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    Phenotypic evaluation of Trifolium repens x Trifolium uniflorum F₁ interspecific hybrids as predictors of BC₁ hybrid progeny : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Plant Breeding at Massey University, Palmerston North, New Zealand
    (Massey University, 2017) Ebbett, Michelle Anne
    Interspecific hybrids between white clover (Trifolium repens) and its close relatives are being created to address the lack of variation within white clover for traits relating to persistence and drought tolerance. This study addresses two concepts related to developing hybrid breeding strategies using Trifolium repens x Trifolium uniflorum hybrids. A first sandframe experiment investigated whether some of the first generation hybrid plants (F1) with common parents were better than others as future parents. A second experiment assessed whether the performance of the first back cross (BC1) progenies could be predicted from the parental F1 phenotypes. The foliage, fertility, roots and dry weight production of four families of F1 hybrids were evaluated following a period of growth in sand. From each family, the F1 hybrids with the highest and lowest dry weight production were selected and back crossed to two contrasting white clover cultivars. The resulting BC1 hybrid phenotypes were evaluated to ascertain whether any F1 hybrids were markedly better as future parents in hybrid breeding programmes, and whether the F1 phenotype could be used to predict that of the BC1 progeny. Differences in expression of more than half of the traits evaluated were found both between F1 families, and among genotypes within F1 families. Evaluation of the subsequent BC1 generation identified large amounts of variation in expression of most traits both within and among hybrid families. However correlations between trait expression of the F1 parent and the corresponding BC1 progeny were weak to non-existent for most traits evaluated. The absence of correlations indicated that the performance of an F1 hybrid genotype is not able to be used as a predictor of the BC1 progeny phenotypes, and that selection out of the F1 generation is futile in the formation of interspecific hybrid breeding populations.
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    Interspecific hybridisation and molecular characterisation of hybrids in the genus Zantedeschia : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Health at Massey University
    (Massey University, 1992) Yao, Jia-Long
    The genus Zantedeschia consists of two sections: a section containing Z. aethiopica, a white-flowered, evergreen species and a second section containing five winter-dormant species. A new species, Z. odorata, was recently described which does not fit into either of the two sections. Chromosome karyotypes of five species and two hybrid cultivars were prepared. Karyotypes are distinct between the two sections but not distinct within the second section. The karyotype of Z. odorata falls between the two sections although it is more closely related to Z. aethiopica. Colchicine treatment of multiplying shoots in vitro produced tetraploid plants from eight cultivars and two species of the second section of the genus. Most of these plants were pure tetraploids. Z. aethiopica did not multiply tn vitro. Colchicine treatment of Z. aethiopica 'Childsiana' germinating seed produced a few tetraploid plants and many diploid/tetraploid chimeric plants. A screening procedure for tetraploids using stomatal measurements, with confirmation by chromosome counting, was demonstrated to be an efficient and accurate way to identify tetraploids. Triploid plants were produced from two diploid/tetraploid crosses with the aid of in vitro embryo culture. Crosses were made between the two sections using a number of species and genotypes at the diploid and tetraploid levels. In these crosses, endosperms were watery and transparent and embryos were small (in most cases less than 0.3 mm). Embryos embedded within endosperms were cultured because the embryos were too small to be cultured separately from the endosperms. From these cultures, over one hundred hybrid embryos were rescued. These hybrids were all albino. In an electron microscopy study, it was found that the plastids of these albino hybrids had no prolamellar body in the dark nor grana in the light. Z. odorata was tested as a bridge for gene transfer between the two sections because it falls in between them. Hybridisation between Z. odorata and Z. aethiopica produced a number of virescent, albino and chimeric (green/albino) hybrids following embryo culture and seed germination. Hybrid production was much easier, however, when Z. aethiopica was used as the maternal parent. All hybrids rescued from crosses between Z. odorata and the second section of the genus were albino. This study also demonstrated that Z. odorata stigmas can receive pollen from the same spadix. Z. odorata embryos become dormant before the seeds matured. Plants of Z. odorata did not produce any flowers unless they were treated with gibberellic acid (GA). However, two or three flowers per plant were produced when 50 ppm GA3 or GA4 + 7 was applied to tubers as a pre-planting treatment. A partial library was constructed with total leaf DNA of Z. aethiopica 'Childsiana'. A species-specific nuclear DNA clone, pZAC3, was isolated by differentially screening this library with radioactively-labelled total DNA of different species. This clone was characterised by restriction enzyme mapping and RFLP (restriction fragment length polymorphism) analysis. By RFLP analysis, an apple rDNA clone differentiated between the sections of the genus. The apple rDNA clone and pZAC3 were successfully used for hybrid identification. From this library, six plastid (pt) DNA clones were also isolated by hybridisation with kiwifruit ptDNA clones. Using these ptDNA clones, RFLP bands were identified to differentiate between species and, in one case, between genotypes within a species. Biparental ptDNA inheritance and a ptDNA deletion were detected in the albino hybrids between the two sections with these ptDNA clones. The data from RFLP analyses gives the first molecular data on the phylogeny of Zantedeschia and indicates that Z. odorata is distinct from the previously-described two sections and falls in between the two sections.
