Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

Pairing and synapsis of homologous chromosomes are required for normal chromosome segregation and the exchange of genetic material during meiosis. Pairing is defined as the recognition and alignment of chromosomes that occurs either pre-meiotically or during early prophase I to ensure that associations via synapsis and recombination occur only between homologues. Synapsis is the intimate juxtaposition of homologous chromosomes that is complete at pachytene following formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is an essential component of the SC that localises along chromosome axes during prophase I and promotes homologous chromosome interactions. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC. In this study, we have characterised the bread wheat homologue of HOP1, TaASY1, and its encoded protein. The full length cDNA and genomic DNA clones of TaASY1 have been isolated, sequenced and characterised. TaASY1 is located on chromosome group 5 and the open reading frame displays significant similarity to OsPAIR2 (84%) and AtASY1 (63%). In addition to OsPAIR2 and AtASY1, the deduced amino acid sequence also displays sequence similarity to ScHOP1, with all four proteins containing a HORMA domain. Transcript and protein analysis showed that expression is largely restricted to meiotic tissue, with elevated levels during the stages of prophase I when pairing and synapsis of homologous chromosomes occurs. Antibodies specific to TaASY1 were used in immuno-fluorescence microscopy and immuno-gold transmission electron microscopy to investigate the localisation of TaASY1 in meiotic cells. Immuno-fluorescence analysis initially detected ASY1 in pollen mother cells (PMCs) during meiotic interphase as foci randomly distributed over the chromatin. The ASY1 signal became increasingly continuous during leptotene, reflecting the changes occurring in chromosome morphology. Throughout zygotene, the signal became progressively more continuous, localising along the entire length of the axial elements as chromosomes synapsed. This signal appeared to persist until pachytene, before disappearing from the chromatin as the SC disassociated through late pachytene and early diplotene. The immuno-gold based electron microscopy displayed that TaASY1 localises to chromatin that is associated with both axial elements before SC formation as well as chromatin of lateral elements within formed SCs. Analysis of RNAi Taasy1 mutants was performed to further define the role of ASY1 in bread wheat meiosis. ASY1 localisation was disrupted in these mutants, with a diffuse and non-continuous signal observed through leptotene and zygotene. Feulgen staining of meiotic chromosomes displayed reduced synapsis during prophase I, as well as multivalents at metaphase I and abnormal chromosome segregation during anaphase I. These observations are consistent with the presence of homoeologous chromosome interactions. TaASY1 expression and localisation was also investigated in the bread wheat pairing mutant, ph1b. Quantitative real-time PCR (Q-PCR) revealed that TaASY1 is significantly up-regulated in ph1b, with greater then 20-fold expression compared to wild-type Chinese Spring, while maintaining the same pattern of expression as wild-type through progressive stages of meiosis. ASY1 localisation was significantly disrupted in ph1b, with irregular loading on axial elements during mid to late zygotene, indicative of abnormal chromatin remodelling and multiple axial element associations that have previously been reported in ph1b. Taken together, these results indicate that TaASY1 is essential for promoting homologous chromosome interactions during meiosis, and that impairment of ASY1 function in bread wheat meiosis results in reduced restriction of chromosome associations to homologues. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1340087 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 3 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Barley (Hordeum vulgare L. subsp. vulgare) is an important crop and ranks fourth in overall production of the major cereal crops in the world. Like other cereal crops, barley suffers from a narrowing of its genetic base and susceptibility to diseases, pests and environmental stresses. H. bulbosum is a possible source of desirable genes for introgressing into barley to restore genetic diversity and improve current cultivars. Sexual hybridisation between barley and H. bulbosum is the main method for interspecific gene transfer in barley breeding but there are several barriers to overcome. Two of these are reduced recombination and the ability to identify recombinants quickly and efficiently. The aim in this thesis was to gain a better understanding of meiotic chromosomal behaviour in the two species and their hybrids and to improve the characterisation of recombinants from the hybrids. To study the events during meiosis, synaptonemal complex (SC) analysis was carried out on the two species and two H. vulgare - H. bulbosum hybrids. The results indicated that there were interspecific and intraspecific variations in SC length. Mean SC length was positively correlated with recombination frequency but not related to genome size. This suggests that the ratios of mean SC length to genome size (SC/DNA) show divergence among these Hordeum examples. An hypothesis based on the conformation of chromatin associated with axial element, which is dependent on SC/DNA ratio, was presented to explain the relationship between SC length and recombination frequency. Chromosome pairing in the two hybrids was determined by observation at pachytene and metaphase I (MI). Mean percentages of synapses were similar but there were different frequencies of MI pairing between these two hybrids, indicating that different mechanisms may regulate synapsis and MI pairing in the hybrids. To investigate meiotic recombination, genomic in situ hybridisation (GISH) was performed on the two hybrids at MI and anaphase I (AI). It was observed that intergenomic pairing and recombination events occur in distal chromosome segments. A great discrepancy between mean pairing and recombination frequencies was observed in both hybrids and several possible reasons for this discrepancy were discussed. Hybrid 102C2 with high MI pairing had a significantly higher recombination frequency than the low pairing 103K5, suggesting that high MI pairing appears to be associated with high recombination in the hybrids. An interesting finding is that the ratio of recombination to MI pairing in 103K5 (l:8.9) is twice as high compared with 102C2 (l:17). However, the mechanism for this difference in the ratio between the two hybrids remains unknown. Sequential fluorescence in situ hybridisation (FISH) and GISH were used successfully to localise the introgressions in selfed progeny from a tetraploid hybrid derived from chromosome-doubled 102C2 (102C2/colch). This procedure is fast, cheap and can efficiently detect and locate introgressions. Several disease-resistant recombinants were analysed in more details and leaf rust and powdery mildew resistance was associated with distal introgressions on chromosomes 2HS and 2HL (leaf rust) and 2HS (powdery mildew). It is possible that the leaf rust and powdery mildew resistances were closely linked in the distal region of 2HS. A considerable variation in introgression size was observed at similar chromosomal sites among the different recombinants, which will provide useful information for map-based cloning of genes.

