Johnson TE (1987) Worm Breeder's Gazette "recombinant inbred lines can be used to conveniently map cloned DNA fragments."

Johnson TE

[Worm Breeder's Gazette, 1987]

We generated recombinant inbred (RI) lines of the worm to answer questions regarding the genetic specification of life span in wild populations. RI lines in both Neurospora and mice are being used to map genes based solely on differences in RFLP's between the parental strains used to generate the RI lines. The lines we generated were derived from Bristol/Bergerac F1 self crosses and so are segregating the several hundred RFLP's that result from Tc1 insertions as well as many others. Any visible single-gene difference segregating in the RI lines can be scored to generate a strain distribution pattern (SDP) for that marker. If a second unmapped gene has a similar SDP, that gene must be near to the previously mapped gene. It has been obvious for some time that the generation of a linkage map based on the SDP's of many RFLP's would be possible, albeit a bit tedious to construct. Such a map might be useful to us in mapping genes specifying life history traits that are segregating in the Bristol/Bergerac background; but the RFIP map might also be useful as a general mapping strategy. Once the RFLP map was well developed one could reveal linkage to within 1 map unit, on the average, by simply probing the 43 RI strains with the clone of interest; these analyses could be carried out on the same set of blots time after time. RFLP's from different contigs could be used to map contigs onto the map relative to each other and relative to mapped RFLP's even if some regions of overlap remain unclonable using yeast cloning vectors. Hi-Recombination Inbred lines could be generated that would allow fine structure mapping to the finest resolution of available RFLP's without necessitating the scoring of more than 25 to 50 strains. The major problem with this idea is that it might be more worm than it's worth at the current time. Nevertheless, I'd appreciate help from the worm community in sending me any information which you may have collected on RFLP's, mapped or unmapped, or any other single-gene trait that is known to differ between Bristol and Bergerac and thus should be segregating in the RI strains.

Johnson TE (1986) Behav Genet "Molecular and genetic analyses of a multivariate system specifying behavior and life span."

Johnson TE

[Behav Genet, 1986]

Recombinant inbred (RI) lines have been constructed by crossing the Bristol and Bergerac BO strains of the nematode Caenorhabditis elegans. The F1 hermaphroditic progeny are allowed to self-fertilize for 20 generations, yielding the RI lines. Heritability estimates for several behavioral traits and for life span, as well as both phenotypic and genetic covariance estimates for these traits, have been obtained. Significant heritablility is detected only for forward movement. Large genetic covariances are detected between different behavioral components, corroborating earlier studies based on observations of sterotypical behaviors in nematodes. A strain distribution pattern (SDP) has been generated using genetic loci defined both by mutation and by restriction fragment length polymorphisms. Estimates of the number of loci affecting the life span range from three to six; these estimates were obtained both by classic quantitative techniques and by mapping, using the SDP for these RI lines. Thus, loci specifying life span are distributed throughout the genome rather than localized to any one region. EMS-induced, long-lived (Age) mutants were also examined. In contrast to an earlier report, these mutants ingest normal amounts of food. The age locus of one strain, MK546, segregates independently of a behavioral

Srinivas Ayyadevara et al. (2003) International Worm Meeting "Fine Mapping of Quantitative Trait Loci Conferring Resistance to Heat-Shock and Ultraviolet Radiation in C. elegans"

Srinivas Ayyadevara, Ping Kang, Robert Shmookler Reis

[International Worm Meeting, 2003]