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    Effect of ploidy on interspecific hybridisation between Trifolium repens L. and related species : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Seed Technology in the Plant Science Department of Massey University, Palmerston North, New Zealand
    (Massey University, 1995) Hussain, Syed Wajid
    Two interspecific Trifolium hybrids were available at the time this project was initiated. The first hybrid (3x H-6909-5) was a sterile triploid obtained from a T. repens x T. nigrescens cross to transfer clover cyst nematode (Heterodera trifolii Goffart) resistance from T. nigrescens to T. repens. The second was a tetraploid hybrid (4x H-435) and its octoploid derivative (8x H-435), developed between 4x T. ambiguum and T. repens to transfer genes conferring longevity and virus resistance from T. ambiguum to T. repens. This was the first partially fertile hybrid reported between the two species. Chromosome doubling had increased pollen stainability in the octoploid clones (8x H-435). The objectives of this project were to generate backcross progenies from these two hybrids at various ploidy levels, and to endeavour to achieve successful flow of genes between the parental species. The triploid H-6909-5 (2n=3x=24) was highly sterile and produced no seed from approximately 3,000 reciprocal crosses to both parental species. It was chromosome doubled by an in vitro colchicine method using 0.1% and 0.05% colchicine for 48-72 h, depending on treatment. Three chromosome doubled plants (approximately 10% of the surviving meristems) from treatments with 0.05% colchicine and 48 or 60 h duration of application were obtained. Chromosome doubling resulted in a marked increase in fertility, as pollen stainability was increased from 9.9% in 3x H-6909-5 to an average of 89.2% (range 87.7-90.9%) in 6x H-6909-5. Subsequent backcrosses of 6x H-6909-5 and interbreeding of backcross derivatives resulted in an array of fertile hybrids at 4x, 5x and 7x levels, and some aneuploids. The occurrence of 7x BC1F1 progeny from the T. repens x 6x H-6909-5 (4x x 6x) cross is the first evidence of functional female 2n gametes in T. repens. The failure or success of different backcrosses, BC1F1 x BC1F1 and BC1F1 x 6x F1 crosses supported a 2 maternal:1 paternal endosperm balance number (EBN) hypothesis to explain seed set in wide crosses. The EBN system will be helpful for predicting the success of future crosses if endosperm failure is the cause of hybrid embryo abortion. Meiotic chromosome pairing in F1 and BC1F1 progeny indicated the presence of allosyndetic pairing, suggesting that genetic exchange between the two species is possible. T. nigrescens appeared to be a useful source of clover cyst nematode resistance. In the initial screening a mean number of 23.4 (range 0-150) cysts per plant was recorded for T. nigrescens in comparison to a mean number of 149.7 cysts per plant for T. repens. Rescreening of sixteen surviving T. nigrescens and five T. repens genotypes confirmed the initial screening results. H-6909-5 (3x and 6x) was also screened for clover cyst nematode resistance, and their resistance equalled that of the resistant T. nigrescens genotype. H-6909-5 (3x and 6x) had significantly fewer cysts/g root dry weight than the susceptible T. nigrescens and T. repens genotypes. The second array of backcross progenies was generated from crosses involving 8x H-435 and T. repens and T. ambiguum. 8x H-435 was only cross fertile with T. repens and resulted in 145 seeds from 1,578 reciprocal crosses. Eleven out of 19 initially grown BC1F1 plants were all hexaploid (2=6x=48). Mean pollen stainability for nine out of the 19 BC1F1's was 32.9% (range 19.1-64.9%). Meiotic chromosome pairing in the 6x BC1F1 plants averaged 2.1 univalents, 18.1 bivalents, 1.1 trivalents and 1.6 quadrivalents. From meiotic configurations it was not possible to conclude whether chromosomes of the two species had paired autosyndetically or allosyndetically but the occurrence of a high frequency of multivalents (up to three trivalents and four quadrivalents) indicated both types of pairing. Backcrosses of 6x BC1F1 plants to T. repens resulted in 134 BC2F1 seeds from 760 crosses. Three out of seven initially grown BC2F1 plants were pentaploids (2n=5x=40). Pollen stainability averaged 59.3% (range 44.4-70.1%) for four 5x BC2F1 plants. On the other hand 6x BC1F1 x 6x T. ambiguum crosses did not produce any seed and only two pentaploid plants were obtained from 6x BC1F1 x 4x T. ambiguum crosses. One of these had 17.6% pollen stainability while the other did not produce normal inflorescences. The difficulty encountered in generating 6x