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Quinoa is an important, highly nutritional grain crop in the Andean region of South America. DNA markers and linkage maps are important tools for the improvement of underdeveloped crops such as quinoa. The objectives of this study were to (i) develop a new set of SSR markers to augment the number of SSR markers available in quinoa, and (ii) construct a new genetic linkage map of quinoa based on SSRs using multiple recombinant-inbred line (RIL) populations. Here we report the development of 216 new polymorphic SSR markers from libraries enriched for GA, CAA, and AAT repeats, as well as 6 SSR markers developed from BAC-end sequences (BES-SSRs). Heterozygosity (H) values of the SSR markers ranged from 0.12 to 0.90, with an average value of 0.56. These new SSR and BES-SSR markers were analyzed on two RIL mapping populations (designated Population 1 and Population 40), each obtained by crossing Altiplano and coastal ecotypes of quinoa. Additional markers, including AFLPs, two 11S seed storage protein loci, a SNP, and the nucleolar organizing region (NOR), were also analyzed on one or both populations. Linkage maps were constructed for both populations. The Population 1 map contains 275 markers, including 200 SSR and 70 AFLP markers, as well as five additional markers. The map consists of 41 linkage groups (LGs) covering 913 cM. The Population 40 map contains 68 markers, including 62 SSR and six BES-SSR markers, and consists of 20 LGs covering 353 cM. Thirty-nine anchor markers common between both maps were used to combine 15 Population 1 LGs with 13 Population 40 LGs. The resulting integrated map consists of 13 LGs containing 140 SSR, 48 AFLP, four BES-SSR, one SNP, and one NOR marker spanning a total of 606 cM. A high level of segregation distortion was observed in both populations, indicating possible chromosomal regions associated with gametophytic factors or QTLs conferring a selective advantage under the particular growing conditions. As these maps are based primarily on easily-transferable SSR markers, they are particularly suitable for applications in the underdeveloped Andean regions where quinoa is grown.

BACKGROUND: Black rot is a destructive bacterial disease causing large yield and quality losses in Brassica oleracea. To detect quantitative trait loci (QTL) for black rot resistance, we performed whole-genome resequencing of two cabbage parental lines and genome-wide SNP identification using the recently published B. oleracea genome sequences as reference. RESULTS: Approximately 11.5 Gb of sequencing data was produced from each parental line. Reference genome-guided mapping and SNP calling revealed 674,521 SNPs between the two cabbage lines, with an average of one SNP per 662.5 bp. Among 167 dCAPS markers derived from candidate SNPs, 117 (70.1%) were validated as bona fide SNPs showing polymorphism between the parental lines. We then improved the resolution of a previous genetic map by adding 103 markers including 87 SNP-based dCAPS markers. The new map composed of 368 markers and covers 1467.3 cM with an average interval of 3.88 cM between adjacent markers. We evaluated black rot resistance in the mapping population in three independent inoculation tests using F₂:₃ progenies and identified one major QTL and three minor QTLs. CONCLUSION: We report successful utilization of whole-genome resequencing for large-scale SNP identification and development of molecular markers for genetic map construction. In addition, we identified novel QTLs for black rot resistance. The high-density genetic map will promote QTL analysis for other important agricultural traits and marker-assisted breeding of B. oleracea.

