Determining the genetic basis of reproductive isolation is a fundamental goal in evolutionary biology. Intrinsic reproductive isolation often arises due to epistasis between divergent interacting genes. The rapid evolution of hybrid male sterility is known to have several causes, including the exposure of recessive X-linked incompatibilities in males and the rapid evolution of male reproductive traits. Despite these insights, little is known about the genetics of reproductive isolation during the early stages of speciation. This deficiency inspired parallel studies on the molecular evolution of male reproduction in house mice and the genetic basis of hybrid male sterility between two mouse species, Mus domesticus and M. musculus. Evolutionary analysis of 946 genes showed that the intensity of positive selection varies across sperm development and acts primarily on phenotypes that develop late in spermatogenesis (Appendix A). Several reciprocal crosses between wild-derived strains of M. musculus and M. domesticus were used to examine F1 hybrid male sterility (Appendix B). These crosses revealed hybrid male sterility linked to the M. musculus X chromosome and a novel sterility polymorphism within M. musculus. A large introgression experiment was used to further dissect the genetic basis of X-linked incompatibilities between M. musculus and M. domesticus (Appendix C). Introgression of the M. musculus X chromosome into a M. domesticus genetic background produced male sterility and involved a minimum of four factors. No sterility factors were uncovered on the M. domesticus X chromosome. These data demonstrate the complex genetic basis of hybrid sterility in mice and provide numerous X-linked candidate sterility genes. The molecular evolution of five rapidly evolving candidate genes was examined using population and phylogenetic sampling in Mus (Appendix D). Four of these loci showed evidence of positive natural selection. One locus, 4933436I01Rik, showed divergent protein evolution between M. domesticus and M. musculus and was one of a handful of testis-expressed genes within a narrow interval involved in hybrid male sterility. In summary, these data demonstrate that hybrid male sterility has a complex genetic basis between two closely related species of house mice and provide a foundation for the identification of specific mutations that isolate these species.

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Thesis (M.S.)--University of Missouri-Columbia, 2003. / Typescript. Includes bibliographical references (leaves 101-107). Also available on the Internet.

Thesis (M.S.)--University of Missouri-Columbia, 2003. / Typescript. Includes bibliographical references (leaves 101-107). Also available on the Internet.

The cactophilic Drosophila mojavensis species group living in the deserts and dry tropical forests of the Southwestern United States and Mexico provides a valuable system for studies in diversification and speciation. My dissertation addresses a variety of evolutionary genetic questions using this system.Rigorous studies of the relationships between host races of D. mojavensis and the relationships among the members of the species group (D. mojavensis, D. arizona, and D. navojoa) are lacking. I used mitochondrial CO1 sequence data to address the phylogenetics and population genetics of this species group (Appendix A). In this study I have found that the sister species D. mojavensis and D. arizonae share no mitochondrial haplotypes and thus show no evidence for recent introgression. I estimate the divergence time between D. mojavensis and D. arizonae to be between 0.66 and 0.99 million years ago. I performed additional population genetic analyses of these species to provide a basis for future hypothesis testing.In Appendix B, I report the first example of substantial intraspecific polymorphism for genetic factors contributing to hybrid male sterility. I show that the occurrence of hybrid male sterility in crosses between Drosophila mojavensis and its sister species, D. arizonae is controlled by factors present at different frequencies in different populations of D. mojavensis. In addition, I show that hybrid male sterility is a complex phenotype; some hybrid males with motile sperm still cannot sire offspring.The large degree of variation between isofemale lines in producing sterile hybrid sons suggests a complex genetic basis to hybrid male sterility warranting quantitative genetic analysis. Since the genes underlying hybrid male sterility in these species are not yet fixed, I am able to perform explicit genetic analysis of this reproductive isolating mechanism. In Appendix C, I present the results of mapping QTL for hybrid male sterility within species. The genetic architecture underlying hybrid male sterility when analyzed directly in the F1 is highly complex. Thus, hybrid male sterility arises as a complex trait in this system and we propose a drift-based model for the evolution of this phenotype.

Cytoplasmic male sterility (CMS) in plants is associated with mitochondrial dysfunction. We have proposed in this study that the mitochondrial-encoded chimeric peptide thought to be responsible for cytoplasmic male sterility in Polima system could function as a dominant male sterility inducer when expressed in the nucleus and targeted to the mitochondria. Transgenic plants expressing such mitochondrial targeting constructs exhibited reduction of pollen production that was characterized in fertile Westar (nap ) and restored fertile Westar (pol) plants by homeotic transformation of floral organs and in male-sterile Westar (pol) plants by a reduction in pollen production with shortening of the stamens. Genetic and molecular analysis has shown that the phenotypic changes were correlated with the effective genetic transmission of the inserted transgene through female gametes. Most significantly, we have found that differences in floral morphology induced by transgene expression between pol CMS and fertile Westar plants might be related to differences in transcriptional activity of the APETALA3 MADS box gene. We suggest that the alterations in floral morphology that accompany CMS in several plant species might be due to effects of mitochondria on transcriptional activity of floral organ identity genes.