We used a novel Tc1-based fine mapping procedure and recombinant inbred (RI) lines to map Quantitative Trait loci (QTLs) for resistance to heat shock and ultraviolet radiation in Caenorhabditis elegans. RI lines, generated from a Bristol-N2 Bergerac-BO cross, were exposed to UV light or heated to 35C, and subsequent survival measured. The RI lines differed markedly with respect to resistance to either stress. Linkage of stress-resistance QTLs to Tc1-based markers was determined by composite interval mapping (Zeng 1994). Quantitative trait loci significantly affecting resistance to UV were positioned on chromosomes I, II and III, with peak LOD scores of 3.3, 9.1 and 3.5, respectively. We found three significant QTLs for heat shock resistance on chromosome IV, and one such QTL on the X chromosome. Two of these loci, one in the middle of chromosome IV (near stP35) and one near the left end of X (between stP41 and stP40) were close or identical to QTLs affecting life span, as determined in an earlier mapping study of recombinant inbred populations (Ayyadevara et al. 2001). All significance thresholds shown (horizontal lines in Figure) were 0.05 genome-wide false positive levels determined empirically by 1000 permutations of genotype with respect to phenotype for each trait mapping (Churchill and Doerge, 1994).

Johnson TE (1982) Worm Breeder's Gazette "use of recombinant inbred strains in the genetic analysis of lifespan."

Johnson TE

[Worm Breeder's Gazette, 1982]

Recombinant inbred (RI) strains have been generated from crosses of Bristol (N2) and Bergerac (Lyon) parental strains. F1 hybrid progeny from this cross are isolated and allowed to self-fertilize. Resultant F2 progeny are then individually isolated and allowed to self- fertilize. After 3-5 days, individual F3 offspring are isolated to new plates. This procedure is repeated through 20 rounds of inbreeding, which leaves the genome homozygous to 1 part in 10+E6. These RI strains show remarkable variations in length of life. Mean lifespans vary from a low of near 11 days to a high of about 30 days. These differences are reproducible. The large variation in lifespan ( both parental strains have lifespans of about 18 days) is presumably due to the recombination and subsequent homozygosis of allelic differences at many genetic loci. There appear to be no major genes that affect lifespan segregating within this population. Since several other genetic characteristics of the Bristol and Bergerac strains (e.g., uncoordinated behavior of Bergerac, lower pharyngeal pump rate of Bergerac, slower rate of larval development of Bergerac, and the presence of a ts gene causing either a Zyg or a Gon phenotype; Wood et al., 1980) are also segregating within these strains, we can estimate the correlations between these factors and lifespan. For example, our studies, which are still in progress, show that about 10% of the variation in lifespan can be explained by the variation in pharyngeal pump rate. We are now using these RI strains for selective breeding experiments designed to obtain longer lived strains of worms.

Shook DR et al. (1996) West Coast Worm Meeting "QTLS FOR LIFE HISTORY TRAITS IN CAENORHABDITIS ELEGANS"

Hinerfeld DA, Shook DR, Johnson TE

[West Coast Worm Meeting, 1996]

The evolution of genes affecting senescence has been hypothesized to depend on the other life history traits (traits affecting survival or fertility). Hence we have used an approach which allows us not only to identify individual loci specifying various life history traits, but also allows us to detect interactions among life history traits at individual loci and interactions between loci for individual traits. 81 recombinant inbred (RI) strains generated from two wild type parental strains, Bergerac (BO) by Bristol (N2), were used. Each RI strain was genotyped for 40 markers, which were based on Tc1 insertions present in one parental genome (generally BO) but not the other. Each RI strain was also assayed for several different life history traits. This data set was used to generate QTL maps for each trait, which allowed us to compare the effects of each locus on several traits. In addition, we tested for epistasis among loci for each trait. We have identified several putative quantitative trait loci (QTLs) for life history traits in self fertilizing hermaphrodites. We have found QTLs for mean and maximum life span, early, mean and maximum fertility, "bagging" (internal hatching of progeny), temperature sensitive fertility, growth rate, and age of onset of reproduction. One locus shows pleiotropic effects on the survival characters mean life span and bagging, while at least two loci show pleiotropic effects among some of the fertility characters. No loci showed pleiotropy, negative or otherwise, for both survival and fertility characters. Several pairs of loci show epistatic interactions for various life span and fertility traits.