O eucalipto é amplamente cultivado em diversos países, dentre os quais, o Brasil se destaca pela sua alta produção. A cultura tem grande importância comercial e atende à uma ampla variedade de setores do mercado, entre eles o de celulose. Apesar disso, a cultura ainda está nas fases iniciais de domesticação devido, principalmente, ao seu longo ciclo reprodutivo e tempo de rotação, uma vez que os cortes são feitos entre 5 e 15 anos. A aplicação de tecnologias de marcadores moleculares é uma proposta promissora para acelerar o melhoramento do eucalipto. Com o desenvolvimento de metodologias modernas de sequenciamento tornou-se acessível a obtenção de grande quantidade de marcadores a baixo custo. Uma das aplicações de tais marcadores é a construção de mapas genéticos de ligação, os quais permitem a caracterização genética de caracteres quantitativos, além de estudos comparativos entre populações e o auxílio na montagem de genomas. No presente trabalho, objetivou-se a construção de um mapa genético integrado em uma progênie F1 segregante com 200 indivíduos, proveniente do cruzamento entre Eucalyptus grandis e Eucalyptus urophylla. Para identificação dos marcadores, foi realizado o ressequenciamento do genoma completo (WGS) dos genitores e a genotipagem por sequenciamento (GBS) da progênie. A metodologia de construção de mapa foi adaptada para o conjunto de dados obtido, que apresenta grande quantidade de marcadores do tipo SNP, pouco informativos e com maior probabilidade de erro comparado com os marcadores tradicionais mais utilizados nos últimos anos. Para isso foram propostas duas estratégias: i) utilização da posição dos marcadores no genoma de referência para auxílio na ordenação dos marcadores no mapa; ii) alteração do parâmetro de probabilidade de erro da abordagem implementada no software Onemap. O mapa obtido apresentou padrão de taxa de recombinação semelhante a outros mapas construídos para eucalipto. O mapa apresentou tamanho total de 1471.91 cM e 1512 marcadores, com distância média entre eles de 1.85 cM. Os marcadores formaram 11 grupos de ligação, que corresponderam aos cromossomos do genoma de referência. Em média, foram cobertos 96.8% dos cromossomos. Também foram agrupados, junto aos 11 grupos, 61 marcadores localizados em outros scaffolds no genoma de referência, os quais podem servir para elucidação na montagem destes. Utilizando as estratégias propostas, foi obtido um mapa integrado adequado para o experimento em questão, considerando o tamanho da população de mapeamento. / The eucalyptus is widely cultivated in several countries, among which Brazil is highlighted by its high production. This culture has great comercial importance and supplies a wide variety of markets, including cellulose. However, the culture is still in the early stages of domestication due, mainly, to its long reproductive cycle and rotation time, since cuts are made between 5 and 15 years. The application of molecular marker technologies is a promising proposal to accelerate the improvement of eucalyptus. By the development of modern sequencing methodologies it was possible to obtain a large quantity of markers at low cost. One of the applications of such markers is the construction of genetic linkage maps, which allow the genetic characterization of quantitative traits, as well as comparative studies between populations and the support in the assembly of genomes. In the present work, the aim was to construct an integrated genetic map in a segregating F1 progeny with 200 individuals, derived from the cross between Eucalyptus grandis and Eucalyptus urophylla. For the identification of the markers, it was performed a complete genome re-sequencing (WGS) of the parents and genotyping-by-sequencing (GBS) of the progeny. The mapping methodology was adapted to the obtained data set, which presents a large amount of SNP-type markers, with little information and with a greater probability of error compared to the most used traditional markers in the last years. For this, two strategies were proposed: i) use of the position of the markers in the reference genome to aid in the ordering of the markers on the map; ii) change of the error probability parameter in the approach implemented in the software Onemap. The obtained map showed recombination rate pattern similar to other maps constructed for eucalyptus. The map presented a total size of 1471.91 cM and 1512 markers, with a mean distance between them of 1.85 cM. The markers formed 11 linkage groups, which corresponded to chromosomes of the reference genome. On average, 96.8 % of chromosomes were covered. 61 markers located in other scaffolds in the reference genome were grouped with the 11 groups. They may serve to elucidate the assembly of these. Using the proposed strategies, a suitable integrated map was obtained for the present experiment, considering the size of the mapping population.

