Dissertationen zum Thema „Barley Genetics“

Barley yellow dwarf virus (BYDV), an aphid transmitted luteovirus, is the most widespread and economically damaging virus of cereal crops. The work in this thesis aims to characterise the basis of the naturally occurring resistance to BYDV in cereals in three ways: Firstly, by facilitating the isolation of the Yd2 gene for BYDV resistance from barley by a map-based approach. Secondly, by determining if a BYDV resistance gene in rice is orthologous to Yd2. Thirdly, by establishing if other BYDV resistance genes in non- Ethiopian barleys are allelic to Yd2. It is hoped that the information generated in this study will ultimately assist in the production of BYDV resistant cereal cultivars. A detailed genetic map of the Yd2 region of barley chromosome 3 was constructed, containing 19 RFLP loci, the centromere and the Yd2 gene. Yd2 mapped on the long arm, 0.5 cM from the centromere, and in the mapping population of 106 F2 individuals, perfectly cosegregated with the RFLP loci XYlp, and Xwg889. This map represents the first stage in a project to isolate the Yd2 gene by a map-based approach. The isolation of Yd2 could help to elucidate the molecular mechanism of the Yd2-mediated BYDV resistance, and may allow the production of BYDV resistant cereals by genetic transformation. The RFLP markers mapped closest to Yd2 could also be useful in barley breeding, by enabling selection for both the presence of Yd2 and the absence of agronomically undesirable traits known to be closely linked to Yd2. Genetically Directed Representational Difference Analysis (GDRDA) is a technique based on subtractive hybridisation, which can be used to identify RFLP markers closely linked to a gene of interest. Two GDRDA experiments were performed with the intention of generating additional RFLP markers close to Yd2. However, the first experiment yielded RFLP probes that were not derived from the barley genome, while the second experiment yielded probes that detected repetitive sequences. It was concluded that GDRDA is of limited use in generating further markers close to Yd2. To isolate the Yd2 gene by a map-based approach, a much larger mapping population will need to be analysed to genetically resolve markers tightly linked to Yd2. If the two morphological markers uzu dwarf and white stripe,,j flank Yd2, then they could assist in this task by enabling the visual identification of F2 seedlings resulting from recombination close to Yd2. However, in this study, both morphological markers were found to be located distal to Yd2. Therefore, these two morphological markers can not be used together to facilitate high resolution genetic mapping of the Yd2 locus. It may be possible to use large-insert genomic DNA clones from the relatively small genome of rice to generate further RFLP markers close to the Yd2 gene in barley, provided that the order of orthologous sequences in barley and rice is conserved close to the Yd2 locus. To assess the feasibility of this approach, RFLP probes used to identify loci close to Yd2 were mapped in rice using a segregating rice F2 population. Five of the RFLP loci mapped together and in the same order as RFLP loci mapped close to Yd2 in barley using the same probes. By comparing the location of RFLPs mapped by other researchers in rice using probes mapped close to Yd2, the region of conserved linkage between rice and the Yd2 region was tentatively identified as the central portion of rice chromosome 1. The collinearity shown by orthologous sequences in barley and rice indicated that it may indeed be possible to use rice to assist in generating RFLP markers close to Yd2. Of all the cereals, rice is the most amenable to map-based gene isolation, due to its small genome, well developed physical and genetic maps, and its ability to be genetically transformed with high efficiency. If a BYDV resistance gene that is orthologous to Yd2 could be identified in rice, this gene could be isolated with relative ease, and then used to identify barley cDNA clones corresponding to Yd2 gene by virtue of the sequence homology expected between these genes. To test if a BYDV resistance gene from an Italian rice line is orthologous to Yd2, recombinant-inbred rice lines previously characterised for this gene were analysed using probes mapped close to Yd2 in barley. No genetic linkage was detected between the RFLP loci and the BYDV resistance gene, indicating that the gene is unlikely to be orthologous to Yd2. BYDV resistance alleles at the Yd2 locus which are of a non-Ethiopian origin may show interesting differences to Ethiopian Yd2 resistance alleles. To identify barleys which may contain resistance alleles of Yd2, ten BYDV resistant barleys not known to contain Yd2 were assessed for their resistance to the PAVadel isolate of BYDV in the glasshouse. CI 1179, Rojo, Perry, Hannchen, Post and CI 4228 were found to be the most resistant under these conditions, and were analysed further. If the resistance from these barleys is controlled by alleles of Yd2, RFLP markers close to Yd2 will be expected to cosegregate with the resistance in F2 families derived from crosses between these resistant barleys and the BYDV susceptible barleys Atlas and Proctor. RFLPs suitable for use in these allelism tests were identified using probes mapped close to Yd2. However, time did not permit the analysis of these F2 populations.
Thesis (Ph.D.) -- University of Adelaide, Dept. of Plant Science, 1996

On title page "B" is superscript. Bibliography: leaves 112-135. The primary aims of the work described in this thesis were to isolate and characterize the cDNAs that correspond to the two B-glucan exohydrolases designated isoenzyme ExoI and isoenzyme ExoII. (abstract)

Bibliography: leaves 183-211. This study evaluates the backcross breeding method for the introgression in barley of agronomically important traits into a malting quality background using molecular markers.

A genome sequence can be conceptualized as a 'book' written with four nucleotide 'letters' in oligonucleotide (oligo) 'words'. These words can be used in genomics, bioinformatics and the development of molecular markers. The whole-genome sequence for rice (Oryza sativa L.) is almost finished and has been assembled into pseudomolecules. For barley ( Hordeum vulgare L.) expressed sequence tags (ESTs) have been assembled into 21,981 tentative consensus sequences (TCs). The availability of such sequence information provides opportunities to investigate oligo usage within and between genomes. For the first of three studies reported in this thesis, a C++ program was written to automatically design oligos that are conserved between two sets of sequence information. In silico mapping between rice coding sequences (CDS) and barley TCs indicated that oligos between 18 and 24 bp provide good specificity and sensitivity (83% and 86%, respectively, for 20mers). Conserved oligos used as PCR primers had a high (91%) success rate on barley lines. Sequencing of PCR products revealed conservation in exon sequence, size and order between barley and rice. Introns were not conserved in sequence but were relatively stable in size. Map locations of eight new markers in barley revealed both genome colinearity and rearrangements between barley and rice. The second study reported in this thesis examined word frequency within the rice genome. A non-random landscape composed of high-frequency and low-frequency zones was observed. Interestingly, high-frequency words seemed to be rice specific while single-copy words were gene specific and conserved across species. As in the first study, oligos of 12 bp or less were not specific, and 18 bp seemed to be a critical length for the specificity of oligos. The third study reported in this thesis involved the development of molecular markers for known genes using public sequence information. Six new polymorphic markers were d

Thesis (MTech (Horticulture))--Cape Peninsula University of Technology, 2018.
This study evaluated the effects of 5 different soaking treatments in conjunction with 5 varying irrigation intervals on the germination, growth and nutritional values of seed of Hordeum vulgare Sv13. The 5 different soaking times consisted of 1, 3, 8, 16 and 24 hours. The barley seed was first cleaned and then placed in a vessel containing 500 ml of distilled water with a 20 % solution of sodium hypochlorite (bleach) at room temperature. Thereafter the pre-soaked seeds were transferred to a perforated container, containing no medium and placed into a growing chamber equipped with drip irrigation. The seed was then irrigated with 1245 ml of water at 5 different intervals namely every 2, 4, 8 10 and 12 hours. The temperature of the hydroponic growing room was kept at a constant 23 °C using a hotoperiod of 16-hour day/ 8-hour darkness. The seed was allowed to germinate and grow for a period of 8 days before being harvested. The objectives of this study were to determine the most beneficial combination of soaking treatment in conjunction with the most beneficial irrigation interval on the germination rate of the seed allowing for radicle emergence and coleoptile production. It was also used to determine which combination of treatments was most beneficial to the growth and nutritional values of the seed post-harvest. Another objective was to ascertain the shortest soaking time for application in a small-scale, hydroponic growing unit as well as the frequency of irrigation required to grow seedlings, thereby determining the amount of water required to produce a seedling mat for a small-scale, subsistence farmer, with the emphasis being on water reduction. Each treatment was replicated 10 times and consisted of 500 grams of seed, which when placed into its container measured 2 centimetres in depth, totalling 25 treatments in all. Germination was measured by observing radicle emergence in the first 2 days of the growing period first after a 24-hour cycle and again after 48 hours. The numbers of leaves present at harvest after an 8-day growing period were also counted to determine germination rate of the seeds. Growth was determined by average leaf height as well as the tallest leaf on day 8 of the growing cycle. Root mat expansion was also measured, post-harvest, which was compared to the initial 2 cm planting depth of seed. Wet and dry weights of the plant material were measured post-harvest. Samples of the harvested material were also sent for nitrogen and protein analysis. It was discovered that most of the results favoured a shorter soaking time and an increase in irrigation frequency, bar a few exceptions. Most favoured a pre-soaking time of only 1 hour together with an irrigation frequency of between 2 and 4 hours. This shows that small-scale farmers would be able to reduce the time spent on soaking of their seed. Although the frequency of the irrigation interval remained high further testing would be required to determine if the amount of water applied at each irrigation interval could be reduced and still produce favourable results. It would also remain to be seen if no irrigation during the 8-hour dark photoperiod would have any negative impact on germination, growth and nutritional values of the seedlings.

