Littérature scientifique sur le sujet « SNP array genotyping »

Single nucleotide polymorphism (SNP) genotyping arrays are powerful tools to measure the level of genetic polymorphism within a population. The coming of next-generation sequencing technologies led to identifying thousands and millions of SNP loci useful in assessing the genetic diversity. The Vitis genotyping array, containing 18k SNP loci, has been developed and used to detect genetic diversity of Vitis vinifera germplasm. So far, this array was not validated on non-vinifera genotypes used as grapevine rootstocks. In this work, a core collection of 70 grapevine rootstocks, composed of individuals belonging to Vitis species not commonly used in the breeding programs, was genotyped using the 18k SNP genotyping array. SNP results were compared to the established SSR (Simple Sequence Repeat) markers in terms of heterozygosity and genetic structure of the core collection. Genotyping array has proved to be a valuable tool for genotyping of grapevine rootstocks, with more than 90% of SNPs successfully amplified. Structure analysis detected a high degree of admixed genotypes, supported by the complex genetic background of non-vinifera germplasm. Moreover, SNPs clearly differentiated non-vinifera and vinifera germplasm. These results represent a first step in studying the genetic diversity of non-conventional breeding material that will be used to select rootstocks with high tolerance to limiting environmental conditions.

We present four studies that illustrate the use of DNA microarrays for the detection and subsequent genotyping of waterborne pathogens. A genotyping array targeting four virulence factor genes in enterohemorrhagic Escherichia coli (EHEC) was tested. The arrays were clearly able to differentiate between E. coli O157:H7 genotypes and E. coli O91:H2. Non-pathogenic E. coli and non-target organisms were not detected on this array. In the second study, an hsp70 gene single nucleotide polymorphism (SNP) array for specific Cryptosporidium parvum detection was constructed to differentiate between principle genotypes. SNPs, and hence differences between genotypes, were easily detected on this type of array. In the third study an array for Helicobacter pylori was tested for simultaneous SNP discrimination and presence or absence of virulence factor genes. Results from this study showed that both SNP discrimination for some conserved genes, and the presence or absence of virulence factor genes was possible. In the fourth study, multiplexing was achieved by direct hybridization and detection of mRNA to the array. For highly expressed genes, visible signal was detected at 312.5 ng of total RNA, indicating that these new methods may have sufficient environmental sensitivity without the need to perform PCR.

Radiata pine (Pinus radiata D.Don) is one of the world’s most domesticated pines and a key economic species in New Zealand. Thus, the development of genomic resources for radiata pine has been a high priority for both research and commercial breeding. Leveraging off a previously developed exome capture panel, we tested the performance of 438,744 single nucleotide polymorphisms (SNPs) on a screening array (NZPRAD01) and then selected 36,285 SNPs for a final genotyping array (NZPRAD02). These SNPs aligned to 15,372 scaffolds from the Pinus taeda L. v. 1.01e assembly, and 20,039 contigs from the radiata pine transcriptome assembly. The genotyping array was tested on more than 8000 samples, including material from archival progenitors, current breeding trials, nursery material, clonal lines, and material from Australia. Our analyses indicate that the array is performing well, with sample call rates greater than 98% and a sample reproducibility of 99.9%. Genotyping in two linkage mapping families indicated that the SNPs are well distributed across the 12 linkage groups. Using genotypic data from this array, we were also able to differentiate representatives of the five recognized provenances of radiata pine, Año Nuevo, Monterey, Cambria, Cedros and Guadalupe. Furthermore, principal component analysis of genotyped trees revealed clear patterns of population structure, with the primary axis of variation driven by provenance ancestry and the secondary axis reflecting breeding activities. This represents the first commercial use of genomics in a radiata pine breeding program.