Identification of allelic variation through quantitative trait loci (QTL) mapping offers possibilities for the improvement of quantitatively inherited traits. This requires a genetic map along with the phenotypic characterization of a mapping population. A doubled haploid (DH) Polo X Topas population consisting of 194 lines and a recombinant inbred line population of 92 lines was developed. Individual genetic maps derived from each population were integrated into a consensus map. The DH-based genetic map was used for QTL mapping. The DH-based map was comprised of 620 loci that were assembled into 19 linkage groups that were anchored to the B. napus chromosomes. The DH-based map covered 2244.1 cM genomic distance with an average marker interval of 3.7 cM. The DH population was phenotyped in four environments with each line replicated twice in a randomized complete block design. Days to flowering was recorded and oil content and fatty acid composition were determined using Near Infrared spectroscopy (NIR) and Gas Chromatography, respectively. Fourteen QTL were identified for oil content, 33 QTL for palmitic acid content, 18 QTL for stearic acid content, 21 QTL for oleic acid content, 20 QTL for linoleic acid content, 23 QTL for linolenic acid content, 16 QTL for arachidic acid content and 14 QTL for flowering time. Oil content QTL were identified on five linkage groups, A3, A10, C1, C5, and C6. An oil content QTL, qOIL-A10c appeared in all four environments, whereas qOIL-A10a appeared in only one environment but explained 26.99% variation. The oil content in the population ranged from 35% to 55.5% with the parents having values of 42% to 46%. Two genomic regions on C3, with map positions at 147.83 cM and 154.55 cM harbored QTL (rQTL) for all the fatty acids studied. The additive effects of the rQTL reveal a correlation pattern which is supported by the phenotypic correlation observed between the fatty acids. This suggests rQTL have role in the fatty acid composition and possibly determine total seed oil content. The rQTL and flanking markers of the identified QTL offer utility in further development of B. napus. / October 2015

Almond (Prunus dulcis) is the most important nut crop in terms of world production. Due to its health benefit and high nutritional value the consumption and world supply of almond is increasing. To remain competitive in the world market, the Australian almond breeding program was established to produce cultivars with better adaptation to Australian conditions. As part of this program an almond mapping population consisting of 93 F₁ progeny derived from a cross between the American cultivar ‘Nonpareil’ (NP) and the European self-compatible cultivar ‘Lauranne’ (LA) was produced to construct the genetic linkage maps. The first almond linkage map developed prior to the commencement of this project failed to produce the eight linkage groups similar to the basic chromosome number of almond (x = 8) and many large gaps were also observed on the linkage groups. Therefore, more markers were needed to saturate the maps. Microsatellite markers are considered one of the best choices for mapping studies. 195 microsatellite markers isolated from Prunus species were obtained from published papers or by personal communication. Polymorphism was revealed by three different methods, and in general, polyacrylamide gel electrophoresis (PAGE) compared to the fluorescent labelled marker detection using an automated DNA sequencer or agarose gel electrophoresis, showed the most efficient and cost effective method of genotyping. A subset of 54 markers which produced reliable and easily interpretable polymorphic bands was selected to screen the whole mapping population. Microsatellites originally isolated from almond species showed the highest rate of amplification and polymorphism followed by peach microsatellites and the least informative markers were isolated from cherry. It seems that the level of transportability and usefulness of microsatellite markers is related to the genetic distance of the closely related species. Almond and peach belong to the same subgenus (Amygdalus) and other Prunus species are classified in Prunophora subgenus. The nut, or kernel, is the commercial part of the almond tree, thus to improve the quality of fruit an understanding of environmental influence, heritability and correlation of traits is required. Pomological and quality characters such as: shell hardness, kernel size, shape, taste, pubescence, colour, and percentage of doubles were measured during three consecutive years (2005-2007) on the total mapping population, but data analysis (ANOVA) was performed only on trees that survived for all three years. Most of the traits showed high broad-sense heritability and kernel shape showed the highest heritability of H² = 0.92 suggesting high genetic control of this trait. Occasionally larger kernels than either parent were found in the progeny indicating potential for improvement of this trait even with smaller kernel size parent that encompass