Laboratory experiments, genetic simulation, and theoretical analyses were performed to address several objectives related to the use of genetic markers in plant breeding programs. Two software packages were developed: GREGOR provides flexible and efficient computer algorithms for performing genetic simulation experiments, and KIN provides improved methods for estimating coancestry from known pedigrees. Random amplified polymorphic DNA (RAPD) markers were investigated in elite barley lines, and estimates of genetic distance based on RAPD markers were compared to estimates based on coancestry. Both types of estimate can provide information that is useful to breeders and geneticists. Genetic simulation was used to investigate the power, accuracy and precision of several methods that are available for analyzing quantitative trait loci (QTL). In most cases, simplified methods of QTL analysis based on linear regression were similar or superior to more complex methods based on mixture models. Methods for genetic analysis using selective genotyping and pooled DNA were also investigated. These methods may allow precise estimates of the positions of markers and QTL to be made.

The control of pollen viability and release is of major commercial importance in the development of crops for hybrid seed production and selective breeding. It has been shown that key transcription factors in Arabidopsis particularly MALE STERILITY1 (MS1), are functionally conserved in rice (Li et al., 2011), therefore extending this comparative analysis and controlling fertility in temperate cereals, such as barley, is the long term goal of this project. Although anther and pollen development of barley seems morphologically similar to Arabidopsis, the genes involved and how they are regulated are currently unknown. Arabidopsis MS1 is a tapetum specific transcription factor which is expressed exclusively from the tetrad stage to early microspores release. Identification and accurate staging of barley anther development is essential for expression analysis and functional characterisation of genes involved in pollen development. Therefore, a complete morphological study of barley development was conducted. External characteristics have been described in parallel to anther development in order to predict anther stages by the observation of external stages phenotypic traits. Characterization of the barley orthologue of MS1 (HvMS1) has been conducted. Recently a new grass genome has been released, Brachypodium distachyion. This new resource has been used to aid primers design alongside the rice OsPTC1 sequence, the orthologue of MS1 (Li et al., 2011). Genome sequencing has indicated that the Brachypodium genus is more closely related to wheat and barley than it is to rice, Due to the close relationship between Brachypodium and barley, this new grass has been used as intermediary to identify the OsPTC1 orthologue in barley as well as downstream MS1 targets. A highly similar sequence to OsPTC1 was found in Brachypodium, Bradi4g31760. This new gene, as a result of its similarities to OsPTC1, was considered as its putative orthologue gene in Brachypodium. Therefore, the most conserved areas between OsPTC1-Bradi4g31760 were used for primers design to successfully amplify equivalent gene in barley (HvMS1). The characterization of this barley gene showed a similar expression pattern to the MS1 putative orthologue in Arabidopsis of tapetum specific expression. In addition, RNAi silencing of this gene has revealed that it is essential for the normal development of pollen, with a lack of viable pollen produced in the putative HvMS1 silenced transgenic lines.

