Karyotyping and in silico characterisation of the chromosomes of Diuraphis noxia (Hemiptera: Aphididae)

Steyn, Louis Johannes (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Diuraphis noxia Kurdjumov (Russian wheat aphid (RWA)), is an economically important agricultural pest that causes substantial losses in small grain production, particularly wheat and barley. Approaches that can be taken to manage this invasive pest include the cultivation of RWA resistant cultivars. The development of new RWA biotypes, virulent against previously classified resistant wheat cultivars presents, an additional problem to the goal of reducing crop losses. Therefore, studying the underlying molecular genetics of the RWA brings us closer to understanding wheat resistance to the RWA and ultimately battling this pest in small grain fields. The objectives of this study were: to study the sex (X) chromosome of the RWA by karyotyping and isolation using flow cytometry; to sequence the X chromosome; and then to map it against the reference genomes of the RWA and Acyrthosiphon pisum (pea aphid). Since aphids reproduce via parthenogenesis, mapping populations reliant on sexual recombination are not available, and therefore information about the locations of genes on chromosomes is completely lacking. To this end, reference mapping against the X chromosome of Drosophila melanogaster (fruit fly) was conducted to identify orthologous regions spanning the X chromosome of RWA. The results confirmed that the RWA karyotype consists of a diploid chromosome number of 10, with a large X chromosome pair and four autosomal chromosome pairs. Flow sorting yielded 2,047,296 X chromosomes and sequencing produced a total read count of 136,814,894 with a Q20 score of 96.32%. The X chromosome had a higher mapping percentage to the RWA genome (82.88%) compared to that of the pea aphid (51.3%). Interestingly, a high mapping coverage across the entire genome of both aphids was observed, suggesting that flow cytometry did not separate the X chromosome from the rest of the chromosomes of the RWA but allowed unintended chromosomes to contaminate the series. Mapping against the fruit fly X chromosome produced eight orthologous regions of which six was confirmed to be present in the RWA karyotype through in situ hybridization, while a protein BLAST of the fruit fly X chromosome against the RWA genome aided in determining approximately 67.42% of the length of the RWA X chromosome.

AFRIKAANS OPSOMMING: Diuraphis noxia Kurdjumov, algemeen bekend as die Russiese koringluis (RWA), is ‘n ekonomiese belangrike landboupes wat ‘n groot afname in klein graangewas produksie, spesifiek koring en gars, veroorsaak. Metodes wat geiimplementeer kan word om hierdie indringer pes te beheer en gewas verliese te verhoed, sluit die kultivering van RWA weerstandbiedende kultivars in. Die ontwikkeling van RWA-biotipes, wat luis populasies is virulent teen voorheen weerstandbiedende koring kultivars, dra addisioneel tot die probleem by. Daarom is dit belangrik om die onderliggende molekulêre genetika van die RWA te verstaan, sodat ons ook koring weerstand tot RWA kan begryp om uiteindelik graangewasse teen die pes te beskerm. Die doel van hierdie studie is: om die seks (X-) chromosoom van die RWA te bestudeer deur kariotipering en te isoleer deur van vloeisitometrie gebruik te maak; die X-chromosoom se volgorde te bepaal; en om dit dan teen die verwysings genome van RWA en Acyrthosiphon pisum (ertjieluis) te vergelyk. Aangesien hierdie luise ongeslagtelik voortplant, bestaan daar nie karteringspopulasies vir RWA nie, en daarom ontbreek inligting oor die ligging van gene op die chromosome. Ten einde die studie doel te bereik, was verwysingkartering teen Drosophila melanogaster (vrugtevlieg) se X-chromosoom gedoen met die doel om ooreenstemmende areas oor die X-chromosoom te identifiseer. Die resultate het bewys dat die RWA-kariotipe uit ‘n diploïde chromosoomgetal van 10 bestaan, met ‘n groot X-chromosoompaar en vier outosomale chromosoompare. Vloeisortering het 2,047,296 X-chromosome opgelewer en volgordebepaling het ‘n totale leesraam-telling van 136,814,894 teen ‘n Q20-telling van 96.32% gelewer. Die X-chromosoom het ‘n hoër ooreenstemming teenoor die genoom van die RWA (82.88%), in vergelyking met die ertjieluis (51.3%) vertoon. Beide luise het n hoë karteringdekking oor hulle hele genoom gehad. Dit was onverwags en dui daarop dat vloeisitometrie nie spesifiek genoeg was vir net die X-chromosoom nie en het dus nie-geteikende chromosome deur gelaat wat die monster gekontamineer het. Kartering teenoor die vrugtevlieg X-chromosoom het agt ooreenstemende streke geproduseer waarvan ses bewys was om voor te kom in die RWA kariotipe deur in situ hibridisering, terwyl ‘n proteïen BLAST van die vrugtevlieg X-chromosoom teenoor die RWA genoom bygedra het tot die bepaling van minstens 67.42% van die lengte van die RWA X-chromosoom.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100215
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