Abstract Motivation Accurate genotyping of DNA from a single cell is required for applications such as de novo mutation detection, linkage analysis and lineage tracing. However, achieving high precision genotyping in the single-cell environment is challenging due to the errors caused by whole-genome amplification. Two factors make genotyping from single cells using single nucleotide polymorphism (SNP) arrays challenging. The lack of a comprehensive single-cell dataset with a reference genotype and the absence of genotyping tools specifically designed to detect noise from the whole-genome amplification step. Algorithms designed for bulk DNA genotyping cause significant data loss when used for single-cell applications. Results In this study, we have created a resource of 28.7 million SNPs, typed at high confidence from whole-genome amplified DNA from single cells using the Illumina SNP bead array technology. The resource is generated from 104 single cells from two cell lines that are available from the Coriell repository. We used mother–father–proband (trio) information from multiple technical replicates of bulk DNA to establish a high quality reference genotype for the two cell lines on the SNP array. This enabled us to develop SureTypeSC—a two-stage machine learning algorithm that filters a substantial part of the noise, thereby retaining the majority of the high quality SNPs. SureTypeSC also provides a simple statistical output to show the confidence of a particular single-cell genotype using Bayesian statistics. Availability and implementation The implementation of SureTypeSC in Python and sample data are available in the GitHub repository: https://github.com/puko818/SureTypeSC Supplementary information Supplementary data are available at Bioinformatics online.

The work described in this PhD thesis aimed to characterize 9 clear cell renal carcinoma primary cultures (RCCpc) and their parental tumor tissues and 5 commercial RCC cell lines, using the Affymetrix GeneChip® SNP array technology (50K and 250K platforms). We performed a genome-wide analysis of copy number alterations (CNAs) and LOH events, together with allele dosage, using CNAG (v3.0) and Affymetrix GTC (v2.0) software. RCCpc and parental tumor tissues were assessed comparing each culture and parental tissue to its corresponding blood sample, while cell lines were analyzed using 48 HapMap CEU samples as normal controls. Also, a comparison was performed between these samples and the typical RCC genomic signature, in order to assess if primary cultures and cell lines were a good in vitro model to study this pathology. The results here obtained indicated that our RCCpc were a reliable model to study RCC pathology, much better than RCC cell lines. Also, comparing RCCpc and parental tumor tissues, we demonstrated that RCCpc provided greater cell homogeneity and an enrichment in tumor cells with respect to heterogeneous bioptic tissues, thus facilitating the characterization of CNAs and the identification of novel genetic elements potentially involved in RCC etiology and useful in clinical applications.

Ogni giorno, circa mille miliardi di nuove cellule vengono prodotte dal midollo osseo e messe in circolazione nel sangue periferico per far fronte alle necessità del nostro organismo. Tutte le cellule che compongono il nostro sangue, derivano da una stessa cellula-madre, chiamata cellula staminale ematopoietica, un precursore primordiale e immaturo che ha la capacità di espandersi e la potenzilità di differenzare a eritrociti, linfociti, leucociti, piastrine. Il processo di differenziamento che va dalla cellula staminale alle cellule mature circolanti è detto emopoiesi. La leucemia è un tipo di tumore circolante che può avere origine da una cellula qualunque del sangue, a un qualsiasi stadio differenziativo, la quale inizia a proliferare all’impazzata, sfuggendo ad ogni controllo, e prende il sopravvento sulle altre cellule sane. Il risultato è l’espansione aberrante di un singolo tipo cellulare, a discapito degli altri compartimenti, che causa una disfunzione del sistema emopoietico. La leucemia non può essere considerata