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Molecular marker analysis of seven putative onion (Allium cepa) doubled haploid (DH) lines developed at Texas A&M University was conducted to verify genetic homozygosity. Analysis was also conducted on five equivalent conventional inbred lines, breeding lines developed from the same parental crosses as the DH lines, and the original parent lines. The markers have revealed polymorphisms within the parental lines and the conventional inbreds, but not in the DH lines. We can conclude therefore that these seven lines are true DH lines. Performance of these DH lines was tested in two field locations and compared to commercial check lines. Bulbs from the various crosses were evaluated for eight bulb traits: diameter, height, centers/bulb, ring thickness, number of rings/bulb, bulb weight, soluble solids content, and pungency. Some crosses were detected that yielded significantly greater bulb weight than the check lines. However, these lines also had significantly greater numbers of centers per bulb. To test how these lines would perform in a breeding program, two full diallel analyses were conducted according to Griffing’s Model I, Method 1. The first consisted of a four parent diallel cross using two red DH lines and two yellow DH lines. Bulbs from the various crosses were evaluated for the same eight bulb traits mentioned above. Significant variation was detected for genotypic, general combining ability (GCA), specific combining ability (SCA), reciprocal (REC), maternal (MAT), and nonmaternal (NMAT) effects for all traits except number of rings/bulb, soluble solids content, and pungency. Significant environmental effects were only detected with number of centers per bulb. The second diallel analysis, a four parent diallel with two DH lines and two inbred lines from the breeding program, showed significant variation for the same effects for all traits except soluble solids content. Generally, GCA effects were more important than SCA effects in explaining the variation observed between crosses. For all traits GCA and SCA were always larger than the reciprocal effects (divided into maternal and nonmaternal components).

Master of Science / Genetics Interdepartmental Program - Agronomy / Allan K. Fritz / Wheat is increasing in importance as the global population rises; therefore, abiotic stresses adversely affecting wheat yield, such as heat stress, are of growing concern. Accessions of Aegilops speltoides, a relative of the ancestral donor of the B genome of hexaploid wheat (Triticum aestivum), have been determined to be highly heat tolerant. Six Ae. speltoides accessions were used as male donors in crosses with six hexaploid wheat lines, in all combinations, to obtain F1 amphiploid seed (ABDS = 28 chromosomes). The F1 seedlings were treated with a colchicine solution to achieve chromosome doubling (AABBDDSS = 56 chromosome doubled amphiploids) and grown out to maturity. S1 seed was collected for optimal or heat treatment after anthesis and SPAD readings were taken daily during treatment until maturity. In addition to physiological measurements, leaf tissue samples were collected from S1 plants and their respective parents for marker sequence analysis. Certain doubled amphiploids survived longer in heat treatment than their wheat parents with similar SPAD readings, but had a longer maturation time, similar to their Ae. speltoides parents. S2 seed was collected from 20 S1 plants, including one plant from the heat treatment. This heat screen demonstrates variation among the amphiploids, the genetic diversity within pedigree warrants further investigation into the viability and heat stress tolerance of the S2 seeds obtained from this experiment.

An exceptionally effective transformation procedure has been established by using class I embryo-derived rice callus. Every treated callus clump yielded multiple independently transformed plants (average 40 plantlets). Analysis of genomic DNA blots and pollen expressing green fluorescent protein (GFP) from T0 plants revealed that 64% bore a single locus T-DNA insertion in which half had one T-DNA copy. Additive transgene expression was observed fromT0 plants with GFP driven by mUbi1 promoter. Transgenic plants could be rapidly characterized by analyzing GFP pollen from T0 plants without the need for further generations or genomic DNA blot analysis. Agrobacterium tumefaciens-mediated transformation of microspore-derived callus for generating large numbers of T-DNA haploid and doubled haploid(DH) plants has also been investigated. The established transformation procedure resulted in 100% transformation frequency for class I microspore-derived rice callus. Each callus typically yields multiple independent transgenic plants. Genomic DNA blot analysis suggested 98% of the transgenic plants are independent events. About half of the transgenic plants were identified as haploid plants, whereas half are DH hemizygous or homozygous transgenic plants. DH homozygous transgenic plants were obtained from T0plants and confirmed by pollen GFP expression and genomic blot analysis in T0transgenic DH plants. In this study, about 60% ofT0transgenic DH plants had a single locus T-DNA insertion of which 45% bore one T-DNA copy. Furthermore, in a population of over 2,000 haploid and doubled haploid T-DNA plants , about 25% showed phenotypic differences from non-transformed haploid plants. Approximately 5% were seriously phenotypically abnormal including lethal or semi-lethal mutants. This highly efficient transformation procedure using microspore-derived callus could be valuable in speeding up plant breeding and in new gene discovery. Diversification of the mUbi1 promoter led to a minimal promoter that has a similar function as the original mUbi1. Transient and stable transformation as measured from gene expression driven by the minimal promoter suggested that it has a similar function as the original wild type promoter.