many desirable characters. High correlation was also found between the in-shell and kernel weight (r = 0.74), kernel length / kernel width (r = 0.67), kernel weight to kernel length (r = 0.78) and kernel width (r = 0.80). This correlation estimation pointed out in this study indicates that the improvement of one character may result the progress in another trait. Neither of the parents in the mapping population had bitter or obvious slightly bitter taste but slightly bitter kernels were observed among the progeny. Amygdalin was assumed to be responsible for bitter taste in almond; therefore we measured the amount of amygdalin in sweet and slightly bitter kernel progeny by HPLC. However, the results showed that amygdalin exists in sweet kernels as well. Although the average amount of amygdalin in slightly bitter kernels (20.34 mg kg⁻¹ FW) was higher than sweet kernels (3.67 mg kg⁻¹ FW), some sweet kernels had higher amounts of amygdalin suggesting the impact of other components on slightly bitter kernel. The highest variability within the traits was observed in the percentage of double kernel, which showed the highest standard error. Strong environmental effects, particularly low temperature at pre-blossom time is speculated to produce much higher double kernels. Three genetic linkage maps, one for each parent and an integrated map were constructed by the addition of 54 new microsatellite markers to the previous dataset. All the data was scored and coded according to the coding system necessary by JoinMap3 which was used for map construction. 131 markers including microsatellite, ISSR, RAPD, SCAR and S-allele markers were placed on the integrated map covering 590.7 cM with the average density of 4.5 cM/marker. The minimum number of six microsatellite markers was placed on linkage group 8 and the linkage group 1 which is the longest linkage group has 14 microsatellite markers. Comparative mapping study with other Prunus maps, especially with the highly saturated reference map showed complete synteny and minor changes in the order of four markers on linkage groups compared with Prunus reference map. The conservation of molecular marker order observed in this study supports the idea of looking at Prunus genome as a single genetic system and practical application of this similarity would be in cross-transportability of microsatellite markers from well developed linkage maps to the less studied species in Prunus. Ten microsatellite loci placed on our map have not been reported before and could be used to improve the density of other Prunus maps, especially the reference map. This study contributed to the better understanding of the mode of inheritance and environmental effect on morphological traits and the effect of amygdalin on kernel taste. The most saturated microsatellite based almond linkage map developed in this study can serve as a framework for future almond breeding program in Australia and benefit Prunus improvement programs internationally. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1348850 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Experiments were conducted to determine the outcrossing rate, the inheritance of markers and establish a basic linkage map in grasspea, <i> Lathyrus sativus </i>L. The outcrossing rate in a white-flowered line of grasspea ranged from 1.7 to 2.7% among eight combinations of gene frequency and location. The outcrossing rate in this study (2.2 ± 0.7%) suggests that individual lines of grasspea should be maintained in isolation to maintain their genetic integrity. Inheritance and linkage were determined for one morphological, 11 isozyme and 72 RAPD markers in five F₂ populations (all RAPD markers were in one F₂ population). The inheritance of flower colour was monogenic with colour dominant over white. The isozymes, ACO-1, ACO-2, AAT-1, AAT-2, EST-6, FDH, LAP-1, PGD-2, SKDH and TPI-1, were codominantly expressed with monogenic inheritance. The isozymes LAP-1 and PGD-2 segregated in a non-Mendelian ratios in the crosses PI 426891.1.3 x PI 283564c.3.2 and PI 426891.1 x PI 172930.4, respectively. The isozymeEST-3 was monogenically inherited and dominantly expressed. Most RAPD markers segregated in a 3:1 ratio. Marker UBC368_425/655 segregated in a co-dominant fashion. The RAPD markers UBC304₈₃₁, UBC304₉₆₄, UBC308₉₉₀, UBC322₁₄₃₂, UBC328₈₃₁, UBC332₁₁₁₈, UBC3321₁₅₈₁, UBC333₆₁₇, UBC349<sub>752<?sub>, UBC365₁₀₁₃ and UBC388₄₅₉ showed distorted segregation. In two F₂ populations, PI 283564c.3 x PI 426885.2 and PI 358601.5 x PI 173714.5, a linkage between AAT-2 and SKDH was reconfirmed. In the cross PI 426891.1.3 x PI 283564c.3.2, one morphological, three isozyme and 71 RAPD markers were mapped resulting in the delineation of 14 linkage groups including 69 markers (1 morphological, 3 isozyme and 65 RAPD markers). The total genome length covered by these 75 markers (69 linked and six unlinked) was about 864 cM. Considering cost, simplicity and abundance, RAPD analysis was more efficient than isozyme analysis in developing linkage map.