un’ unica malattia, poichè ne esistono molti diversi tipi, a seconda del tipo cellulare interessato, e del tipo di mutazione genica che la caratteizza. Nel nostro laboratorio, presso il Centro di Ricerca M. Tettamanti dell’Opedale S. Gerardo di Monza, ci occupiamo delle leucemie del bambino. In particolare, nel mio periodo di dottorato, mi sono occupata di studiare una particolare forma di leucemia linfoblastica acuta, detta infant LLA, ad insorgenza nel primo anno di vita, che presenta il riarrangiamento del gene MLL. Questa leucemia rappresenta la forma prevalente in età neonatale, ed è purtoppo una malattia molto aggressiva con prognosi infausta che in Italia colpisce circa 10-12 nuovi nati all’anno. In particolare, i casi che presentano la traslocazione t(4;11) con espressione del prodotto di fusione MLL-AF4 rappresentano il sottogruppo genetico a incidenza maggiore in età neonatale. Per moltissimi aspetti, sia dal punto di vista clinico che biologico, la LLA infant rappresenta una malattia peculiare, diversa dalle altre forme di leucemia dell’età pediatrica e adulta. In questi quattro anni di dottorato mi sono occupata nello specifico di due progetti. Nel primo, in collaborazione con il Prof. Jacobsen in Svezia, ci siamo proposti di identificare, isolare e caratterizzare dal punto di vista biologico e funzionale la ‘cellula staminale leucemica’ della LLA infant con riarrangiamento del gene MLL. Nel secondo studio, pubblicato su ‘Leukemia’ abbiamo voluto valutare se il solo riarrangiamento del gene MLL fosse di per se sufficiente per dare origine alla malattia, oppure se alterazioni genetiche ulteriori, secondarie al riarrangiamento di MLL che costituisce il primo evento, fossero necessarie per la manifestazione clinica della leucemia. La composizione cellulare di ogni singolo tumore umano è altamente eterogenea, e non tutte le cellule che lo compongono sono capaci di sostenerne la crescita. Per spiegare queste osservazioni è stata postulata l’esistenza di un sottogruppo ristretto di cellule, dette staminali tumorali (CSC), o staminali leucemiche (LSC) nel caso si tratti di leucemia, che hanno la potenzialità di auto-rigenerarsi e di riprodurre l’intera massa tumorale. Secondo il modello della ‘cellula staminale tumorale’, in tutti i tumori solo un ristretto gruppo di cellule è capace di generare l’intera massa tumorale e sostenerne la crescita all’infinito. Poter bersagliare in modo specifico questa cellula significa eradicare definitivamente la malattia, che altrimenti inevitabilmente si rigenererebbe. Questa ipotesi è in contrasto con il precedente modello stocastico secondo cui ogni cellula che compone il tumore è potenzialmente capace di sostenerne la crescita: tuttavia la eterogenicità tumorale, e la sua bassa tumorigenicità sono dovute alla scarsa probalilità per una cellula di iniziare il ciclo cellulare e proliferare. Tuttavia recenti studi mettono in discussione l’ipotesi stessa dell’esistenza di una CSC, ed attualmente l’opinione scientifica è divisa riguardo al fatto se il modello gerarchico della CSC, piuttosto che il classico modello stocastico, siano più adatti per spiagere il meccanismo biologico alla base dell’insorgenza dei tumori umani. Infatti, benchè il concetto di CSC sia intuitivamente immediato, la sua effettiva identificazione e caratterizzazione si è rivelata inaspettatamente complicata, e ad oggi, non esiste una definizione universale di CSC. Il termine ‘leucemia’ comprende in realtà tantissimi tipi differenti di tumore, e tale eterogeneità si riflette sulla LSC. La specifica alterazione genetica, la cellula di origine (cioè la cellula inizialmente colpita dal primo evento trasformante da cui il tumore ha avuto luogo), le diverse metodologie in vivo applicate, l’influenza del microambiente midollare e l’alta variabilità fenotipica tra diversi pazienti affetti dalla medesima forma di leucemia, hanno portato inevitabilmente alla generazione di risultati apparentementente contrastanti tra i vari gruppi di ricerca che indipendentemente si sono riproposti di identificare la LSC. Il modello sperimentale d’elezione per identificare la LSC è il trapianto