In the developing mouse oocyte, as well as in the preimplantation embryo, betaine (N,N,N-trimethylglycine) plays an important role first as a mechanism for cell volume regulation and second as a major methyl donor. Thus, the presence of betaine has implications both during development, and throughout the lifespan. It has previously been observed that betaine accumulates in the mouse oocyte as it matures, however its origin in the egg is unknown. Here I explore the enzyme choline dehydrogenase (CHDH; EC 1.199.1) as a method by which the mouse oocyte synthesizes the betaine that we observe prior to initiation transport activity in the preimplantation embryo. I carefully monitored betaine transport throughout meiotic maturation to confirm that no other previously unobserved membrane transport existed in the maturing oocyte. However, no betaine transport into oocytes was detected during meiotic maturation suggesting de novo synthesis. Previous data suggests that the enzyme is expressed (at the transcript level) in the developing oocyte, and becomes active during meiotic maturation. I demonstrated the presence of CHDH protein in the oocyte and preimplantation embryo. I then examined whether the mouse oocyte synthesizes betaine autonomously and addressed whether CHDH is a requirement for this process. Chdh knockout oocytes did not accumulate betaine in vivo, while normal betaine levels were observed in Chdh wildtype oocytes. CHDH-mediated synthesis of betaine was directly confirmed by detection of increased betaine in oocytes matured in vitro in the presence of choline. Chdh-/- oocytes failed to produce betaine when similarly cultured in choline. This establishes the production of betaine as an autonomous process in maturing oocytes. Overall, I have built upon previous data to demonstrate that betaine accumulation is a feature of meiotic maturation that occurs by de novo synthesis of the molecule, a process that requires transient activation of the enzyme choline dehydrogenase.

The maintenance of genomic stability through suppression of retrotransposon activity is vital for the avoidance of potentially mutagenic genomic disruption caused by retrotransposition. Germline development is a particularly important phase for retrotransposon silencing as retrotransposition events here have the potential for transmission to the entire embryo, threatening the health of offspring. A collection of germline genome defence genes are required for the suppression of retrotransposons in the developing germline of male mice (e.g. Tex19.1, Dazl, Mili, Miwi2, Gasz, Mov10l1, Mael, Dnmt3l), all of which trigger meiotic prophase arrest when mutated. I have analysed the meiotic defects which arise in Tex19.1-/- male mice to contribute to the understanding of the fundamental mechanisms required for successful completion of meiosis and to investigate the involvement of retrotransposon silencing in this process. The absence of TEX19.1 in male mice causes infertility; with failed chromosome synapsis in ~50% of pachytene nuclei and associated apoptosis, as well as individual univalent chromosomes in 67% of remaining nuclei progressing to metaphase I. Where studied, failed chromosome synapsis is a common feature of germline genome defence mutant spermatocytes. One aim of my studies has been to better understand the mechanism responsible for this failed chromosome synapsis. I have demonstrated that unlike Mael-/- spermatocytes, additional SPO11-independent DNA damage potentially attributable to retrotransposition is not detectable in Tex19.1-/- spermatocytes. Rather, the formation of meiotic DNA double strand breaks (DSBs) is dramatically reduced in early prophase to around 50%, resulting in a reduction in nuclear γH2AX signal, production of SPO11- oligonucleotide complexes and foci formation by early recombination proteins RPA, DMC1 and RAD51. Despite this early reduction, DSB frequency recovers to more normal levels shortly after in zygotene. I have shown that defective pairing of homologous chromosomes by meiotic recombination is likely responsible for the asynapsis previously reported. The initial reduction in DSB frequency could be sufficient to cause failed chromosome synapsis in this mutant, assuming that late-forming DSBs cannot participate effectively in promoting homologous pairing. Alternative hypotheses include altered positioning of DSBs in response to altered chromatin organisation relating to retrotransposon upregulation, misguiding the pairing of homologous chromosomes. Such a model of disruption could also extend to other germline genome defence mutants. I have demonstrated that despite successful pairing of homologous chromosomes in a sub-population of Tex19.1-/- spermatocytes, subsequent progression of these cells through pachytene is delayed. Numerous diverse features of progression are all delayed, including recombination, ubiquitination on autosomes and sex chromosomes, expression of the mid-pachytene marker H1t, and chromosome organisation. The delay identified is related to recombination therefore this feature is likely to stem from the initial defect in DSB formation early in prophase. While some delayed features are probably directly related to recombination, others are not. The coordinated delay observed may suggest the