Dissertation (PhD(Agric)) -- University of Stellenbosch, 2001.
ENGLISH ABSTRACT: Net blotch of barley, caused by Pyrenophora teres, is one of the most important diseases of this cereal in the south Western Cape Province of South Africa. This fungus exists as two different types (forms), namely a nettype and a spot-type that are distinguished by differential symptom expression on barley leaves. Based on this specific plant pathological difference a series of studies of agricultural importance were executed to investigate the effects of sexual recombination between these two types. In addition, studies were done to determine the difference between local net- and spot-type populations with regards to population structure and fungicide sensitivity. This dissertation therefore, consists of a collection of separate publications and as a result a certain degree of redundancy has been unavoidable. Recombination is one of the most important evolutionary forces involved with sexual reproduction. In plant-fungal agricultural ecosystems this may result in pathogenic fungal populations adapting more rapidly to control programs such as fungicide applications. The first section of the review in part 1 of this dissertation covers different aspects of sexual reproduction in ascomycetes, specifically focussing on mating-type genes, vegetative incompatibility and recombination. The major part of the review is then dedicated to various plant pathological aspects of P.teres, specifically addressing the differences between the two types, and in various cases highlighting the significance of sexual recombination within and between the net- and spot-type. Using morphological criteria for identification purposes there have been many conflicting reports concerning the identity of leaf spot isolates in the Western Cape Province of South Africa. In part 2, the correct identity was eventually achieved employing mating studies and molecular markers .: This was accomplished after single ascospores were obtained from pseudothecia after in vitro mating had occurred between a verified P. teres net-blotch isolate from Denmark and a representative Pyrenophora leaf spot isolate from South Africa. Using amplified fragment length polymorphism (AFLP) and RAPD markers, recombination was demonstrated in the progeny that had DNA banding patterns different from the two parental isolates. Pathogenicity trials also confirmed that recombination had taken place during mating. Inoculations were conducted on the differential cultivars susceptible to the net-blotch and leaf spot forms. The two parents induced typical net-blotch or leaf spot symptoms whereas the progeny mostly induced a jagged spot symptom on each cultivar. Fungicide sensitivity tests using the ergosterol biosynthesis inhibitors showed that, due to recombination, some progeny could have increased resistance to these fungicides. Due to mating and subsequent recombination between a net blotch isolate of P. teres and a representative leaf spot isolate, it was concluded that the latter was P. teres f. maculata. Fifteen of the net-spot hybrid progeny (F1) produced from the mating study in Part 2 were screened in Part 3 to assess their viability and genetic stability. Hybrid progeny (F1) inoculated onto barley seedlings consisting of the cultivars Stirling (differentially susceptible to net-type isolates), B87/14 and Clipper (both differentially susceptible to spot-type isolates) produced intermediate symptoms on all cultivars. Axenic cultures (F1-1) isolated from foliar lesions, followed by repeated inoculation and isolation (F1-2) onto a healthy set of seedlings produced similar intermediate symptoms. RAPDs conducted with two 1Q-mer primers on all isolates of F1-1and F1-2progeny revealed profiles similar to those obtained for F1 isolates. RAPD molecular data, therefore, indicated that hybrid progeny of this net x spot mating were genetically stable after having been subjected to two repetitive inoculation and reisolation cycles. Phylogenetic analysis of DNA sequences of the internal transcribed spacers (ITS1 and ITS2) flanking the 5.8S nuclear ribosomal RNA gene and the 5' end partial histone-3 gene confirmed the genetic stability of the hybrid progeny. These results also indicated that the hybrid progeny produced consistent symptoms throughout the series of experiments, and maintained their virulence to the differential cultivars screened. Both types of P. teres are prevalent in the south Western Cape Province of South Africa, found on susceptible cultivars often grown within close proximity of each other. In Part 4, a net- and spot-type population were characterised in terms of their population structure using RAPD markers. Samples were collected from infected barley leaves from two separate quadrants in each field, the two quadrants positioned in corners of the fields, diagonal to one another. A total of 65 loci were produced of which 54 were polymorphic. Total gene diversities determined for all loci resulted in mean indices of 0.063 and 0.082 being obtained respectively for the net- and spottype populations. A coefficient of genetic differentiation (Gs) of 0.0149 was obtained between sites within populations while a coefficient (GT) of 0.63 was obtained between the two populations. Genotypic variation revealed 13 distinct multilocus genotypes (haplotypes) in the net-type population while there were 12 in the spot-type population. UPGMA cluster analysis done on the two populations together with six progeny from the mating between a netand spot-type isolate resulted in three main clusters being produced, one for each population and one for the progeny. One isolate collected from the nettype population also contained a unique spot-type RAPD fragment. This suggested that sexual recombination may be taking place between isolates of the net- and spot-type under field conditions. Fungicide application is the most important method used in the control of net blotch in South Africa. In Part 5 the fungicide sensitivities (ICsD values) of 89 monoconidial isolates (46 net-type and 43 spot-type) of P. teres to sterol demethylation inhibiting fungicides were determined, based on the inhibitory effect on radial mycelial growth. The fungicides evaluated were triadimenol, bromuconazole, flusilazole, propiconazole and tebuconazole. Both net- and spot-type isolates revealed strong resistance to triadimenol while flusilazole was shown to be the strongest inhibitor of fungal growth. Spot-type isolates showed a higher resistance than net-type isolates to all five fungicides screened. The ICsD values indicated significant differences between four of the fungicides (triadimenol, tebuconazole, flusilazole and propiconazole). The ICsD values between propiconazole and bromuconazole were not significant. This study suggested that spot-type isolates showed a higher degree of resistance to commercially used fungicides than net-type isolates. The overall conclusion of this study is that the spot-type of P. teres is the pathogen associated with leaf spots of barley in the south western Cape province of South Africa and not P. japonica as earlier reported. Together with the net-type, both types exist as genetically variable populations in this barley production region. Mating between the two types results in sexual progeny that are genetically stable. This implies that barley fields adjacent to one another in which either net- or spot-type susceptible cultivars are being cultivated may lead to sexual progeny being produced. This in turn may lead to an increased rate at which fungal populations may become resistant to commercially used fungicides. It is furthermore suggested that an alternative fungicide seed treatment is used instead of triadimenol due to high resistance of P. teres to this fungicide.
AFRIKAANSE OPSOMMING: Netvlek op gars is een van die belangrikste siektes van hierdie graansoort in die suidelike deel van die Westelike Kaapprovinsie. Dié siekte word veroorsaak deur die swam Pyrenophora teres. Hierdie swam kom voor as twee verskillende tipes, naamlik 'n net-tipe en 'n kol-tipe wat onderskei word op grand van die voorkoms van hulle simptome op garsblare. Hierdie planpatologiese verskil in ag genome, is 'n reeks studies van landboukundige waarde uitgevoer om die effek van geslagtelike rekombinasie tussen die twee tipes te ondersoek. Daarbenewens is ook studies uitgevoer om om die verskil te bepaal tussen plaaslike net- en koltipe populasies ten opsigte van populasiestruktuur en fungisiedsensitiwiteit. Hierdie verhandeling bestaan dus uit 'n versameling afsonderlike publikasies en as gevolg daarvan is daar onvermydelik'n mate van oorvleueling. Rekombinasie is een van die belangrikste evolusionêre kragte betrokke by geslagtelike voortplanting. In plant-swam landboukundige ekostelsels kan dit veroorsaak dat patogene swampopulasies vinniger aanpas by beheerpragramme soos fungisiedtoediening. Die eerste gedeelte in deel 1 van hierdie verhandeling dek die verskillende aspekte van geslagtelike voortplanting van ascomycetes, met spesifieke verwysing na paringstipe gene, vegetatiewe onverenigbaarheid en rekombinasie. Die grootste gedeelte van die oorsig word gewyaan verskeie plantpatologiese aspekte van P. teres,en wys veralop die verskille tussen die twee tipes. In verskeie gevalle word die betekenis van geslagsrekombinasie binne en tussen die net- en koltipe uitgelig. Deur morfologiese kenmerke vir identifikasiedoeleindes te gebruik, is daar baie teenstrydige verslae rakende die identifikasie van blaarvlekisolate in die Westlike Kaapprovinsie van Suid-Afrika. In deel 2 is die korrekte identifikasie eventueel verkry deur gebruik te maak van paringstudies en molekulêre merkers. Dit is bereik nadat enkel ascospore verkry is uit pseudothecia gevorm na in vitro paring plaasgevind het tussen 'n bevestigde P. teres netvlek isolaat uit Denemarke en 'n verteenwoordigende Pyrenophora blaarvlekisolaat van Suid- Afrika. Deur gebruik te maak van versterkte fragmentlengte polimorfisme [AFLP] en RAPD merkers, is rekombinasie gedemonstreer in die nasate wat DNA bandpatrone gehad het wat verskil het van dié van die "ouer" isolate. Patogenisiteitstoetse het ook bevestig dat rekombinasie tydens paring plaasgevind het. Inokulasies is uitgevoer op die verskillende cultivars wat vatbaar is vir die netvlek en blaarvlek vorme. Die twee ouers het tipiese netvlek of blaarvlek simptome veroorsaak, terwyl die nasate hoekige vlekke veroorsaak het op elke cultivar. Toetse vir fungisiedsensitiwiteit deur gebruik van die ergosterol biosintese inhibeerders het gewys dat a.g.v. rekombinasie sekere nasate verhoogde weerstand teen hierdie fungisiedes het. As gevolg van paring en daaropvolgende rekombinasie tussen 'n netvlek isolaat van P. teres en 'n verteenwoordigende blaarvlek isolaat is afgelei dat laasgenoemde P. teres f. maculata is. Vyftien van die netvlek hibried nakomelinge (F1) verkry van die paringstudie in deel 2 is ondersoek in deel 3 om hul lewensvatbaarheid en genetiese stabiliteit te bepaal. Hibried nasate (F1) geïnokuleer op garssaailinge bestaande uit die volgende cultivars: Stirling (soms vatbaar vir net-tipe isolate) , B87/14 en Clipper (albei soms vatbaar vir kol-tipe isolate) het intermediêre simptome op al die cultivars veroorsaak. Akseniese kulture (F1-1) geïsoleer uit blaarletsels gevolg deur herhaalde inokulasie en isolasie (F1-2) op 'n gesonde stel saailinge het dieselfde intermediêre simptome veroorsaak. RAPDs uitgevoer met twee 10-mer inleiers op al die isolate van F1-1 en F1-2 nasate het profiele opgelewer soortgelyk aan dié wat vir F1 isolate verkry is. RAPD molekulêre data het dus gewys dat die hibried nasate van hierdie net x kol paring geneties stabiel was nadat dit onderwerp is aan twee inokulasie en reïsolasie siklusse. Genetiese stabiliteit van die hibried nageslag is bevestig deur filogenetiese analise van die DNA volgorde van die interne getranskribeerde spasieerders (ITS1 en ITS2) reg langs die 5.8S nukluêre ribosomale RNA geen en die 5' end gedeeltelike histoon-3 geen. Hierdie resultate het ook gewys dat die hibried nasate konstante simptome getoon het tydens die hele reeks eksperimente en hulle virulensie behou het vir die kultivars wat getoets is. Beide tipes van P. teres kom algemeen voor in die suidelike deel van die Westelike Kaapprovinsie en word gevind op vatbare cultivars wat dikwels naby mekaar groei. In deel 4 is 'n net- en kol-tipe populasie gekarakteriseer in terme van hulle populasiestruktuur deur gebruik van RAPD merkers. Monsters is versamel van geïnfekteerde garsblare van twee aparte kwadrante in elke saailand. Die twee kwadrante is geplaas in die hoeke van die saailand, diagonaal tot mekaar. 'n Totaal van 65 lokusse is gevorm, waarvan 54 polimorfies was. Die algehele genetiese verskeidenheid bepaal vir alle lokusse, het gelei tot gemiddelde indekse van 0.063 en 0.082 soos gevind vir die net- en kol-tipe populasies. 'n Koëffisiënt van genetiese differensiasie (Gs ) van 0.0149 is gevind tussen gebiede tussen populasies, terwyl 'n koëffisiënt (GT) van 0.63 gevind is tussen die twee populasies. Genotipiese variasie het 13 duidelike multilokus genotipes (haplotipes) getoon in die net-tipe populasie, terwyl daar twaalf was in die kol-tipe populasie. UPGMA groeperingsanalises wat gedoen is op die twee populasies tesame met ses nasate van die paring van 'n net- en koltipe isolaat het tot gevolg gehad dat drie hoof groepe gevorm is, een vir elke populasie en een vir die nasate. Een isolaat wat versamel is, van die net-tipe populasie het 'n unieke kol-tipe RAPD fragment bevat. Dit wys daarop dat geslagtelike rekombinasie in veldomstandighede mag voorkom tussen isolate van die net- en kol-tipe. Fungisiedtoediening is die belangrikste metode wat gebruik word om netvlek in Suid-Afrika te beheer. In deel 5 is die fungisiedsensitiwteit (Ieso waardes) van 89 enkelkonidiale isolate (46 net-tipe en 43 kol-tipe) van P. teres teen sterol demetielasie inhiberende fungisiedes bepaal, op die basis van die onderdrukkende effek op die radiale groei van die miselium. Die volgende fungisiedes is geëvalueer: triadimenol, bromuconazole, flusilazole, propiconazole en tebuconazole. Beide net- en kol-tipe isolate het 'n sterk weerstand teen triadimenol openbaar, terwyl flusilazole gevind is as die sterkste onderdrukker van swamgroei. Kol-tipe isolate het 'n hoër weerstand as die net-tipe isolate teen al vyf fungisiedes wat getoets is, gehad. Die lesowaardes het aangedui dat daar beduidende verskille tussen vier van die fungisiedes IS (triadimenol, tebuconazole, flusilazole en propiconazole). Die leso waardes tussen propiconazole en bromuconazole was nie beduidend nie. Die gevolgtrekking van hierdie studie is dus dat die kol-tipe isolate 'n hoër graad van weerstand teen kommersiëel gebruikte fungisiedes as die net-tipe isolate gehad het. Die algehele gevolgtrekking van hierdie studie is dat die kol-tipe van P. teres, die patogeen is wat geassosieer word met blaarvlekke op gars in die suidwestelike Kaapprovinsie van Suid-Afrika, en nie P. japonica soos voorheen gerapporteer nie. Tesame met die net-tipe, kom altwee tipes voor as geneties veranderlike populasies in hierdie gars verbouingstreek. Paring tussen die twee tipes lei tot geslagtelike nasate wat geneties stabiel is. Dit impliseer dat aangrensende garsvelde waarop net- óf kol-tipe vatbare kultivars verbou word, mag lei tot die produksie van geslagtelike nasate. Dit kan weer lei tot 'n verhoogde tempo waarteen swampopulasies weerstandbiedend teenoor kommersiële fungisiedes raak. Daar word verder ook voorgestel dat alternatiewe fungisied saadbehandelings gebruik word in plaas van triadimenol as gevolg van verhoogde weerstand van P. teres teenoor laasgenoemde.