The fungus Fusarium graminearum (Schwabe) causes Gibberella ear rot in maize, resulting in accumulation of harmful mycotoxins in the grain. Disease severity and pericarp/aleurone dehydrodiferulic acid content are negatively correlated. Furthermore, quantitative trait locus mapping (QTL) identified colocalization between QTL for both traits. A candidate gene approach was employed to identify the genes responsible for the observed colocalization. Candidate genes selected on the basis of their putative involvement in various aspects of cell wall DFA accumulation were mapped in silico using the maize genome sequence. Polymorphisms were discovered in putative genes and converted to molecular markers. The in silico mapping effort was successful in predicting map locations of the analyzed sequences, and the segregation of certain marker alleles could explain variation for Gibberella ear rot severity and pericarp-aleurone DFA content.

Brassica carinata is being actively pursued as a new industrial oil crop platform for the Canadian Prairies. A genetic assessment of B. carinata was performed to elucidate its evolutionary origins and create a genetic map to assist in locating genes and traits of interest that would help in marker-assisted breeding. First, genetic analysis using simple sequence repeat (SSR) markers, previously tested on B. juncea and B. napus, was performed, to examine the genetic diversity of 37 B. carinata lines. SSR analysis revealed world accessions were more diverse than lines conditioned to grow in the prairies. Diversity analysis revealed that the parental lines of a double haploid (DH) population, 179 and 345, obtained from the John Innes Centre (JIC), were among the more genetically diverse lines, supporting the use of this population for linkage mapping. Genetic markers created from 3’ targeted SNP discovery between 179 and 345, were tested on the DH population resulting in the generation of a B. carinata genetic linkage map essentially with no prior sequence data knowledge. This genetic map contained 341 SNP and 86 SSR loci identifying eight linkage groups belonging to the B genome, nine belonging to the C genome and two unidentified groups spanning 2041 cM. Comparative mapping of polymorphic markers identified in the amphidiploid B. carinata indicated the orientation of B and C genomes coincide with that of other Brassica species, and the two genomes have remained essentially unaltered, with no major chromosomal rearrangements since the formation of B. carinata. A lesser number of polymorphic markers were detected in the C genome, which suggested the B genome is more genetically diverse in B. carinata. Limited field trials of the 179 x 345 DH population were performed during the 2011 and 2012 growing seasons. Preliminary quantitative trait loci (QTLs) for agronomic traits including flowering time (FT), plant height (PH), and seed quality were identified.