in vivo di blasti leucemici in topi umanizzati immunocompromessi, capaci di accettare cellule umane senza rigettarle e permetterne l’attecchimento. Nel periodo all’estero trascorso nel laboratorio del Porf. Jacobsen a Lund, mi sono occupata di mettere a punto il modello di trapianto in vivo di cellule leucemiche da pazienti affetti da infant LLA in topi unamizzati. Previa irradiazione subletale del topo ricevente, che ha il duplice scopo di eliminare le cellule circolanti murine mature e creare spazio per le cellule umane trapiantate, i blasti leucemici vengono iniettati nel topo per via sistemica attraverso la vena della coda.. Se l’attecchimento è avvenuto, già dopo poche settimane, i topi mostreranno alti livelli di ricostituzione nel midollo e nei compartimenti extramidollari (come milza, fegato e sangue periferico), con conseguente manifestazione dei sintomi clinici della malattia (quali splenomegalia, pelo arruffato, letargia, anoressia, perdita di peso e decesso). L’attecchimento viene valutato eseguendo periodiche biopsie midollari (dopo 6, 9, 12 o più settimane dal trapianto) dalla sede femorale, in anestesia generale, e successivamente la presenza di cellule leucemiche umane è rilevata nell’aspirato midollare tramite analisi successive. Nel primo lavoro inviato a ‘Cancer Cell’ abbiamo osservato che nella LLA infant MLL-AF4 positiva la LSC risiede nella frazione CD19+, costituita da linfociti B a diversi stadi maturativi. Infatti, la frazione CD19- è composta sia da cellule staminali normali residue (non leucemiche) che danno luogo a una ricostituzione normale in vivo, che da cellule leucemiche (MLL riarrangiate), tuttavia prive di potenziale leucemogenico, poiché incapaci di ripopolare il midollo di un topo irradiato e dare origine alla leucemia. Inoltre abbiamo dimostrato che all’interno della frazione CD19+, esistono molteplici sottopolazioni LSC con diverso potenziale leucemogenico e diverse caratteristiche biologiche e funzionali in vivo (es. penetranza, latenza di insorgenza della malattia e fenotipo della leucemia risultante). In conclusione, i nostri risultati dimostrano che, a differenza del modello convenzionale, esistono molteplici sottopopolazioni LSC con caratteristiche biologiche e funzionali distinte, che possono essere distinte tra loro e dalla restante popolazione leucemica (non tumorigenica) sulla base dell’espressione di determinati antigeni di superficie. Nel secondo studio pubblicato su ‘Leukemia’, ci siamo chiesti se la sola presenza della traslocazione del gene MLL fosse di per se sufficiente per la manifestazione clinica della malattia, o se mutazioni accessorie cooperanti fossero necessarie. Per fare ciò abbiamo analizzato a livello genomico un gruppo consistente di pazienti affetti da LLA infant MLL-AF4 positiva, tramite tecniche sofisticate di screening su tutto il genoma ad alta risoluzione, e abbiamo osservato che, contrariamente ai pazienti affetti da altri tipi di LLA pediatrica ad insorgenza dopo l’anno, nella LLA infant MLL-AF4 positiva non si rilevano mutazioni aggiuntive. Questi risultati dimostrano che il solo riarrangiamento di MLL è necessario e sufficiente per la manifestazione, in tempi brevi, della malattia conclamata, a differenza di altre forme di leucemia del bambino e dell’adulto, in cui mutazioni genetiche aggiuntive sono indispensabili. Inoltre tali evidenze ribadiscono ulteriormente che la LLA infant con riarangiamento di MLL è una malattia nica dal punto di vista biologco, diversa dagli altri tipi di leucemia, e che che il meccanismo di leucemogenesi può essere distinto. Considerando l’alta mortalità, l’alto rischio di ricaduta, la prognosi infausta della LLA infant MLL positiva e l’urgente necessità di avere a disposizione nuove ed efficaci terapie antitumorali, questi studi sono di rilevante importanza, in quanto non solo aiutano a comprendere meglio i meccanismi di insorgenza della LLA infant e fare luce sulla biologia e l’aggressività di questa malattia, ma rappresentano anche il punto di partenza per identificare nuove strategie terapeutiche al fine bersagliare la LSC ed eradicare la malattia in modo definitivo.