Malting quality of barley is a complex but important trait for the malting and brewing industries. Several malting quality QTLs have been located on the chromosome 4H of barley. However, the genes associated with these QTLs regions are unknown. The Ac/Ds transposon system was used to dissect these malting quality QTLs. New single-copy Ds insertion lines (TNPs) were generated through sequential re-activation of Ds transposon in the candidate parental lines – TNP-29 and -79, in which the Ds insertion sites were mapped in the vicinity of the malting quality QTLs on chromosome 4H. Reactivation of Ds was carried out by crossing these TNPs with AcTPase expressing plants as well as through in-vitro expression of AcTPase in immature barley embryos. Furthermore, a new PCR based approach – HE-TAIL PCR was devised to expedite the detection of new transposition events. This study will contribute to a better understanding of genes involved in the barley malting quality.
La qualité du malt de l'orge est un trait complexe mais important pour les secteurs du maltage et de l'industrie brassicole. Plusieurs QTLs associés à la qualité du malt sont localisés sur le chromosome 4H de l'orge. Cependant, les gènes associés à ces QTLs sont inconnus. Par conséquent, nous avons utilisé le système de transposons Ac/Ds afin de caractériser ces QTLs. De nouvelles lignées comprenant une insertion unique de l'élément Ds (TNPs) ont donc été produites grâce à la réactivation séquentielle du transposon Ds chez des lignées reconnues comme ayant un élément Ds unique à proximité de ces QTLs. La réactivation de l'élément Ds a été réalisée en croisant les lignées parentales TNP-29 et TNP-79 avec une lignée exprimant l'AcTPase ainsi que par l'insertion par transformation de l'AcTPase chez des embryons immatures obtenus à partir de ces mêmes lignées. De plus, nous avons développé l'approche HE-TAIL PCR afin d'accélérer la détection de nouveaux événements de transposition. Par conséquent, mes travaux contribuent à améliorer notre compréhension des mécanismes impliqués dans la régulation de la qualité du malt de l'orge.

Thesis (PhD)--Stellenbosch University, 2015
ENGLISH ABSTRACT : Diuraphis noxia Kurdjumov (Russian Wheat Aphid; RWA) is a pest of wheat and barley that has spread from its home range in the fertile crescent to most wheat producing countries except Australia. Since its first introduction to South Africa and the USA in the late 20th century, breeding programs for wheat phenotypes resistant to the aphid were put in place. Conventional breeding practices rely on phenotypic screening to verify traits carried by offspring and genetic tools such as marker assisted selection (MAS) have greatly aided this process in speed and accuracy. The size and complexity of the wheat genome, its allopolyploid nature and repetitive elements have, however, posed a challenge to studies on the genetics of this cereal crop. Many studies have focused on chromosome 3B which is the largest of the wheat chromosomes and easily separated from the redundant genomic background by techniques such as flow cytometry. The similarity in size of the remaining chromosomes however, limits the application of flow cytometry to their isolation. Databases such as Grain-Genes (http://wheat.pw.usda.gov/GG2/index.shtml) house marker data from various mapping studies for all wheat chromosomes and in 2014 the International Wheat Genome Sequencing Consortium (IWGSC) completed the draft genome sequence of wheat categorized by chromosome. Sources of resistance (Dn resistance genes) against RWA are located on chromosome 7D. but despite the marker and sequence data available currently, mapping studies specific for the Dn resistance genes are few. Additionally, sequence data available is derived from cultivars susceptible to RWA and is not comprehensively annotated and assembled in many cases. In this study, we demonstrate a novel, combined approach to isolate and characterize the Dn resistance genes through the use of a genetic map constructed from Amplified Fragment Length Polymorphism (AFLP), Expressed Sequence Tag (EST) and microsatellite markers and a physical map constructed from Next Generation Sequencing (NGS) data of ditelosomic chromosomes (7DS and 7DL) isolated by microdissection on the PALM microbeam system. A 122.8 cM genetic map was produced from 38 polymorphic AFLP markers and two ESTs with the microsatellite Xgwm111 as anchor to related genetic maps. Through comparison to maps available on GrainGenes the location of the Dn1 resistance gene was narrowed down to a deletion bin (7DS5-0.36-0.62) on the short arm of chromosome 7D with an AFLP marker (E-ACT/M-CTG_0270.84) mapping closely at 3.5 cM and two ESTs mapping at 15.3 cM and 15.9 cM from Dn1. Isolation of individual chromosome arms 7DS and 7DL using the PALM Microbeam system allowed sequencing of the chromosome without the redundancy of the remainder of the hexaploid genome. Through isolating the chromosome arms in this way, a >80-fold reduction in genome size was achieved as well as a major reduction in repetitive elements. Analysis of the sequencing data confirmed that 7DL is the physically shorter arm of the chromosome though it contains the majority of protein coding sequences.
AFRIKAANSE OPSOMMING : Diuraphis noxia Kurdjumov (Russiese koring-luis; RWA) is « pes wat op koring en gars voorkom. Die pes het vanaf sy tuiste in die midde Ooste na meeste koringproduserende lande behalwe Australië versprei. Sedert die eerste bekendstelling van RWA in Suid Afrika en die VSA in die vroeë 20ste eeu is teelprogramme ten gunste van koring lyne met weerstand teen RWA begin. Tradisionele teelprogramme maak op fisieise observasie van die fenotipe staat om te verifieer of plante in die nageslag die gewenste eienskap dra. Genetiese metodes soos merkerondersteunde seleksie (MAS) versnel hierdie selekteringsproses grootliks. Die grootte en kompleksiteit van die koring genoom asook die polyploïde en herhalende natuur daarvan is « groot hindernis vir genetiese studies van hierdie graangewas. Baie studies het op chromosoom 3B gefokus wat die grootste van die koring chromosome is en dus maklik vanaf die res van die oorbodige genomiese agtergond deur tegnieke soos vloeisitometrie geskei word. Die ooreenkoms in grootte tussen die res van die chromosome bemoeilik die toepassing van vloeisitometrie om hulle te isoleer. Databasisse soos GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml) bevat merker data vanaf verskeie karterings-studies vir al die chromosome en in 2014 het die "International Wheat Genome Sequencing Consortium"(IWGSC) die voorlopige basispaarvolgorde van die koring genoom bekendgestel, gekategoriseer volgens chromosoom. Weerstandsbronne (Dn weerstandsgene) teen RWA kom meestal op chromosoom 7D voor. Ten spyte van merker en basispaarvolgorde data tans beskikbaar is karterings-studies spesifiek tot die Dn gene skaars en basispaarvolgorde data is vanaf kultivars afkomstig wat nie weerstandbiedend teen RWA is nie en waarvan die annotasie en samestelling baie keer nie goed is nie. In hierdie studie demonstreer ons « nuwe, gekombineerde aanslag om die Dn weerstandsgene te isoleer en karakteriseer deur van « genetiese kaart opgestel met "Amplified Fragment Length Polymorphism"(AFLP), "Expressed Sequence Tag"(EST) en mikrosatelliet merkers asook « fisiese kaart saamgestel deur die volgende-generasiebasispaarvolgordebepaling van ditelosomiese chromosome (7DS en 7DL) geïsoleer deur mikrodisseksie met die "PALM Microbeam"sisteem gebruik te maak. « Genetiese kaart van 122.8 cM was met 38 polimorfiese AFLP merkers en twee EST merkers geskep. Die mikrosatelliet, Xgwm111, is ook ingesluit en het as anker vir verwante genetiese-kaarte gedien. Deur vergelyking met genetiese-kaarte op GrainGenes is die posisie van die Dn1 weerstandsgeen vernou na « delesie bin (7DS5-0.36-0.62) op die kort arm van chromosoom 7D met « AFLP merker (EACT/ M-CTG_0270.84) wat ongeveer 3.5 cM vanaf die geen karteer. Die twee EST merkers is 15.3 cM en 15.9 cM vanaf die geen gekarteer. Isolering van die individuele chromosoom arms, 7DS en 7DL, deur van die "PALM Microbeam"sisteem gebruik te maak het basispaarvolgordebepaling van die chromosoom toegelaat sonder die oortolligheid van die res van die hexaploïde genoom. Deur die chromosoom so te isoleer is « >80-maal verkleining in genoom grootte bereik insluitend « groot reduksie in herhalende elemente. Analise van die data vanaf basispaarvolgordebepaling het bevestig dat chromosoom 7D die fisiese kleiner chromosoom is maar dat dit die meerderheid van proteïn koderende basispaarvolgordes bevat.

