Fig 1.
Establishment of genetically homogeneous strains from NRW-WE.
Mortality data of first instar larvae during the inbreeding procedure of NRW-WE are shown. Offspring of each generation F1-F5 were selected on artificial diet containing either CpGV-M (white bars) or CpGV-S (gray bars) at a concentration of 2 x 105 OB/ml. Surviving larvae from each generation were reared to adulthood, followed by subsequent mass cross and selection procedure, resulting in the F5 strains CpR5M and CpR5S. The Abbott-corrected mean mortality and standard errors (bars) were determined 16 days post infection [25]. Generation number (F1-F5), total number of tested individuals (n) and the percentage of surviving male moths (m [%]) are given below the chart, n.d., not determined.
Fig 2.
Resistance testing in CpS, CpR5M andCpR5S.
Mortality of neonate larvae of CpS, CpR5M or CpR5S tested for resistance on artificial diet containing either CpGV-M (continuous line) or CpGV-S (broken line) at a discriminating concentration of 5.8 x 104 OB/ml. Mean mortality and standard errors after seven (black bars) and 14 days (gray bars) post infection are shown. All mortality data were corrected for control mortality [25]. The total numbers of replicates (N) and tested individuals (n) of the CM strains are given below the chart.
Fig 3.
Scheme of hybrid crosses and backcrosses of resistant CpR5M and CpR5S with susceptible CpS.
The female crosses started with female moths (f) of CpR5M and CpR5S mated with CpS males (m); and vice-versa in the male crosses. Resulting neonate larvae of the first generation (F1) were divided into three cohorts. Two cohorts were tested for resistance with the discriminating concentration 5.8 x 104 OB/ml of CpGV-M and CpGV-S and mortality was observed after seven days. A third cohort of F1 offspring was reared as an untreated control on diet without virus until adulthood. For the backcrosses, resulting male moths of the F1 control group were mated with females of CpR5M or CpR5S and the backcross (BC) offspring was again tested for resistance using the discriminating concentration.
Table 1.
Crosses and backcrosses of CpR5M or CpR5S with CpS.
Mortality rates of CM larvae determined in resistance tests with neonate larvae seven days post infection (p.i.), exposed to CpGV-M or CpGV-S at a discriminating concentration of 5.8 X 104 OB/ml are shown. The mortality was determined for the offspring of the different strains, CpS, CpR5M, and CpR5S, of the hybrid crosses between CpR5M or CpR5S with CpS, and of the backcrosses. Progeny genotypes are shown for two hypotheses: a single Z-linked, dominant resistance gene (Z) or a single autosomal, dominant resistance gene (A). Abbreviations are BC (backcross), R (resistant allele), S (susceptible allele), F1 (offspring of the first generation), f (female), m (male), M (mean), SD (standard deviation), N (number of replicates), and n (total number of tested larvae). Given are the expected (Exp) mortality rates for dominant Z-linked (Z) or autosomal (A) inheritance hypotheses of the F1 generation and of the backcrosses. Differences in the expected mortality predicted by Z-linked vs. autosomal inheritance hypotheses are highlighted in gray.
Table 2.
Dominance values and direct test for monogenic inheritance of type II resistance in CpR5M and CpR5S.
Dominance values were calculated according to Bourguet [26] using the mortality rates given in Table 1. A direct test of monogenic inheritance for resistance to CpGV-M or CpGV-S [27] was conducted by comparing expected and observed mortality of the backcrosses subjected to CpGV-M and CpGV-S, respectively. Asterisks indicate significant differences from a monogenic model (χ2 test, df = 1; *P value < 0.05).
Fig 4.
Examples of BAC-FISH mapping of Z-linked genes on the Cydia pomonella neo-Z chromosome.
Cy3-dUtP-labeled (red hybridization signals) and Fluorescein-12-dUTP-labeled (green) BAC probes were used for BAC-FISH mapping in spread chromosome preparation from testes, counterstained with DAPI (blue). A: CpS-Krym mitotic spermatogonial metaphase showing 2n = 56 chromosomes; two neo-Z chromosomes marked with hybridization signals of BAC probes containing per (green) and nan (red) genes. B: Detailed view of pachytene ZZ bivalent of CpRR1 with hybridized ap (terminal red, arrow), Tpi (green) and Idh-2 (interstitial red) probes. C: CpR5M pachytene complement showing a long ZZ bivalent with hybridized ap (terminal red), ABCF2 (green) and Notch (interstitial red, arrow) probes.
Fig 5.
Gene-based schemes of the neo-Z chromosome of CpR5M, CpRR1 and CpS-Krym, integrating all BAC-FISH mapping results.
The relative positions of 13 marker gene loci were generated by measuring the physical distance between hybridization signals and the ap-labeled chromosome end of at least seven ZZ bivalents of each strain. The measured distances were normalized to the total length of the ZZ bivalent. Genes of CpR5M labeled with one asterisk showed a statistically significant difference in their mean position of the normalized gene loci to CpRR1; genes with two asterisks are significantly different to both CpRR1 and CpS-Krym (ANOVA, Scheffé test, P = 0.05).
Table 3.
Overview of the 13 Z-linked genes and summary of statistical analyses of their mapping on the neo-Z chromosome of different codling moth (CM) strains by BAC-FISH.
The relative position of hybridization signals of BAC probes to the total length of the neo-Z chromosome in at least seven ZZ bivalents was calculated in each CM strain. Different letters indicate statistical differences in the mean relative position of gene loci (ANOVA, Scheffé test, P = 0.05).