DNA markers tightly-linked to nuclear fertility restorer genes for cytoplasmic male sterility (CMS) are valuable tools for breeders and researchers working with these genes. Two different targeting approaches were used to identify markers linked to the Rfp1 restorer gene for the pol CMS of canola (Brassica napus L.): nearly isogenic line (NIL) comparison and bulked segregant analysis. These methods were equally efficient in identifying markers linked to Rfp1; combining them allowed a targeting efficiency of 100% to be achieved. The efficiency of bulked segregant analysis was found to be limited by the inadvertent occurrence of shared homozygosity at specific chromosomal regions in the bulks, in contrast with the efficiency of NIL comparison which was limited by the occurrence of residual DNA from the donor cultivar at scattered sites around the genome of the NILs. Eleven DNA markers linked to the Rfp1 gene were identified, one of which perfectly co-segregates with Rfp1. The linkage group on which Rfp1 is localized contains 17 DNA markers. Two restorer genes of the pol CMS, Rfp1 and Rfp2, and a Rfn restorer gene of the nap CMS were found to be at least tightly linked to one another and may all reside at the same locus. A fourth restorer gene, the Rfo restorer for the ogu CMS, was, however, found to be unlinked to the other restorer genes. Different restorer genes for the nap CMS were found in the lines 'Westar-Rf and 'Karat'. A linkage map of the B. napus genome containing 146 markers organized into 23 linkage groups covering a total length of 850.2 cM was constructed from a BC$ sb1$ population. This map contains 63 loci previously localized on the B. napus genome through analysis of an F$ sb2$ population. Comparative analysis indicates that the total length of the BC$ sb1$-derived map is smaller than that of the F$ sb2$-derived map, which suggests that a reduction in recombination frequency is occurring in male gametes. The preferential use of two or three probe-

In order to identify organizational differences between sterile Polima (pol) and fertile Campestris (cam) mitochondrial genomes that could be linked to cytoplasmic male sterility (CMS), the physical map of the pol mitochondrial genome was constructed and compared to the physical map of the cam mitochondrial genome. The only structural differences between the two genomes are confined to a region encompassed by a 4.5 kb segment, present in pol mtDNA but absent in cam mtDNA. This 4.5 kb CMS-associated pol segment contains a chimeric gene called orf224 that is cotranscribed with atpG and comprises the single mtDNA region expressed differently in fertile, sterile and fertility restored plants which makes it a good candidate for specifying the sterility trait. Sequence analysis of the pol 4.5 kb segment has shown that orf224 was the only significant open reading frame (ORF) within the segment that gives rise to abundant transcripts, strengthening the view that the orf224/atp6 gene region is conferring pol male sterility. The pol 4.5 kb segment is also present and similarly organized in the common Brassica napus nap mtDNA but the sequences flanking the two segments are unrelated. Thus, the 4.5 kb segment appears to have transposed during the evolution of the pol and nap mitochondrial genomes and appears to have been lost in the cam mitochondrial genome. Sequence analysis of the nap segment revealed the presence of an ORF related to but divergent from orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino-acids with 79% homology to the predicted product of orf224. orf222 is co-transcribed with the third exon of the trans-spliced gene, nad5, and another ORF of unknown function. Expression of the orf222 gene region is tightly associated with nap CMS since the levels of orf222 transcripts are significantly reduced upon restoration while the expression of 22 other mitochondrial genes do not consistently correlate with nap CMS. Antibodies were rai

Understanding reproductive isolation and divergence is the focus of speciation research. Recent evidence suggested that some Dendroctonus ponderosae populations produced hybrids with reproductive incompatibilities, a reproductive boundary undetected by phylogeographic analyses using molecular markers. Additionally, the unique bifurcated distribution of D. ponderosae and the proposed isolation-by-distance gene flow pattern around the Great Basin Desert provided a unique opportunity to investigate the evolution of postmating (postyzygotic) isolation while also understanding phenotypic divergence along latitudinal (climatic) gradients. First, I characterized the strength, biological pattern, and geographic pattern of postzygotic isolation in D. ponderosae by crossing increasingly divergent populations in a common garden environment. There was little evidence of hybrid inviability in these crosses, yet geographically distant crosses produced sterile males, consistent with expectations under Haldane's rule. Hybrid male sterility appeared at a threshold among increasingly divergent populations, was bidirectional (reciprocal crosses were affected), and less geographically distant crosses did not show significant gender-specific decreases in fitness. Second, a separate investigation of two critical phenotypic traits (body size and development time) was conducted on intrapopulation F2 generation offspring from a common garden experiment. Genetic differences contributing to phenotypic variance were interpreted within the context of the previously described reproductive incompatibilities, gene flow patterns, and latitudinal gradients. Genetic differences in development time were striking between faster developing and more synchronized northern populations and slower developing, less synchronized southern populations. Differences in development time were not detected between populations at similar latitudes. Body size, although more variable than developmental time, generally conformed to expectations, with northern populations being smaller than southern populations. Average adult size was found to be quite different between many populations and did vary between populations at similar latitudes, yet relative sexual size dimorphism was rather consistent. There was no evidence of correspondence between phenotypic traits (body size and development time) and either reproductive boundaries or gene flow patterns. The results suggest that latitudinally imposed climatic differences are likely driving phenotypic divergence between populations.

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