Nitrite reduction is the third step of the nitrate assimilation pathway in higher plants and is catalysed by nitrite reductase. The whole-plant barley mutants STA1010, STA2760 and STA4169 accumulate nitrite in the leaf after treatment with nitrate and, like the nir1 mutant STA3999 (Duncanson et al, 1993), lack detectable nitrite reductase cross-reacting material in the leaf and root. STA1010, STA2760 and STA4169 carry a recessive mutation in a single nuclear gene, identified as the Nir1 locus. RFLP analysis of the nir1 mutant STA3999 has allowed the Nir1 locus to be mapped to within 0.3cM of the nitrite reductase apoprotein gene, Nii. Studies to confirm the identity of the Nir1 locus as Nii, by establishing the full-length Nii cDNA sequences from STA3999 and from its wild-type cv Tweed for comparative purposes, were unsuccessful as attempts to isolate a Nii cDNA clone from a barley cv Tweed cDNA library yielded only partial-length Nii clones. These nirl mutants display greatly reduced nitrite reductase activity and increased NADH-nitrate reductase activity in the leaf, as compared to wild-type plants, suggesting a regulatory perturbation in the expression of the Nar1 gene. Northern analysis shows that the nir1 mutants possess nitrite reductase apoprotein (nii) transcript of wild-type size (2.3kb) and at approximately wild-type levels. Since nir1 mutants possess a phenotype that might be anticipated for a Nii mutant, it is likely that the nir1 mutation is present in the nitrite reductase apoprotein gene Nii and affects translation of the nii transcript. Studies of barley wild-type cv Golden Promise have demonstrated that nitrite reductase in leaf tissue is up-regulated by a coaction of nitrate and light which acts, at least partly, at the transcriptional level.

Grain protein content (GPC) in wheat has been found to be regulated by the gene NAM-B1. Homologues to the NAM-B1 gene have been found in barley, HvNAM-1 and HvNAM-2. Previous studies have found that base mutations in the NAM-1 gene at base position 544 might have an impact on GPC. Previous studies also found that landrace of barley showed higher GPC than cultivated barley, indicating that plant improvement might have affected base mutations and therefore GPC. I wanted to study if there are any nutritional differences in Nordic barley and if those differences might correlate with haplotypes. Comparisons of barley varieties from four Nordic countries, and two varieties from the US used as low and high GPC controls, did not show any significant differences depending on their origin country and no differences regarding plant improvement status between the countries. When sequencing Nordic barley varieties, five haplotypes were found for the gene HvNAM-1, and two haplotypes for the gene HvNAM-2. A low polymorphism for both genes indicate a strong natural selection for the consensus haplotype which might be preferable for Nordic climate with a short growing season and cold temperatures. Even though it is not clear what is the cause of the low polymorphism in Nordic barley varieties, they showed a generally higher nutrient content than barley varieties of the high GPC and may be suitable for breeding for a yield with a high nutrient content.

Possible maintainer lines were selected from CC XXXII and crossed onto cytoplasmically male-sterile plants. Complete male sterility was maintained in both the F₁ and BC₁ generations of 46.4% of the lines. Four cultivars with maintainer genotypes that were in both normal and msm1 cytoplasm were intercrossed using the male-sterile forms as the female parents. All F₁'s were completely male-sterile. Restoration of male fertility by 22 lines selected from CC XXXII was shown in each case to be due to a single dominant gene. In some lines, restoration was influenced by environment and genetic background. Partial restoration was observed in cultivars in the World Collection and lines selected from CC XXXII. Partial restoration appeared to be due to several genes that were subject to environmental influence. Accumulation of some of these genes increased the amount of restoration. There was no evidence that cytoplasmic factors were passed through the pollen. Twenty-two F₁ hybrids were produced by crossing restorer lines onto male-sterile msm1 lines. The 22 hybrids, their 44 parental restorer and maintainer lines and six check cultivars were grown in a four-replication yield trial. Total yield, 1000-seed weight and hectaliter weight were measured for each plot. All the F₁ hybrids outyielded their midparent values and 17 of the hybrids outyielded their high parents. Half of the F₁'s outyielded the high check cultivar, which yielded about 9,130 kg/ha. Twenty-one F₁'s had greater 1000-seed weights than their midparent values while only 11 F₁'s had greater 1000-seed weights than their high parents. The high check cultivar had the greatest 1000-seed weight, 49.0 gm. The hybrids with the greatest 1000-seed weights were not the hybrids with the greatest yields. Eighteen of the F₁'s had greater hectaliter weights than the midparent values, but only seven had greater hectaliter weights than their high parents. The high check cultivar had the greatest hectaliter weight, 75 kg. The hybrids with the greatest hectaliter weights were not the highest yielding hybrids.

Leaf rust caused by Puccinia hordei is one of the most common and widely distributed rust diseases of barley. Deploying genetic resistance is considered as the most effective and environmentally friendly approach to control this disease. Among 27 designated loci conferring resistance to P. hordei, three genes, namely Rph1, Rph15, and Rph22, have been previously isolated. In this study, the Rph3 gene was isolated using the positional cloning approach. A high-resolution map of the gene constructed using 10,411 F2 plants from six segregating populations delimited the Rph3 locus between two markers covering an interval of 0.024 cM. The physical map revealed that these two closest markers delimited a region of 8.5kb bp in Rph3-carrying cultivars such as cv. Barke and 98kb bp in cultivars lacking Rph3 such as cv. Morex. Two putative genes were determined within the 8.5kb region using FGENESH. The candidate gene for Rph3 was validated by mutational analysis, where four independent point mutations confirmed that ORF2 is Rph3. The resistance gene Rph3 encodes a protein of unknown function that does not belong to any known plant disease resistance protein family. The amino acid sequence showed no sequence homology to any domains/motifs of known function. Predictions using various tools revealed five to seven transmembrane helices in the structure of RPH3. Gene expression was locally induced at the infection site by pathotypes avirulent for Rph3. The current results suggest that the Rph3 gene acts like an executor gene that is regulated by transcriptional activator-like (TAL) effectors. A second study determined that resistance to P. hordei pathotype 5477 P- in the barley line 67:ZBS15 was conferred by two genes interacting in a complementary manner. One of these two genes is Rph3 and the other one was supposed to be Rph5 or an allele of Rph5 or an independent locus in the same genomic region as that of Rph5.

Barley grain is an important commercial crop, being used mainly as an animal feed and in the production of malt for the brewing and distilling industries. The protein and carbohydrate composition of the endosperm (the major storage tissue) determines the grain quality and suitability for different end uses. The differentiation and maturation stages of endosperm development have been extensively studied. However, little is known about the cellular and molecular biology of the syncytial and cellularisation stages of development which occur within the first 8 days post anthesis (DPA). Events occurring during this period of development are particularly important as the overall pattern for the development and structure of the grain is laid down. Patterns of gene expression during the syncytial and cellularisation stages were investigated. A cDNA library was constructed from whole caryopses aged between 1 and 10 DPA. This cDNA library was then differentially screened using mRNA from 3 and 10 DPA caryopses. Northern and dot blot analysis led to the isolation of a number of clones which appear to show variation in level of expression. Partial sequencing of some of these clones and FASTA analysis (Genetics Computer Group, 1991) has shown four clones to have significant identity to sequences in the databases. These clones were clone 27B which showed identity to Ketol acid reductoisomerase (KARI) sequences, clone 16D which showed identity to Caffeoyl CoA-O-methyltransferase (CCoAOMT) sequences, clone 3B which showed identity to sucrose synthase sequences and clone 16B which showed identity to blue copper-binding protein sequences. A further 4 clones which were sequenced showed no significant identity to data base entries following FASTA analysis (Genetics Computer Group, 1991). The temporal and spatial distribution of these clones within tissues of barley caryopses was then analysed by in-situ hybrdisation. None were found to be associated uniquely with the endosperm tissues of barley caryopses. However, there were indications that the expression of the genes represented by the cDNA clones might vary during the course of development. Immunolocation studies utilising a set of JIM (John Innes Monoclonal) antibodies (and MAC207), which recognise carbohydrate epitopes of arabinogalactan proteins (AGP) were also carried out. AGPs have been associated with the plant cell surface and have been ascribed a number of possible functions related to developmental processes. The temporal and spatial distribution of AGPs within barley endosperm was analysed using sections from fixed and embedded barley caryopses and immunolocalisation techniques at the light microscope level. This revealed that at least one AGP epitope, recognised by JIM13, was expressed during early barley grain formation. JIM 13 binding was observed in developing barley caryopses at the beginning of endosperm cellularisation. It was localised to the first anticlinal and then periclinal endosperm cell walls, to the crease region and the nucellar/endosperm boundary. It was not observed in any caryopsis tissue at the earlier stage of syncytial endosperm and unfortunately its distribution could not be studied at later stages of endosperm development because of poor structural integrity within the sections.

Bibliography: leaves 118-132. This thesis examines the structure and gene expression of barley yellow dwarf viruses (BYDVs)-PAV in order to gain a better understanding of the interaction between the virus and the Yd2 resistance gene. The protein products of open reading frame (ORF)3, ORF4 and ORF5 are expressed in bacterial cells, in order to characterise the BYDV-PAV virion-associated proteins. The effect of the Yd2 resistance gene on the expression of the BYDV-PAV viral proteins in infected cells is also studied.