This study comprised three major parts: a comparative physiological study of drought responses under controlled conditions; a genetic study to construct the skeleton map of a doubled haploid (DH) population; and a quantitative trait loci (QTL) analysis to identify QTLs associated with drought tolerance traits in the field. In the first part (Chapter 3), three cultivars of wheat (Triticum aestivum L.) adapted to South Australian conditions were tested for drought tolerance under cyclic drought in growth rooms and glasshouse. Extensive physiological traits, including stomatal conductance, chlorophyll content and fluorescence, ABA content, water status traits (e.g. osmotic adjustment, RWC and leaf water potential), water soluble carbohydrates (WSC) and carbon isotope discrimination (Δ¹ ³C) were measured during experiments. Through these experiments, the drought responses of the three cultivars were physiologically dissected and the likely processes contributing most to drought tolerance were identified. In the South Australian wheatbelt, cyclic drought is a frequent event, represented by intermittent periods of rainfall which can occur around anthesis and post-anthesis in wheat. Three South Australian bread wheat cultivars, Excalibur, Kukri and RAC875, were evaluated in two growth room experiments under cyclic water-limiting conditions. In the first experiment, where plants were subjected to severe water stress, RAC875 and Excalibur (drought tolerant) showed significantly (P < 0.05) higher grain yield under cyclic water availability compared to Kukri (drought susceptible), producing 44% and 18% more grain yield compared to Kukri, respectively. In the second growth room experiment, where plants were subjected to a milder drought stress, the differences between cultivars were less pronounced, with only RAC875 showing significantly higher grain yield under the cyclic water treatment. Grain number per spike and the percentage of aborted tillers were the major yield components that affected yield under cyclic water stress. Excalibur and RAC875 adopted different morpho-physiological traits and mechanisms to reduce water stress. Excalibur was most responsive to cyclic water availability and showed the highest level of osmotic adjustment (OA), highest stomatal conductance, lowest ABA content and most rapid recovery from stress under cyclic water stress. RAC875 was more ‘conservative’ in its responses, with moderate OA, high leaf waxiness, high chlorophyll content and slower recovery from stress. Within this germplasm, the capacity for osmotic adjustment was the main physiological attribute associated with tolerance under cyclic water stress, which enabled plants to recover from water deficit. In the second part (Chapter 4), the genetic linkage map of a DH population including 368 lines, which was developed from a cross between ‘RAC875’ and ‘Kukri’, was constructed. The genetic linkage map consisted of about 500 molecular markers including ~300 DArT (Diversity array technology) and ~200 SSR (Microsattelite markers). In the third part (Chapter 5), Quantitative Trait Loci (QTLs) linked to plant phenology and production traits under irrigated and drought stress conditions were mapped by means of a DH population. To phenotype the population, 368 DH lines were cultivated in two replicates in five environments (three sites across South Australian wheatbelt in collaboration with Australian Grain Technology (AGT) in 2006, and two trials in Mexico in collaboration with CYMMIT, 2007). Data of grain yield, yield components, maturity related traits and some morpho-physiological traits such as leaf chlorophyll content, leaf waxiness, plant height, peduncle length, flag leaf and spike length were measured. Raw data were then analysed for spatial variation for each single trial using the REML procedure in GenStat (version 6). The DH lines showed significant variation for plant phenology, grain yield and yield components under irrigated and drought stress conditions. QTL analyses were performed using QTLCartographer and QTLNetwork for each trait in each site. Two major QTL for maturity traits were identified on chromosomes 2BS and 2DS corresponding to Ppd-B1 and Ppd-D1, respectively. A region was identified on chromosome 7A that harbored major QTL for grain yield, number of grains per square meter, number of grain per spike and spike fertility under drought stress. For yield data in the irrigated trial, two major QTL were identified on chromosome 3B which were not detected in drought stress environments. By using different datasets in the QTL analysis (splitting the population into two subpopulation based on heading time and also adjusting the phenotypic data for heading time to eliminate heading time effect), a QTL for grain yield was consistently detected on chromosome 7A in drought-affected environments. The coincidence of a drought response index QTL on this chromosome indicated that it might be a QTL for yield response under drought. This study demonstrated that the region on the long arm of chromosome 7A identified for grain yield and yield components is a drought response QTL which is closely linked to, but separate from, a heading time QTL. This QTL cluster on chromosome 7A could be used as a good target for positional cloning and gene isolation. However further work would be required to confirm and validate the identified QTLs in this preliminary QTL analysis. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1340056 / Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008