Centeno, Christian C (2017-08). DEVELOPMENT OF PCR-BASED TRANSPOSABLE ELEMENT ASSAYS FOR VERIFICATION OF PEARL MILLET-NAPIERGRASS HYBRIDS (Pennisetum glaucum [L.] R. BR X Pennisetum purpureum SCHUMACH.). Master's Thesis.
Thesis
Interspecific hybridization can serve a number of purposes in plant breeding. When significant investments are required to produce and/or use hybrids, it is important to accurately differentiate between hybrid and non-hybrid seed or progeny. However, recognition of hybrids versus non-hybrids is problematic for some parental species combinations, e.g., hybrids between pearl millet (Pennisetum glaucum, 2n=2x=14: AA) and napiergrass (P. purpureum, 2n=4x=28: A'A'BB). This cross can be made reciprocally to produce pearl millet-napiergrass ('PMN') hybrid taxa (2n = 3x = 21 chromosomes; AA'B genome) from P. glaucum x P. purpureum, or kinggrass (KG) hybrid taxa (2n = 3x =21; AA'B genome) from P. purpureum x P. glaucum. Identification of these hybrids is reportedly complicated by homeology among parental genomes, similarity of parental C-values, genetic similarity between parental species, morphological similarities and insufficient molecular methods. In this research, we explored hybrid identification through several approaches -- morphological, cytogenetic, flow cytometric and molecular genetics. Based on ANOVA and t-tests after replicated sampling from a genotypic panel that included one P. glaucum, two P. purpureum, four PMN hybrid taxa and one KG hybrid taxa, the morphological traits -- spikelet primary bristle length, average length of bristles, number of bristles, and length of spikelets -- were found to be insufficient as individual indicators of hybridity and did not allow for the construction of a taxonomic key. In contrast, chromosome number determinations from spreads of root-tip mitotic cells sufficed to distinguish PMN (2n=21), pearl millet (2n=14), napiergrass (2n=28) types, but the overall procedures were time- and resource-consuming. C-values were determined by flow cytometry but the differences between triploid hybrids and the parental species were non-significant. This research explored a molecular approach based on the Tuareg MITE in Pennisetum, which is widely distributed and highly polymorphic in Pennisetum genomes. Quantitative differences in PCR-based amplification should be diagnostic of genotype, at least for some Tuareg sequence-specific primers. Possible Tuareg sequence targets were determined using a sequence-based approach. Candidate sequences were assessed by electrophoretic analysis of PCR amplicons for presence/absence, band number and size distributions. Two markers, PgTb1 and Tr54, were individually assessed in detail. Among the sampled materials, the PgTb1 provided the most time and cost-efficient method of accurately identifying the interspecific Pennisetum hybrid taxa. PgTb1 was effective at separating the PMN and KG hybrids from both parents in a reliable manner. Tr54 was able to separate the hybrids from the napiergrass mean, and was able to separate the average of a group of such hybrids from pearl millet, but due to variation among individual hybrids, Tr54 assays were unable to reliably separate individual hybrids from the pearl millet PEGL 09TX04. Thus, PgTb1 is deemed the superior test marker for hybrid identification.
Interspecific hybridization can serve a number of purposes in plant breeding. When significant investments are required to produce and/or use hybrids, it is important to accurately differentiate between hybrid and non-hybrid seed or progeny. However, recognition of hybrids versus non-hybrids is problematic for some parental species combinations, e.g., hybrids between pearl millet (Pennisetum glaucum, 2n=2x=14: AA) and napiergrass (P. purpureum, 2n=4x=28: A'A'BB). This cross can be made reciprocally to produce pearl millet-napiergrass ('PMN') hybrid taxa (2n = 3x = 21 chromosomes; AA'B genome) from P. glaucum x P. purpureum, or kinggrass (KG) hybrid taxa (2n = 3x =21; AA'B genome) from P. purpureum x P. glaucum. Identification of these hybrids is reportedly complicated by homeology among parental genomes, similarity of parental C-values, genetic similarity between parental species, morphological similarities and insufficient molecular methods. In this research, we explored hybrid identification through several approaches -- morphological, cytogenetic, flow cytometric and molecular genetics.
Based on ANOVA and t-tests after replicated sampling from a genotypic panel that included one P. glaucum, two P. purpureum, four PMN hybrid taxa and one KG hybrid taxa, the morphological traits -- spikelet primary bristle length, average length of bristles, number of bristles, and length of spikelets -- were found to be insufficient as individual indicators of hybridity and did not allow for the construction of a taxonomic key. In contrast, chromosome number determinations from spreads of root-tip mitotic cells sufficed to distinguish PMN (2n=21), pearl millet (2n=14), napiergrass (2n=28) types, but the overall procedures were time- and resource-consuming. C-values were determined by flow cytometry but the differences between triploid hybrids and the parental species were non-significant.
This research explored a molecular approach based on the Tuareg MITE in Pennisetum, which is widely distributed and highly polymorphic in Pennisetum genomes. Quantitative differences in PCR-based amplification should be diagnostic of genotype, at least for some Tuareg sequence-specific primers. Possible Tuareg sequence targets were determined using a sequence-based approach. Candidate sequences were assessed by electrophoretic analysis of PCR amplicons for presence/absence, band number and size distributions. Two markers, PgTb1 and Tr54, were individually assessed in detail. Among the sampled materials, the PgTb1 provided the most time and cost-efficient method of accurately identifying the interspecific Pennisetum hybrid taxa. PgTb1 was effective at separating the PMN and KG hybrids from both parents in a reliable manner. Tr54 was able to separate the hybrids from the napiergrass mean, and was able to separate the average of a group of such hybrids from pearl millet, but due to variation among individual hybrids, Tr54 assays were unable to reliably separate individual hybrids from the pearl millet PEGL 09TX04. Thus, PgTb1 is deemed the superior test marker for hybrid identification.