The responses of 92 barley genotypes to selected P. hordei pathotypes was assessed in greenhouse tests at seedling growth stages and in the field at adult plant growth stages to determine known or unknown resistances. On the basis of multipathotype tests, 35 genotypes were postulated to carry Rph2, Rph4, Rph5, Rph12, RphCantala alone or combinations of Rph2 + Rph4 and Rph1 + Rph2, whereas 52 genotypes lacked detectable seedling resistance to P. hordei. Five genotypes carried seedling resistance that was effective to all pathotypes tested, of which four were believed to carry uncharacterised resistance based on pedigree information. Field tests at adult plant growth stages indicated that while 28 genotypes were susceptible, 57 carried uncharacterised APR to P. hordei. Pedigree analysis indicated that APR in the test genotypes could have been derived from three different sources. The resistant responses of seven cultivars at adult plant growth stages were believed to be due to the presence of seedling resistance effective against the field pathotypes. Genetic studies conducted on 10 barley genotypes suggested that ‘Vada’, ‘Nagrad’, ‘Gilbert’, ‘Ulandra (NT)’ and ‘WI3407’ each carry one gene providing adult plant resistance to P. hordei. Genotypes ‘Patty’, ‘Pompadour’ ‘Athos’, ‘Dash’ and ‘RAH1995’ showed digenic inheritance of APR at one field site and monogenic inheritance at a second. One of the genes identified in each of these cultivars provided high levels of APR and was effective at both field sites. The second APR gene was effective only at one field site, and it conferred low levels of APR. Tests of allelism between resistant genotypes confirmed a common APR gene in all genotypes with the exception of ‘WI3407’, which based on pedigree information was genetically distinct from the gene common in ‘Vada’, ‘Nagrad’, ‘Patty’, ‘RAH1995’ and ‘Pompadour’. An incompletely dominant gene, Rph14, identified previously in an accession of Hordeum vulgare confers resistance to all known pathotypes of P. hordei in Australia. The inheritance of Rph14 was confirmed using 146 and 106 F3 lines derived from the crosses ‘Baudin’/ ‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks from F3 lines using diversity array technology (DArT) markers located Rph14 to the short arm of chromosome 2H. Polymerase chain reaction (PCR) based marker analysis identified a single simple sequence repeat (SSR) marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cM in the populations ‘Baudin’/ ‘PI 584760’and ‘Ricardo’/‘PI 584760’, respectively. Seedlings of 62 Australian and two exotic barley cultivars were assessed for resistance to a variant of Puccinia striiformis, referred to as BGYR, which causes stripe rust on several wild Hordeum species and some genotypes of cultivated barley. With the exception of six Australian barley cultivars and an exotic cultivar, all displayed resistance to the pathogen. Genetic analyses of six Australian barley cultivars and the Algerian barley ‘Sahara 3771’, suggested that they carried either one or two major seedling resistance genes to the pathogen. A single recessive seedling resistance gene, Bgyr1, identified in ‘Sahara 3771’ was located on the long arm of chromosome 7H and flanked by restriction fragment length polymorphism (RFLP) markers wg420 and cdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using a doubled haploid population derived from the cross ‘Clipper’/‘Sahara 3771’ identified two major QTLs on the long arms of chromosomes 3H and 7H that explained 26 and 18% of total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to the seedling resistance gene Bgyr1. The second QTL was concluded to correspond to a single adult plant resistance gene designated Bgyr2, originating from cultivar ‘Clipper’.

Doctor of Philosophy
The responses of 92 barley genotypes to selected P. hordei pathotypes was assessed in greenhouse tests at seedling growth stages and in the field at adult plant growth stages to determine known or unknown resistances. On the basis of multipathotype tests, 35 genotypes were postulated to carry Rph2, Rph4, Rph5, Rph12, RphCantala alone or combinations of Rph2 + Rph4 and Rph1 + Rph2, whereas 52 genotypes lacked detectable seedling resistance to P. hordei. Five genotypes carried seedling resistance that was effective to all pathotypes tested, of which four were believed to carry uncharacterised resistance based on pedigree information. Field tests at adult plant growth stages indicated that while 28 genotypes were susceptible, 57 carried uncharacterised APR to P. hordei. Pedigree analysis indicated that APR in the test genotypes could have been derived from three different sources. The resistant responses of seven cultivars at adult plant growth stages were believed to be due to the presence of seedling resistance effective against the field pathotypes. Genetic studies conducted on 10 barley genotypes suggested that ‘Vada’, ‘Nagrad’, ‘Gilbert’, ‘Ulandra (NT)’ and ‘WI3407’ each carry one gene providing adult plant resistance to P. hordei. Genotypes ‘Patty’, ‘Pompadour’ ‘Athos’, ‘Dash’ and ‘RAH1995’ showed digenic inheritance of APR at one field site and monogenic inheritance at a second. One of the genes identified in each of these cultivars provided high levels of APR and was effective at both field sites. The second APR gene was effective only at one field site, and it conferred low levels of APR. Tests of allelism between resistant genotypes confirmed a common APR gene in all genotypes with the exception of ‘WI3407’, which based on pedigree information was genetically distinct from the gene common in ‘Vada’, ‘Nagrad’, ‘Patty’, ‘RAH1995’ and ‘Pompadour’. An incompletely dominant gene, Rph14, identified previously in an accession of Hordeum vulgare confers resistance to all known pathotypes of P. hordei in Australia. The inheritance of Rph14 was confirmed using 146 and 106 F3 lines derived from the crosses ‘Baudin’/ ‘PI 584760’ (Rph14) and ‘Ricardo’/‘PI 584760’ (Rph14), respectively. Bulk segregant analysis on DNA from the parental genotypes and resistant and susceptible DNA bulks from F3 lines using diversity array technology (DArT) markers located Rph14 to the short arm of chromosome 2H. Polymerase chain reaction (PCR) based marker analysis identified a single simple sequence repeat (SSR) marker, Bmag692, linked closely to Rph14 at a map distance of 2.1 and 3.8 cM in the populations ‘Baudin’/ ‘PI 584760’and ‘Ricardo’/‘PI 584760’, respectively. Seedlings of 62 Australian and two exotic barley cultivars were assessed for resistance to a variant of Puccinia striiformis, referred to as BGYR, which causes stripe rust on several wild Hordeum species and some genotypes of cultivated barley. With the exception of six Australian barley cultivars and an exotic cultivar, all displayed resistance to the pathogen. Genetic analyses of six Australian barley cultivars and the Algerian barley ‘Sahara 3771’, suggested that they carried either one or two major seedling resistance genes to the pathogen. A single recessive seedling resistance gene, Bgyr1, identified in ‘Sahara 3771’ was located on the long arm of chromosome 7H and flanked by restriction fragment length polymorphism (RFLP) markers wg420 and cdo347 at genetic distances of 12.8 and 21.9 cM, respectively. Mapping resistance to BGYR at adult plant growth stages using a doubled haploid population derived from the cross ‘Clipper’/‘Sahara 3771’ identified two major QTLs on the long arms of chromosomes 3H and 7H that explained 26 and 18% of total phenotypic variation, respectively. The QTL located on chromosome 7HL corresponded to the seedling resistance gene Bgyr1. The second QTL was concluded to correspond to a single adult plant resistance gene designated Bgyr2, originating from cultivar ‘Clipper’.

Small RNA are short, 18-25 nt molecules that regulate gene expression in plants and animals. Two main types, microRNA (miRNA) and short interfering RNA (siRNA) perform this regulation by transcript silencing, translation inhibition, DNA methylation and chromatin remodeling. This thesis is an investigation into small RNA activity in archaeological plant material, specifically barley grain from Qasr Ibrim, a multi-period archaeological site in southern Egypt. It is of particular interest due to its unusual phenotype, suggestive of stunted development that is unexpected in a staple, domesticated cultivar, and the unusual level of DNA and RNA preservation attributable to the extremely arid climate at the site. The research presented here is a comparative analysis of small RNA profiles and epigenetic states of Qasr Ibrim barley and modern, unstressed counterparts. It concludes that differential microRNA and epigenetic profiles are the result of stress response, adaptation, dormancy and / or viral infection unique to the archaeological grain. The primary method of investigation was generation of small RNA sequence data using the Illumina GAIIx platform. This was followed by extensive bioinformatic analysis (RNA diagenesis patterns, miRNA prediction, siRNA target prediction and small genome in silico reconstruction) the results of which were in turn validated experimentally (genomic methylation states, locus-specific methylation analysis and direct miRNA detection). The research represents a twofold contribution to knowledge: first, proof-of-principle that biologically meaningful archaeological RNA can be extracted despite its relative instability to DNA, and second that a unique miRNA profile and epigenetic state is detectable in this particular cultivar of archaeological barley.