<p>In this project relational and Lotus Notes database technology are evaluated with regard to their suitability in providing computer-based support in plant breeding in general and specifically in the production of doubled haploids. The two developed databases are compared based on a set of requirements produced together with the DH-group which is the main users of the databases. The results indicate that both Lotus Notes and the relational databases are able to fulfil all needs documented in this project, although both systems have their limitations. An often expressed opinion is that it is difficult to combine biology and databases. The experience gained in this project however suggests that it does not need to be the case in instances where data is not as complicated as often discussed. Observations made during this project indicate that data warehousing with integrated data mining and OLAP tools are surprisingly similar to how the DH-group at Svalöf Weibull works and could be a suitable solution for the production of doubled haploids.</p>

Doubled haploidy (DH) is a plant breeding technique that is often utilized by plant breeders to minimize the time required to reach homozygosity in breeding lines. The first objective of this study was to compare two methods of generating DH lines in tobacco (Nicotiana tabacum L.). Inbred burley tobacco varieties TN 90LC and GR 149LC were used to produce both androgenic derived doubled haploids (ADDH) and maternally derived doubled haploids (MDDH). The relative agronomic performance of TN 90LC and GR 149 LC ADDH and MDDH lines was compared when used either as pure-line cultivars or when used for the production of the KT 204LC and TN 97LC hybrid cultivars, respectively. The ADDH method was more efficient than the MDDH method in generating large numbers of haploid plants. On average the ADDH TN 90LC population was statistically inferior to the inbred TN 90LC for several agronomic traits; this inferiority of the ADDH method was not observed in the GR 149LC populations. For both genotypes, the MDDH populations were comparable to the inbred parental genotypes. The ADDH method was inferior for TN 90LC, but several individual TN 90LC ADDH lines were equal or superior to the inbred source. The agronomic variability observed in both ADDH and MDDH lines was decreased when they were used to produce hybrid cultivars. Less variation was observed in the DH-derived hybrids KT 204LC and TN 97LC compared to the ADDH and MDDH TN90LC and GR149LC parental lines, respectively. The significant inferiority of ADDH TN 90 lines in comparison to inbred TN 90LC was not observed in the ADDH derived KT 204 population compared to KT 204LC. The second objective of this study was to compare DH Lines derived from an F1 breeding population versus DH lines derived from a segregating F2 population where plants used for DH were pre-screened for quantitatively inherited resistance to soil-borne diseases black shank (Phytophthora nicotianae) and/or Fusarium wilt (Fusarium oxysporum f. nicotianae). There was a clear difference in susceptibility to black shank between the F1 and F2 derived DH populations, both in terms of average disease incidence, and more importantly, in the percentage of individual lines displaying high disease resistance. For two different burley crosses, DH lines derived from the F1 generation were considerably more susceptible to black shank than DH lines derived from the F2 generation. No differences in the incidence of Fusarium wilt were observed between DH lines of F1 and F2 generations; this was likely due to low overall disease incidence. Although delaying the DH process in tobacco from the F1 to the F2 generation could add time to the development of homozygous breeding lines, the delay may be offset by having to screen fewer finished DH lines to identify superior lines.

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

Fusarium head blight (FHB), a fungal disease caused principally by Fusarium graminearum, and barley yellow dwarf (BYD) caused by BYD luteoviruses are two serious fungal and viral diseases of wheat resulting in high economic losses annually. Wuhan, a Chinese wheat cultivar resistant to FHB, and Maringa, a Brazilian cultivar tolerant to BYDV were inter-crossed and crossed with Roblin, a Canada western red spring wheat susceptible to both FHB and BYDV, to determine the genetic basis of resistance/tolerance and to combine the two traits. Four hundred ninety nine F1-derived doubled haploid (DH) lines were generated from reciprocal crosses using corn pollen-mediated DH technology. The DH lines and the parents were evaluated for disease symptoms, reduction in height and spike mass for BYD and for disease incidence, disease severity and Fusarium-damaged kernels for FHB in field and controlled environments. A subset (20/150) of the best performing DH lines from Wuhan/Maringa populations for both BYD and FHB were further evaluated. Plants were point inoculated with F. graminearum in greenhouse experiments, and macroconidial spray inoculations and spread of corn inoculum were used in field environments to evaluate FHB. BYDV inoculations were performed by placing ten to fifteen viruliferous aphids (Rhopalosiphum padi infected with BYDV-PAV isolate 9301PAV), at the one to two leaf stage for both greenhouse and field trials. The studies showed that both FHB and BYDV are quantitatively inherited. Transgressive segregants were observed and the broad sense heritability was high (0.90 to 0.97) for all traits evaluated. Results from independent testing of diseases on Wuhan/ Maringa populations showed fourteen DH lines were as, or more resistant than Wuhan for FHB and Maringa for BYDV tolerance and have combined both BYDV tolerance and FHB resistance. Identifying such lines facilitates the pyramiding of independent genes to obtain adequate levels of enduring resistance. A further experiment was conducted on the 14 lines by inoculating them with BYDV and F. graminearum successively on the same plant. Six out of 14 selected DH lines demonstrated high resistance to FHB and tolerance to BYDV. These six lines can be used in FHB/BYDV resistance/tolerance breeding programs.