DNA copy number variations (CNVs), which involve the deletion or duplication of subchromosomal segments of the genome, have become a focus of genetics research. This dissertation develops Bayesian HMMs for finding CNVs from single nucleotide polymorphism (SNP) arrays. A Bayesian framework to reconstruct the DNA copy number sequence from the observed sequence of SNP array measurements is proposed. A Markov chain Monte Carlo (MCMC) algorithm, with a forward-backward stochastic algorithm for sampling DNA copy number sequences, is developed for estimating model parameters. Numerous versions of Bayesian HMMs are explored, including a discrete-time model and different models for the instantaneous transition rates of change among copy number states of a continuous-time HMM. The most general model proposed makes no restrictions and assumes the rate of transition depends on the current state, whereas the nested model fixes some of these rates by assuming that the rate of transition is independent of the current state. Each model is assessed using a subset of the HapMap data. More general parameterizations of the transition intensity matrix of the continuous-time Markov process produced more accurate inference with respect to the length of CNV regions. The observed SNP array measurements are assumed to be stochastic with distribution determined by the underlying DNA copy number. Copy-number-specific distributions, including a non-symmetric distribution for the 0-copy state (homozygous deletions) and mixture distributions for 2-copy state (normal), are developed and shown to be more appropriate than existing implementations which lead to biologically implausible results. Compared to existing HMMs for SNP array data, this approach is more flexible in that model parameters are estimated from the data rather than set to a priori values. Measures of uncertainty, computed as simulation-based probabilities, can be determined for putative CNVs detected by the HMM. Finally, the dissertation concludes with a discussion of future work, with special attention given to model extensions for multiple sample analysis and family trio data.

A current trend in biological science is the increased use of computational tools for both the production and analysis of experimental data. This is especially true in the field of genomics, where advancements in DNA sequencing technology have dramatically decreased the time and cost associated with DNA sequencing resulting in increased pressure on the time required to prepare and analyze data generated during these experiments. As a result, the role of computational science in such biological research is increasing. This thesis seeks to address several major questions with respect to the development and application of single nucleotide polymorphism (SNP) resources in non-model organisms. Traditional SNP discovery using polymerase chain reaction (PCR) amplification and low-throughput DNA sequencing is a time consuming and laborious process, which is often limited by the time required to design intron-spanning PCR primers. While next-generation DNA sequencing (NGS) has largely supplanted low-throughput sequencing for SNP discovery applications, the PCR based SNP discovery method remains in use for cost effective, targeted SNP discovery. This thesis seeks to develop an automated method for intron-spanning PCR design which would remove a significant bottleneck in this process. This work develops algorithms for combining SNP data from multiple individuals, independent of the DNA sequencing platforms, for the purpose of developing SNP genotyping arrays. Additionally, tools for the filtering and selection of SNPs will be developed, providing start to finish support for the development of SNP genotyping arrays in complex polyploids using NGS. The result of this work includes two automated pipelines for the design of intron-spanning PCR primers, one which designs a single primer pair per target and another that designs multiple primer pairs per target. These automated pipelines are shown to reduce the time required to design primers from one hour per primer pair using the semi-automated method to 10 minutes per 100 primer pairs while maintaining a very high efficacy. Efficacy is tested by comparing the number of successful PCR amplifications of the semi- automated method with that of the automated pipelines. Using the Chi-squared test, the semi-automated and automated approaches are determined not to differ in efficacy. Three algorithms for combining SNP output from NGS data from multiple individuals are developed and evaluated for their time and space complexities. These algorithms were found to be computationally efficient, requiring time and space linear to the size of the input. These algorithms are then implemented in the Perl language and their time and memory performance profiled using experimental data. Profiling results are evaluated by applying linear models, which allow for predictions of resource requirements for various input sizes. Additional tools for the filtering of SNPs and selection of SNPs for a SNP array are developed and applied to the creation of two SNP arrays in the polyploid crop Brassica napus. These arrays, when compared to arrays in similar species, show higher numbers of polymorphic markers and better 3-cluster genotype separation, a viable method for determining the efficacy of design in complex genomes.