[Truncated abstract] Under prolonged wet and damp conditions, barley grain with low dormancy can germinate precociously, a condition known as preharvest sprouting that causes a number of detrimental effects in grain quality. In particular, preharvest sprouting renders the grain unsuitable for malting. The aim of this study was to take a genomics approach to identify and characterise candidate genes that could be linked to the control of seed dormancy in barley. This thesis developed a bioinformatic strategy that exploited the availability of gene sequences with functional evidence in the model species of Arabidopsis and rice. The bioinformatic strategy integrated phenotypic data (QTL data) and comparative genomics for a targeted approach in identifying candidate genes with a high probability of having a conserved function in cereals. This bioinformatic study identified two candidate genes ERA1 and ABI2 with strong evidence for a role in seed dormancy based on their function in Arabidopsis in abscisic acid (ABA) signal transduction and their co-location to seed dormancy QTLs in Arabidopsis, rice and wheat. In order to establish whether the candidate genes mapped to seed dormancy QTLs in barley, QTL analyses were performed on a double haploid population, not previously studied, developed from a cross between Stirling, a major Australian malting cultivar, and Harrington, a major Canadian malting cultivar. This cross was specifically chosen for this study, as elucidation of chromosomal regions associated with seed dormancy in the background of a malting cultivar would make a significant contribution for the malting industry. '...' Identification of a seed dormancy QTL on the long arm of 3H, in a region syntenic to the wheat chromosome locations of ESTS aligning to the ERA1 and ABI2 genes, laid the foundation for physical and genetic mapping of the candidate genes to investigate whether the genes co-located to the QTL on 3H. Physical mapping of the genes in wheat barley addition lines confirmed their positions on the long arm of 3H. Genetic mapping of the ERA1 gene was performed using a CAPS marker developed in this thesis. The genetic mapping of the ERA1 gene did not place the gene within either of the minor QTLs on 3HL, although segregation distortion may have influenced the map position of this gene. Further investigation is required to resolve the positioning of the ERA1 and ABI2 genes in relation to the 3H seed dormancy QTL. The main outcomes of this study have been 1) identification of candidate genes for further study; 2) identification of QTLs on the long arm of 3H that were previously unknown; 3) demonstration of the potential differences in dormancy that can be achieved through the use of specific gene combinations, highlighting the importance of minor genes and the epistatic interactions that occur between them and; 4) the development of a CAPS marker for the ERA1 gene, which can be used to track the gene in barley breeding programs to observe its association with important agronomic traits. This thesis also pioneered the implementation of several new technologies including multiplex-ready PCR (Hayden et al. 2008) for fluorescence–based SSR genotyping and QTLNetwork (Yang et al. 2008) for statistical analysis of QTLs. Seed dormancy is a complex trait and is likely to involve the interplay of a number of genes that have a role in other developmental and regulatory processes.

Plants that are heterozygous for genetic markers but do not produce segregating progeny may be suspected of carrying a mutation conditioning apomixis. Seed stocks in which heterozygous plants could be identified phenotypically were treated with a chemical mutagen. These seed stocks were heterozygous for recessive genetic markers, and/or heterozygous for a chromosome translocation. Spikes from heterozygous M1 plants were harvested and seeded in bulk. Spikes from heterozygous M2 plants were harvested and planted in M3 rows. The M3 rows were observed for the absence of segregating progeny and/or were observed cytologically for the presence of a heterozygous translocation. M3 rows not segregating for the genetic markers were crossed onto plants homozygous for the genetic markers. The F1 progenies were observed for an expected ratio of 1 normal: 1 recessive plant. All nonsegregating lines were found to be non-heterozygous. These lines most likely occurred due to seed and pollen contamination or were the result of crossing over between genetic markers. Fertile M2 plants were harvested from the treated heterozygous translocation seed stock. Normally, barley plants heterozygous for a translocation will produce semisterile spikes. Plants that would normally be semisterile but are fertile could be carrying a mutation conditioning apomixis. Progeny of the fertile M2 plants were examined cytologically for the presence of the heterozygous translocation. All selected lines contained the normal seven pairs of chromosomes and were the result of seed or pollen contamination. Seed stocks which could eliminate the problem of contamination in future experiments were developed and discussed. Haploviable mutants closely linked with the male sterile locus, msg2, were isolated in these seed stocks. Haploviable mutants are recognized by upset genetic ratios of alleles linked with the mutant. Selfed progenies of plants carrying a haploviable mutation contained fertile and male sterile plants in about a 1:1 ratio. Mostly male sterile progenies were obtained when plants heterozygous for the haploviable mutant and the male sterile allele were crossed onto male sterile plants. Four lines containing haploviable mutants were evaluated for their usefulness in producing all male sterile lines for hybrid barley production.

Barley row-type describes the number of grains present at a node on the barley spike. Two forms exist amongst cultivated barley: two-rowed with only the central of three spikelets fertile producing a single grain at a node and six-rowed with all three spikelets fertile, producing three grain at a node. Twelve regions of the barley genome have been associated with the row-type character with specific genes identified at three loci, VRS1, VRS4 and INT-C. Advancements in the understanding of the genetic control underpinning barley row-type enables the identification of potential mechanisms for improving yield and yield architecture within the cereals.  This study used genetic linkage mapping in segregating F2 populations to refine the genetic location of the row-type locus, VRS3, to 16 candidate genes on barley chromosome 1H. Sequencing candidate loci in 32 vrs3 induced mutant alleles identified VRS3 to be a highly conserved JmjC histone demethylase, with two natural alleles within European cultivated barley. VRS3 was further characterised as a potential means of improving grain uniformity within cultivated six-rowed barley, through phenotypic assessment of grain size in varying allele combinations of VRS3, VRS1 and INT-C. The addition of six-rowed alleles at these loci was found to improve balance between central and lateral grain parameters, resulting in a more uniform grain sample. Analysis of gene expression found Vrs3 to be constitutively expressed across a diverse panel of barley tissues. Moreover, detailed study within the developing inflorescence suggests a role for Vrs3 in the regulation of the row-type genes VRS1 and INT-C.

The fungal pathogen Puccinia hordei Otth. is the causal agent of the leaf rust disease on barley. It is an economically important disease worldwide. The pathogen occurs in all barley growing regions of Australia. The present study assessed a diverse African barley germplasm for resistance to this disease in the seedling (10 pathotypes) and adult (one pathotype) growth stages which resulted in identification of new sources of high and moderate adult plant resistance (APR) different from the known APR genes Rph20 and Rph23. In addition, known seedling resistance genes were also postulated. The African barley collection was also assessed for seedling resistance to three pathotypes of the stem rust pathogen P. graminis tritici (Pgt). New seedling resistance for Pgt that lacked the molecular markers for the known Pgt resistant genes Rpg1 and Rpg5 were identified in 18 accessions. Genetic and molecular characterisation was conducted in new APR to P. hordei identified in six barley lines, four from Africa and two from Uruguay. Using the Diversity Array Technology (DArT) molecular markers a monogenic resistance locus identified in a line was mapped on chromosome 6H based on selective genotyping. Using DArT molecular markers, the first designated seedling leaf rust resistance gene Rph1 was mapped at 8.85 – 10.48 cM interval on the short arm of chromosome 2H using two mapping populations. Genetic, cytogenetic and molecular characterisation was carried out on two barley lines displayed resistance to the wheat yellow rust pathogen P. striiformis tritici (Pgt) and the barley grass yellow rust (BGYR) pathogen P. striiformis pseudo-hordei revealed that each line carried two and three effective genes against Pst and BGYR, respectively, with two common genes effective against both pathogens in each genotype. A locus conferring hypersensitive resistance to BGYR was mapped between 11.76 and 18.57 cM in chromosome 1H.