Fusarium head blight (FHB), a fungal disease caused principally by Fusarium graminearum, and barley yellow dwarf (BYD) caused by BYD luteoviruses are two serious fungal and viral diseases of wheat resulting in high economic losses annually. Wuhan, a Chinese wheat cultivar resistant to FHB, and Maringa, a Brazilian cultivar tolerant to BYDV were inter-crossed and crossed with Roblin, a Canada western red spring wheat susceptible to both FHB and BYDV, to determine the genetic basis of resistance/tolerance and to combine the two traits. Four hundred ninety nine F1-derived doubled haploid (DH) lines were generated from reciprocal crosses using corn pollen-mediated DH technology. The DH lines and the parents were evaluated for disease symptoms, reduction in height and spike mass for BYD and for disease incidence, disease severity and Fusarium-damaged kernels for FHB in field and controlled environments. A subset (20/150) of the best performing DH lines from Wuhan/Maringa populations for both BYD and FHB were further evaluated. Plants were point inoculated with F. graminearum in greenhouse experiments, and macroconidial spray inoculations and spread of corn inoculum were used in field environments to evaluate FHB. BYDV inoculations were performed by placing ten to fifteen viruliferous aphids (Rhopalosiphum padi infected with BYDV-PAV isolate 9301PAV), at the one to two leaf stage for both greenhouse and field trials. The studies showed that both FHB and BYDV are quantitatively inherited. Transgressive segregants were observed and the broad sense heritability was high (0.90 to 0.97) for all traits evaluated. Results from independent testing of diseases on Wuhan/ Maringa populations showed fourteen DH lines were as, or more resistant than Wuhan for FHB and Maringa for BYDV tolerance and have combined both BYDV tolerance and FHB resistance. Identifying such lines facilitates the pyramiding of independent genes to obtain adequate levels of enduring resistance. A further experiment was conducted on the 14 lines by inoculating them with BYDV and F. graminearum successively on the same plant. Six out of 14 selected DH lines demonstrated high resistance to FHB and tolerance to BYDV. These six lines can be used in FHB/BYDV resistance/tolerance breeding programs.

Resumo: Estudos de associação baseados em desequilíbrio de ligação (DL) são importantes ferramentas para construção de mapas de ligação e utilização em programas de melhoramento assistidos por marcadores. Em geral, são utilizadas populações segregantes ou linhagens isogênicas para composição destes programas. Com o objetivo de comparar o desequilíbrio de ligação em linhagens duplo-haplóides (DH) e linhagens obtidas por meio de autofecundação, duzentas e quarenta e cinco linhagens - cento e setenta e cinco convencionais e setenta DHs - foram submetidas à análise de marcadores do tipo Single Nucleotide Polymorphism (SNP), utilizando-se mil duzentos e trinta e quatro marcadores distribuídos pelos dez cromossomos. O resultado da regressão entre as distâncias mostrou um R2 de 0,6546 para linhagens duplo-haplóides e uma equação de tendência logarítmica, sendo y= -0,024ln(x) + 0,159. Para as linhagens convencionais, o R2 foi de 0,5727, com a equação que explica a tendência, sendo y = -0,008ln(x) + 0,0659. Os dados de DL foram analisados individualmente, por cromossomo; e, assim como na análise conjunta, individualmente, todos os cromossomos tiveram o mesmo comportamento quando se comparam o DL de linhagens DH e os convencionais, sendo que os valores de DL nas linhagens DH foram em geral mais altos que nas convencionais. Os resultados indicam que, para a obtenção de linhagens DH, a recombinação ocorre em blocos maiores quando comparado com as linhagens convencionais. / Abstract: Association studies based on linkage disequilibrium (LD) are important tools for linkage maps construction and to use in marker-assisted breeding programs. Typically, segregating populations or isogenic lines are used to compose them. With objective of compare the linkage disequilibrium on double haploid lines (DH) and lines obtained by self pollination (Conventional), two hundred and forty five inbred lines, where one hundred seventy five conventional and seventy DHs where submitted to the analysis of Single Nucleotide Polymorphism (SNP) molecular markers, using one thousand two hundred and thirty four markers random distributed over the ten chromosomes. The regression output among the distances showed R2 of 0.6546 for double haploids lines and one logarithmic trend equation, being y = -0.024ln(x) + 0.159 For conventional lines, R2 was 0.5727, with an equation that explains the trend, being y = -0.008ln(x) + 0.0659. LD data were analyzed by chromosomes individually and as such in the joint analysis, all individual chromosomes showed the same patternr when comparing the LD of DH lines versus conventional lines, being the LD of DH lines generally higher that the conventional lines. Results show that to obtain DH lines the recombination occurs in larger blocks when compared against the conventional lines. / Orientador: José Roberto Moro / Coorientador: Heyder Diniz Silva / Banca: Ricardo Machado da Silva / Banca: Herberte Pereira da Silva / Banca: Antonio Carlos Silva / Banca: Sandra Helena Unêda Trevisoli / Doutor