presence of a recombination-sensitive cell-cycle checkpoint operating to regulate progression through pachytene. My research has also aimed to establish the cause of elevated univalent chromosomes not connected by chiasmata in metaphase I Tex19.1-/- spermatocytes. I have demonstrated that that absence of chiasmata is not due to failed crossover formation between synapsed chromosomes. Rather, the frequent observation of individual unsynapsed chromosomes during crossover formation suggests that some spermatocytes with low-level asynapsis are leaking through meiotic checkpoints and are unable to form a crossover before reaching metaphase. Therefore, again this later meiotic defect appears to stem from the initial defect in meiotic DSB formation, the consequences of which vary widely in severity. Remarkably the unsynapsed chromosomes present during crossover formation include both sex chromosomes, and autosomes. Tolerance of an unsynapsed autosome from pachytene into metaphase is an unusual observation in mice and this observation may aid the understanding of spermato cyte quality control mechanisms during this progression. Together these findings have greatly advanced the understanding of the infertility incurred during meiosis in Tex19.1-/- male mice. These findings may also extend to benefit the understanding of other germline genome defence mutants. Diverse observations made during my investigations also reveal a potential system of coordinated progression through pachytene relating to meiotic recombination. The variable severity of the synapsis defects incurred in this mutant appears to have variable effects on spermatocyte survival and could also inform the understanding of meiotic checkpoint sensitivity.

Meiotic recombination, known as crossover, is a vital mechanism for generating genetic diversity in sexually reproducing populations. Recombination events are non-uniform across the genome, due to a variety of influences including chromatin structure, DNA-sequence, epigenetic marks and interference from other recombination events. These known factors do not fully explain the distribution of recombination events, and additionally do not account for all the variability in recombination frequency observed both between and within species. Furthermore, of the mechanisms that have been identified, many are not yet fully understood. In Arabidopsis thaliana, considerable variation is observed in recombination frequency and distribution between natural accessions. By investigating recombination events in A.thaliana, this project aimed to identify trans-acting modifiers of recombination frequency that varied between natural accessions. Identification of meiotic recombination modifiers was performed through Quantitative Trait Loci (QTL) mapping in A.thaliana natural-accession cross populations. Populations were generated from crosses between two accessions which differed significantly for recombination frequency as measured across a defined region of the genome flanked by a fluorescent-reporter system. F1 plants were then self-fertilised to produce segregating mosaic F2 populations for mapping. Recombination frequency for specific genomic intervals was determined for each individual in the population through measurement of the segregation of flanking fluorescence-genes expressed in the products of meiosis - seeds or pollen. Individuals were also genotyped using accession-specific markers across the genome, at a marker density of one marker per 2-5Mb, depending on the chromosome. Association of variation in recombination frequency with specific sections of the genome differing between the parental accessions through QTL mapping revealed significant modifiers of meiotic recombination segregating within the populations. This resulted in the identification of three significant large-effect modifiers that differed between Col-0 and Cvi-0 accessions, on chromosomes 1 ,2 and 5, affecting recombination in an interval in the sub-telomere region of chromosome 3. An additional modifier on chromosome 4 affecting the same sub-telomeric interval was identified that differed between the Col-0 and Can-0 accessions. Further fine-mapping of modifiers to improve location resolution was performed by repeated backcrosses into the Col-0 genetic background to remove the influence of other large-effect QTL and possible unknown small-effect modifiers. Improving the resolution provided a number of potential candidates for genes underlying the recombination phenotype for each QTL. Candidate testing was then performed, either through transformation of different accession alleles into the fluorescent-reporter system, or through analysis of T-DNA insertion lines that interrupted candidate genes. Preliminary results from T-DNA insertion mutants crossed to the fluorescent-reporter system suggest a potential role for the AT2G31510 gene in modification of meiotic recombination frequency, though the mode of action remains unknown. These results demonstrate the presence of large-effect modifiers of meiotic recombination frequency that vary between the natural A.thaliana accessions Col-0, Cvi-0 and Can-0. Confirmation of underlying genes or sequence elements and characterisation of their mechanism of action are opportunities for exploration in future experiments.