Rhynchosporium commune is one of the most destructive fungal pathogens of barley worldwide. It causes scald, responsible for reduced grain quality and yield losses of up to 40%. This project aimed to identify genetic resistance in barley using two different approaches: an effector approach through the identification of important pathogen virulence factors and their barley targets, and a genomics association approach. Numerous secreted effectors have been identified in many phytopathogens including R. commune. Rrs1 resistance, recognising the R. commune avirulence protein - AvrRrs1 (NIP1) has been deployed in the field to prevent infection but has soon proven ineffective. R. commune has managed to overcome this resistance by alteration or deletion of the NIP1 gene as it is not essential for pathogenicity. However, our field trial data suggests that Rrs1 remains an important component of resistance to R. commune in the field. Resistance genes recognising more essential Avr genes are likely to be more durable and as a consequence, the discovery of novel R. commune Avr genes is fundamental for the implementation of an integrated pest management approach to prevent this disease. Recent sequencing of the R. commune genome allowed identification of putative effectors. Expression of 26 potential effectors with low sequence variability in 9 sequenced R. commune strains have been analysed during barley infection. The best genes were selected for gene disruption and individual expression in barley cultivars and landraces using the Barley Stripe Mosaic Virus (BSMV) – based expression system to see if they are recognised by the plant. The work also focused on candidate effectors with putative functions. A putative protease inhibitor was chosen for functional characterisation but its function and importance for pathogenicity could not be confirmed. In addition, high amount of the candidate protein appeared to be toxic for barley and Nicotinana benthamiana. Two SA (salicylic acid)-related putative effectors were also chosen for further characterisation and revealed a direct link between the SA pathway of barley and R. commune. The results of this project suggest that R. commune might be able to manipulate the SA pathway of the host confirming the existence of a biotrophic phase of the fungus. The genomics association approach to identify resistance genes against R. commune in barley used a Genome Wide Association Scan (GWAS) using a combination of three years of disease nursery field trial data for a collection of over 500 elite spring barley cultivars. This analysis identified a number of quantitative trait loci (QTL) in barley genome regions previously shown to contain major resistance genes such as Rrs1 on chromosome 3H, Rrs2 on chromosome 7H, Rrs3 on chromosome 4H, Rrs4 on chromosome 3H, Rrs13 on chromosome 6H, Rrs14 on chromosome 1H and Rrs16 on chromosome 4H, as well as novel QTL. The work was focused on Rrs1 resistance. R. commune strains producing a type of NIP1 effector, recognised by barley lines containing Rrs1, were used to confirm the resistance in predicted Rrs1 barley cultivars. The Rrs1 interval has been narrowed down to 3 Mbp, and high resolution mapping led to the identification of 3 SNP markers which perfectly discriminated Rrs1Rh4 lines from susceptible lines. These diagnostic markers will provide a useful breeding tool for improving the design of new varieties allowing the incorporation of the Rrs1 resistance. This research takes us a step closer towards cloning the first barley major resistance (R) gene against R. commune, which is likely to be present only in Rrs1 lines and have a kinase domain very similar to the one in a putative wall associated kinase found within the Rrs1 interval in the genome assembly of susceptible cultivar Morex. It will also help us to better understand R. commune-barley pathosystem and to identify further R genes.

Rust diseases caused by Puccinia spp. are specialised to different economically important crops such as wheat and barley and can cause significant crop failure when severe. For many decades, genetic resistance has proven effective in protecting crops against rust pathogens. However, the ability of rust pathogens to evolve rapidly can lead to the emergence of new virulent races that can overcome genetic resistance. Therefore, the identification of new durable sources of resistance is essential for crop production and food security. Barley is a diploid crop with a reference genome sequence that is a near non-host to some non-adapted rust pathogens. Thus, the barley-Puccinia pathosystem is amenable for studies of the genetic architecture of resistance to rust pathogens. This thesis aims to determine the genetic basis of specificity of resistance in different barley-Puccinia pathosystems and to identify valuable sources of resistance in barley that can be used for genetic improvement of cereal crops. Chapter one contains a detailed review of the literature, addressing aspects of biology of barley and rust pathogens as well as mechanisms of host and non-host resistance. Chapter two investigates the genetic architecture of non-host resistance in barley using diverse Puccinia isolates and identifies non-host resistance (NHR) QTL that are suitable for either map-based or rapid cloning approaches. Chapter three examines the isolate specificity and polygenic inheritance of resistance in barley to different P. striiformis isolates. Chapter four generates a high-density linkage map using DArT-Seq markers with the aim of mapping loci for seedling and adult plant resistance to adapted stripe, leaf and stem rust pathogens. Chapter five delivers an overall conclusion based on the findings of the thesis and includes future paths and perspectives.

In commercial barley cultivars meiotic crossover (CO) distribution is skewed to the distal regions of the paired chromosomes. This restricts recombination to these regions thereby reducing the potential genetic variation that can be exploited in plant breeding programs. The aim of this project was to develop experimental strategies that will enable the frequency and distribution of meiotic crossovers to be modified in order to generate progeny with novel gene combinations. Treatment with the histone deacetylase inhibitor trichostatin A, led to significant modifications in crossover frequency in a concentration-dependent manner with lower concentrations not greatly impacting fertility, allowing for the extraction of fertile seeds. The genetic screening of a treated marker population at The James Hutton Institute (JHI), demonstrated subtle but significant shifts in the distribution of meiotic recombination, indicating that modifying recombination through chemicals applied via the transpiration stream is indeed feasible in barley and hence, possibly in other cereals. The cytological study of a barley desynpatic mutant \(des8\) in collaboration with JHI revealed that synapsis is normal despite reduced chiasma frequency. Genetic mapping studies are in progress to identify the mutant gene responsible for this phenotype which will help us to improve our current knowledge of meiosis in barley.

Pyrenophora teres f. teres is a fungal pathogen that causes barley net form net blotch. To evaluate the genetics of resistance in barley, a RIL population was developed using resistant barley lines CI5791 and Tifang and tested against a global collection of nine P. teres f. teres isolates. QTL analysis indicated that CI5791 resistance mapped to chromosome 6H and was effective against all isolates. Additionally, CI5791 harbored resistance on chromosome 3H effective against two Japanese isolates. Tifang also had resistance that mapped to 3H and was effective against four of the isolates. To evaluate the genetics of virulence in P. teres f. teres, a fungal population was developed and evaluated against ten barley lines. 19 unique QTL were identified on 12 different linkage groups. 1 or 2 major loci were identified for a few of the barley lines whereas for most lines, virulence was contributed by several loci.

Plants have a remarkable ability to resist the majority of pathogenic microbes they encounter. As such, they are described as nonhosts. Nonhost resistance is often conceptualised as a qualitative separation from host resistance. Classification into these two states is generally facile, as they fail to fully describe the range of states that exist in the transition from host to nonhost. This poses a problem when studying pathosystems that cannot be classified into either of these categories due to their intermediate status relative to the two extremes. Therefore, the terms intermediate host and intermediate nonhost have been proposed to describe pathosystems in the evolutionary transition between host and nonhost status. At present, a significant amount of research exists into the molecular genetics of host and nonhost pathosystems but very little is known about intermediate systems. The work in this Ph. D. thesis focuses on the interaction of barley with Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, as an intermediate host pathosystem. The first research chapter describes the development of two microscopic phenotypic assays used to quantify P. striformis f. sp. tritici in barley leaves challenged with the pathogen. These assays are then used to screen a large panel of barley accessions to define the intermediate host status of barley relative to a host pathosystem. Subsequently, these assays play a key role in determining that the genetic architecture of resistance in barley is underpinned by three major effect resistance loci: Rpst1, Rpst2, and Rpst3. Using a combination of classical map-based genetics and contemporary genomics information I identify a candidate NLR gene underlying Rpst2 resistance on chromosome 7HL. Furthermore, I show that distinct genes condition host and nonhost resistance in barley by mapping the host resistance gene, rps2 to chromosome 2HL.

[Truncated abstract] Zinc deficiency in crops is the most widespread micronutrient deficiency, with about 50% of the cereal-growing areas worldwide containing low levels of plant-available Zn. Zinc plays multiple key roles in different metabolic and physiological processes; its deficiency in crops reduces not only grain yield, but also the nutritional quality of grains. Insufficient micronutrient intake, particularly Zn and Fe, afflicts over 3 billion people in the world, mainly in developing countries. Increasing the amount of Zn in food crops can contribute to improving the Zn status of people. Furthermore, Zn-dense seeds have agronomic benefits, resulting in greater seedling vigour, bigger root system and higher crop yield when sowed to soils with low plant-available Zn. Enhancing nutrient content and nutritional quality of crops for human nutrition is a global challenge currently, but it was mostly ignored during the breeding process in the past. There is a significant genotypic variation for seed Zn accumulation in several crops (including barley) which could be exploited in the breeding programs to produce genotypes with higher seed Zn concentration and content. However, the progress in Zn efficiency until now has mainly relied on conventional plant breeding approaches that have had limited success. Therefore, reliable alternative methods are required. Enhancing mineral nutrition through plant biotechnology may be a sustainable and beneficial approach in developing Zn-dense seeds in the staple crops. ... This DNA band was sequenced and converted into a simple sequence-specific PCR-based marker, which was designated as SZnR1 (seed Zn-regulator1). The developed marker is very easy to score, is inexpensive to run and amenable for a large number of plant samples. The successful development of SZnR1 molecular marker linked to chromosome region associated with seed Zn concentration and content using MFLP in this study illustrates the advantage of this technique over some other DNA fingerprinting methods used for identification of molecular markers for marker-assisted selection (MAS). In conclusion, the greater Zn efficiency of Sahara over Clipper under sufficient Zn supply may be attributed to its higher uptake of Zn. It appears that soil-based pot experiments under controlled condition may offer potential improvements over field experiments in screening for seed Zn accumulation. Shoot and seed Zn concentration and content can be used to diagnose the Zn statues of barley genotypes, and may be a useful selection criterion for Zn efficiency in large populations like doubled-haploid populations aimed at developing molecular markers for Zn efficiency. Identified QTLs influencing seed Zn concentration were repeatable in the field and glasshouse conditions, suggesting their robustness across environments as well as their value in marker-assisted selection. The developed PCR-based marker SZnR1 and other molecular markers associated with the QTLs on the short and long arms of chromosome 2H have the potential to be used for marker-assisted selection in breeding for Zn-dense seed in barley.