclature for Cultivated Plants, namely, the Pyrus Ussurian pear group, the Pyrus Chinese white pear group, the Pyrus Chinese sand pear group, and the Pyrus Japanese pear group.     

Specific biological features and genetic variation of Chinese sleeper <Emphasis Type="Italic">Perccottus glenii</Emphasis> (Odontobutidae) in aquatic bodies of Tyumen oblast

Specific biological features, morphology, and genetic variation of an invasive species, the Chinese sleeper Perccottus glenii, in several lake and river ecosystems of the Tyumen oblast have been studied. This species displays relatively high rate of linear and weight growths. The main feed of the Chinese sleeper in both the river and lake ecosystems is the chironomid larvae. The diet is richer in the river ecosystem; predation plays an important role; and cannibalism is observed. As has been shown, the qualitative and quantitative characteristics of the Chinese sleeper infestations with local helminth species are by one order of magnitude lower as compared with the native fish species. A significant infestation rate is observed only for Nippotaenia mogurndae, a parasite specific for the Chinese sleeper. The population of this introduced species displays a high genetic variation for DNA markers. The rate of polymorphic ISSR bands is 73% and Nei’s genetic diversity is 0.26. The Chinese sleeper populations in the not directly connected river and lake (distance, 90 km) display a genetic identity of 99.5%, suggesting the absence of genetic differentiation of local populations, explainable by their recent origin from a single source of invasion. Morphological differences of the Chinese sleeper in different ecosystems appear on the same genetic basis and suggest a wide reaction norm characteristic of this species, underlying its ecological plasticity.     

An Induced Sorghum Mutant Population Suitable for Bioenergy Research

The sorghum [Sorghum bicolor (L.) Moench] inbred line BTx623 has served as a parent for development of several mapping populations, also providing a source for the generation of DNA libraries for physical mapping, and as the inbred line selected for sorghum genome sequencing. Since genetic mapping, physical mapping and genome sequencing are all based on the same inbred line, these genetic resources have made the genome study of sorghum very efficient. However, in comparison with other model species, there is one important genetic resource still missing in the sorghum research community, a mutant population. A systematically annotated mutant population will facilitate many avenues of research, especially those focusing on functional genomics and bioenergy research. Here we report the generation of a sorghum mutant population derived from the inbred line BTx623 by treatment with the chemical agent ethyl methanesulfonate (EMS). The mutant population consists of 1,600 pedigreed M₃ families; each of them was derived from an independent M₁ seed. Many lines displayed traits such as brown midrib (bmr), erect leaves (erl), multiple tillers (mtl), and late flowering (lfl), characteristics useful for bioenergy research. Results from our phenotyping and genotyping studies indicate that this mutant population will be a valuable and useful genetic resource for both sorghum functional genomics and bioenergy research.     

Needle-in-a-haystack detection and identification of base substitution mutations in human tissues

Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 10⁷ wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 10⁶ wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10⁻⁵ and 10⁻⁶. These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10⁻² to 10⁻⁷) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.