Ebert RHA et al. (1995) International C. elegans Meeting "ANTIOXIDANT ENZYME ACTIVITIES AND MAPPING OF H2O2 RESISTANCE IN THREE STRAINS OF C. ELEGANS"

Ebert RHA, Shammas MA, Sohal BH, Sohal RS, Shmookler Reis RJ

[International C. elegans Meeting, 1995]

One factor determining the rate of senescence of an organism may be the level of oxidative stress it experiences throughout its lifespan. Recent studies of the long-lived mutant age-1 (Larsen, PNAS 90, 8905, 1993; Vanfleteren, Biochem J 292, 605, 1993) reveal that activity levels of both catalase and Cu-Zn SOD are elevated in old worms compared to controls. Our work with N2 BO (Ebert et al., Genetics 135, 1003, 1993) and N2 DH424 (submitted) recombinant-inbred (RI) populations indicates that there are genes with allelic differences between the parental strains, which have differential effects on longevity. Here we have tested whether SOD and catalase activities vary in an age- or strain- dependent manner. Middle-aged worms (~70-85% of the cohort still alive) were compared with old worms (~4-6% alive). SOD activity levels decreased with age for cohorts of strains DH424 and RW7000 (BO) by ~50%, but remained at the same level for an N2 population. Catalase activity levels dropped with increasing age for populations of all three strains tested and the decrease was largest for N2 (to 38% of the earlier value). Currently, we are determining the sensitivity to acute hydrogen peroxide (H2O2) exposure for these strains and for a cohort of N2 BO RI progeny. We are also analyzing RI survivors of acute H2O2 exposure by single-worm PCR (Williams et al., Genetics 131, 609, 1992) to map any polymorphic genes responsible for their resistance to this pro-oxidant.

Johnson TE et al. (1986) Worm Breeder's Gazette "developmental effects of loci lengthening life-span."

Bryant M, Johnson TE, Conley WL

[Worm Breeder's Gazette, 1986]

Both recombinant inbred (RI) lines and single gene mutants (see other abstracts in this issue) have been produced which have dramatic changes in life-span. Stocks average a 60% increase in mean and maximal life-span. We have been interested in knowing whether these changes also result in a change in developmental events (length of the embryonic period, timing of cell divisions, rate of larval growth, timing of larval molts) or in the length of the reproductive period. We have assayed the length of the embryonic period by isolating individual eggs at the 2-cell stage and transferring these eggs to incubation chambers at 16 C (chosen for convenience of monitoring). About 20 hours later the eggs hatch. We find no change in the length of this period in either the RI lines or in age-1(hx542) or age-1( hx546). Surprisingly, N2 seems to take about 1 hour longer to complete this phase than any other stock analyzed, including Bergerac BO. We see variation among the RI lines in the rate of larval growth, in the timing of larval molts, and in the time from hatch to 50% adult fecundity. These variations do not seem to correlate with the adult life-span but limited numbers of stocks have been assayed as of yet and we remain unclear about our level of statistical significance. It is clear that both(?) age-1 alleles are without effect on any aspect of larval development rate. There is marginal variation in the length of the reproductive period and in the total fecundity, among the RI lines. The quantitative variations in total fecundity showed statistically significant correlations with the length of life in one repeat which has not yet been confirmed. There is no correlation between the length of the fecund period and life-extension. For the age-1 locus, there is a significant reduction in total brood size but no significant change in the temporal distribution of progeny production, after normalizing to total brood size. As always, we welcome helpful and provocative suggestions of additional questions to ask and directions to proceed.

Iwato, A. et al. (2019) International Worm Meeting "Identification of genes required for natural variation affecting temperature acclimation."

Ohta, A., Okahata, M., Mitani, S., Iwato, A., Kuhara, A., Koyama, H., Yoshina, S., Minakuchi, Y., Toyoda, A.

