Mapping QTLs for blight resistance and morpho-phenological traits in inter-species hybrid families of chestnut (Castanea spp.).

Chestnut blight (caused by Cryphonectria parasitica), together with Phytophthora root rot (caused by Phytophthora cinnamomi), has nearly extirpated American chestnut (Castanea dentata) from its native range. In contrast to the susceptibility of American chestnut, many Chinese chestnut (C. mollissima) genotypes are resistant to blight. In this research, we performed a series of genome-wide association studies for blight resistance originating from three unrelated Chinese chestnut trees (Mahogany, Nanking and M16) and a Quantitative Trait Locus (QTL) study on a Mahogany-derived inter-species F2 family. We evaluated trees for resistance to blight after artificial inoculation with two fungal strains and scored nine morpho-phenological traits that are the hallmarks of species differentiation between American and Chinese chestnuts. Results support a moderately complex genetic architecture for blight resistance, as 31 QTLs were found on 12 chromosomes across all studies. Additionally, although most morpho-phenological trait QTLs overlap or are adjacent to blight resistance QTLs, they tend to aggregate in a few genomic regions. Finally, comparison between QTL intervals for blight resistance and those previously published for Phytophthora root rot resistance, revealed five common disease resistance regions on chromosomes 1, 5, and 11. Our results suggest that it will be difficult, but still possible to eliminate Chinese chestnut alleles for the morpho-phenological traits while achieving relatively high blight resistance in a backcross hybrid tree. We see potential for a breeding scheme that utilizes marker-assisted selection early for relatively large effect QTLs followed by genome selection in later generations for smaller effect genomic regions.

Frozen in time: Rangewide genomic diversity, structure, and demographic history of relict American chestnut populations.

American chestnut (Castanea dentata) was once the most economically and ecologically important hardwood species in the eastern United States. In the first half of the 20th century, an exotic fungal pathogen-Cryphonectria parasitica-decimated the species, killing billions of chestnut trees. Two approaches to developing blight-resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted restoration populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight-resistant varieties. Population structure analyses suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defence against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant genetic populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

Microsatellite analysis for determination of the mutagenicity of extremely low-frequency electromagnetic fields and ionising radiation in vitro.

Extremely low-frequency electromagnetic fields (ELF-EMF) have been reported to induce lesions in DNA and to enhance the mutagenicity of ionising radiation. However, the significance of these findings is uncertain because the determination of the carcinogenic potential of EMFs has largely been based on investigations of large chromosomal aberrations. Using a more sensitive method of detecting DNA damage involving microsatellite sequences, we observed that exposure of UVW human glioma cells to ELF-EMF alone at a field strength of 1 mT (50 Hz) for 12 h gave rise to 0.011 mutations/locus/cell. This was equivalent to a 3.75-fold increase in mutation induction compared with unexposed controls. Furthermore, ELF-EMF increased the mutagenic capacity of 0.3 and 3 Gy gamma-irradiation by factors of 2.6 and 2.75, respectively. These results suggest not only that ELF-EMF is mutagenic as a single agent but also that it can potentiate the mutagenicity of ionising radiation. Treatment with 0.3 Gy induced more than 10 times more mutations per unit dose than irradiation with 3 Gy, indicating hypermutability at low dose.

A mortality gene(s) for the human adenocarcinoma line HeLa maps to a 130-kb region of human chromosome 4q22-q23.

Human chromosome 4 was previously shown to elicit features of senescence when introduced into cell lines that map to complementation group B for senescence, including HeLa cells. Subsequently, a DNA segment encoding the pseudogene Mortality Factor 4 (MORF4) was shown to reproduce some of the effects of the intact chromosome 4 and was suggested to be a candidate mortality gene. We have identified multiple MORF4 alleles in several cell lines and tissues by sequencing and have failed to detect any cancer-specific mutations in three of the complementation group B lines (HeLa, T98G, and J82). Furthermore, MORF4 was heterozygous in these lines. These results question whether MORF4 is the chromosome 4 mortality gene. To map other candidate mortality gene(s) on this chromosome, we employed microcell-mediated monochromosome transfer to introduce either a complete copy, or defined fragments of the chromosome into HeLa cells. The introduced chromosome 4 fragments mapped the mortality gene to a region between the centromere and the marker D4S2975 (4q27), thus excluding MORF4, which maps to 4q33-q34.1. Analysis of microsatellite markers on the introduced chromosome in 59 immortal segregants identified a frequently deleted region, spanning the markers BIR0110 and D4S1557. This defines a new candidate interval of 130 kb at 4q22-q23.

Functional evidence for a squamous cell carcinoma mortality gene(s) on human chromosome 4.

Squamous cell carcinoma (SCC) immortality is associated with p53 and INK4A dysfunction, high levels of telomerase and loss of heterozygosity (LOH) of other chromosomes, including chromosome 4. To test for a functional cancer mortality gene on human chromosome 4 we introduced a complete or fragmented copy of the chromosome into SCC lines by microcell-mediated chromosome transfer (MMCT). Human chromosome 4 caused a delayed crisis, specifically in SCC lines with LOH on chromosome 4, but chromosomes 3, 6, 11 and 15 were without effect. The introduction of the telomerase reverse transcriptase into the target lines extended the average telomere terminal fragment length but did not affect the frequency of mortal hybrids following MMCT of chromosome 4. Furthermore, telomerase activity was still present in hybrids displaying the mortal phenotype. The MMCT of chromosomal fragments into BICR6 mapped the mortality gene to between the centromere and 4q23. Deletion analysis of the introduced chromosome in immortal segregants narrowed the candidate interval to 2.7 Mb spanning D4S423 and D4S1557. The results suggest the existence of a gene on human chromosome 4 whose dysfunction contributes to the continuous proliferation of SCC and that this gene operates independently from telomeres, p53 and INK4A.