backcross progeny with 6x T. ambiguum was overcome by the creation of a fertile "bridging population". However the "fertile bridge" did not eventuate until after two generations of crossing. The 6x BC1F1 plants were intercrossed and produced 114 BC1F2 seeds from 663 crosses. Two of the six initially grown BC1F2 plants were studied for somatic chromosome counts and were found to be hexaploid (2n=6x=48). The average pollen stainability was 40.8% for all six BC1F2 plants. One of these 6x BC1F2 plants was cross compatible as a female with 6x T. ambiguum and resulted 17 seeds from 318 reciprocal crosses. Most of the 6x BC1F1 plants combined the rhizomatous and stoloniferous growth habit of the parental species and two of the ten 6x BC1F1 showed significant improvement in stolon number, stolon length, shoot dry weight and nodulation over 8x H-435. However, 6x BC1F2 are likely to be superior to 6x BC1F1 progeny, as they have exhibited better expression of the combined stoloniferous and rhizomatous growth habit, improved fertility, frequent nodal rooting and heavier nodulation than the BC1F1 progeny. Consequently the 6x BC1F1 plants can either be used directly in the selection programme or as a "fertile bridge" between the two parental species. This work has resulted in the development of two arrays of fertile backcross progenies by manipulation of chromosome numbers and the production of a range of hybrid plants combining agronomic characteristics of the parent species in varying genome balances and at a range of ploidy levels. It is therefore concluded that initial sterility of the primary interspecific hybrids need not be a barrier to successful interbreeding.
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    Using supernetworks to distinguish hybridization from lineage-sorting
    (BioMed Central, 2008) Holland BR; Benthin S; Lockhart PJ; Moulton V; Huber KT
    BackgroundA simple and widely used approach for detecting hybridization in phylogenies is to reconstruct gene trees from independent gene loci, and to look for gene tree incongruence. However, this approach may be confounded by factors such as poor taxon-sampling and/or incomplete lineage-sorting.ResultsUsing coalescent simulations, we investigated the potential of supernetwork methods to differentiate between gene tree incongruence arising from taxon sampling and incomplete lineage-sorting as opposed to hybridization. For few hybridization events, a large number of independent loci, and well-sampled taxa across these loci, we found that it was possible to distinguish incomplete lineage-sorting from hybridization using the filtered Z-closure and Q-imputation supernetwork methods. Moreover, we found that the choice of supernetwork method was less important than the choice of filtering, and that count-based filtering was the most effective filtering technique.ConclusionFiltered supernetworks provide a tool for detecting and identifying hybridization events in phylogenies, a tool that should become increasingly useful in light of current genome sequencing initiatives and the ease with which large numbers of independent gene loci can be determined using new generation sequencing technologies.
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    Wellington geckos meet Wairarapa geckos : hybridisation between two genetically and morphologically distinct populations of the New Zealand common gecko complex (Hoplodactylus maculatus) : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Zoology at Massey University, Palmerston North, New Zealand
    (Massey University, 2010) Fitness, Josephine
    The purpose of this study was to use molecular techniques and morphological measurements to set out to find whether a hybrid zone exists between two coastal populations of the common gecko (Hoplodactylus maculatus), on the Wellington south coast. I collected geckos from five sites in a coastal transect from the population of small geckos to the large geckos. Using four genetic loci, one mitochondrial (16S) and three nuclear (Rag-1, Rag-2, C-mos), I was able to determine that the coastal populations do have geneflow, however each population maintains some unique alleles. Morphological evidence reveals a significant difference in gecko sizes from Turakirae Head and those caught at Ocean Beach, separated by just 15 km. Adult geckos at Turakirae Head are on average 10mm smaller (snout-to-vent) than adult geckos at Ocean Beach, representing almost a doubling in average weight. The centre of the steep frequency clines of four characters is coincident and the widths are concordant. The narrower morphological clines indicate stronger selection on the size of the gecko, than on genetic loci.