[International Worm Meeting, 2019]

Temperature is one of the critical environmental factors for creatures. We are studying natural variation in temperature acclimation of C. elegans. 15 degree-cultivated N2 can survive at 2 degree, meanwhile 25 degree cultivated N2 can not survive at 2 degree. Only 3 hours after cultivation temperature-shift from 25 to 15 degree, cold acclimation was newly established(Ohta et al., Nature common, 2014; Okahata et al., Science Advances, 2019). AB1 from Australia can rapidly acclimate to new temperature. On the other hand, CB4856 from Hawaii can acclimate slowly. To identify the gene involved in the natural variation of temperature acclimation, we decoded whole genome DNA sequences of three natural variants including AB1 by using next generation sequencer (Okahata et al., JCPB, 2016). We made recombinants inbred(RI) lines by crossing AB1 with CB4856, and isolated 71 RI lines. The gene responsible for rapid temperature acclimation in AB1 was mapped on specific region of chromosome I, by performing SNP analysis (Okahata et al., JCPB, 2016). To narrow the mapping region, we obtained more RI lines and compared their cold acclimation phenotype and genotype with next generation sequencer. The candidate genes responsible for rapidtemperature acclimation in AB1were narrowed down to 15 genes. We then measured cold acclimation of knockout mutants of them, so far, VH15N14R.1 mutant lacking a novel gene showed the abnormal cold acclimation. VH15N14R.1 protein in CB4856 strain has the substitutions of two amino acid residues and the deletion of an arginine, comparing to AB1 strain. VH15N14R.1 gene was expressed in head neurons including small pairs of sensory neurons. The expression levels of VH15N14R.1 gene was increased when animals were cultivated at 25 degree compared with animals cultivated at 15 degree. We are going to identify the cell where VH15N14R.1 regulate cold acclimation, and determine a molecular function of VH15N14R.1 protein.

Vaupel JW et al. (1994) Science "Rates of mortality in populations of Caenorhabditis elegans."

Johnson TE, Fukui HH, Curtsinger JW, Capra WB, Vaupel JW, Xiu L, Lithgow GJ, Khazaeli A, Pletcher S, Wang JL, Carey JR, Muller HG

[Science, 1994]

A. Brooks et al. report that mortality increased exponentially for a cohort of 180,000 nematodes of the species Caenorhabditis elegans of the single genotype TJ1060 [spe-9(hc88) fer-15(b26)]. A closer look at the data reveals that from days 5 through 8 mortality increased at a rate of 0.58 +/- 0.0004, which is more than twice the rate thereafter (0.21 +/- 0.02). Such a "biphasic pattern," with death rates increasing rapidly at younger ages and more slowly at older ages, was also found in a genetically heterogeneous population of 79 recombinant-inbred (RI) strains.

Barnes TM et al. (1996) Worm Breeder's Gazette "Daring rescue! Corruption! in.... CLONEWATCH"

Pak J, Hekimi S, Vouloumanos A, Barnes TM

[Worm Breeder's Gazette, 1996]

In the course of narrowing the unc-14 rescuing region to a 5.2 kb Hind III - Sal I fragment that is mostly occupied by part of the YK363 gene (see also Ogura and Ohshima WBG 14(1) p51), we came across two unusual clones. We had established a restriction map across a 60 kb region, with particular attention to Eco RI sites, and had obtained 8 clones from this region from the LMB, which we mapped and tested for rescue. The first unusual clone, P1a74 (which uses a P1 phage-derived vector), was a clone which should have rescued. This clone has a wild-type restriction map, and the 11 kb Eco RI fragment which contains unc-14(+) is in the middle of this clone. However, 3/3 lines (and 18 F1 Rol) carrying this clone were not rescued, not even partially (since Unc-14 adults are strongly paralyzed this is a sensitive test). This suggests that the clone is not faithful to the genomic sequence and contains a mutation which completely inactivates unc-14(+). Of course this could just be a sporadic case, but we wonder if anyone else has noticed any odd behaviour of P1 clones?