Rapid progress in genotyping technologies, including the scaling up of assay technologies to genome-wide levels and next generation sequencing, has motivated a burst in methods development and application to detect genotype-phenotype associations in a wide array of diseases and other phenotypes. In this chapter, the authors review the study design and genotyping options that are used in association mapping, along with the appropriate methods to perform mapping within these study designs. The authors discuss both candidate gene and genome-wide studies, focused on DNA level variation. Quality control, genotyping technologies, and single-SNP and multiple-SNP analyses have facilitated the successes in identifying numerous loci influence disease risk. However, variants identified have generally explained only a small fraction of the heritable component of disease risk. The authors discuss emerging trends and future directions in performing analysis for rare variants to detect these variants that predict these traits with more complex etiologies.

The primary goals of this project were: (1) development of a genetically characterized association panel of wild emmer for high resolution analysis of the genetic basis of complex traits; (2) characterization and mapping of genes and QTL for seedling and adult plant resistance to stripe rust in wild emmer populations; (3) characterization of LD patterns along wild emmer chromosomes; (4) elucidation of the multi-locus genetic structure of wild emmer populations and its correlation with geo-climatic variables at the collection sites. Introduction In recent years, Stripe (yellow) rust (Yr) caused by Pucciniastriiformis f. sp. tritici(PST) has become a major threat to wheat crops in many parts of the world. New races have overcome most of the known resistances. It is essential, therefore, that the search for new genes will continue, followed by their mapping by molecular markers and introgression into the elite varieties by marker-assisted selection (MAS). The reservoir of genes for disease and pest resistance in wild emmer wheat (Triticumdicoccoides) is an important resource that must be made available to wheat breeders. The majority of resistance genes that were introgressed so far in cultivated wheat are resistance (R) genes. These genes, though confering near-immunity from the seedling stage, are often overcome by the pathogen in a short period after being deployed over vast production areas. On the other hand, adult-plant resistance (APR) is usually more durable since it is, in many cases, polygenic and confers partial resistance that may put less selective pressure on the pathogen. In this project, we have screened a collection of 480 wild emmer accessions originating from Israel for APR and seedling resistance to PST. Seedling resistance was tested against one Israeli and 3 North American PST isolates. APR was tested on accessions that did not have seedling resistance. The APR screen was conducted in two fields in Israel and in one field in the USA over 3 years for a total of 11 replicates. We have found about 20 accessions that have moderate stripe rust APR with infection type (IT<5), and about 20 additional accessions that have novel seedling resistance (IT<3). We have genotyped the collection using genotyping by sequencing (GBS) and the 90K SNP chip array. GBS yielded a total 341K SNP that were filtered to 150K informative SNP. The 90K assay resulted in 11K informative SNP. We have conducted a genome-wide association scan (GWAS) and found one significant locus on 6BL ( -log p >5). Two novel loci were found for seedling resistance. Further investigation of the 6BL locus and the effect of Yr36 showed that the 6BL locus and the Yr36 have additive effect and that the presence of favorable alleles of both loci results in reduction of 2 grades in the IT score. To identify alleles conferring adaption to extreme climatic conditions, we have associated the patterns of genomic variation in wild emmer with historic climate data from the accessions’ collection sites. The analysis of population stratification revealed four genetically distinct groups of wild emmer accessions coinciding with their geographic distribution. Partitioning of genomic variance showed that geographic location and climate together explain 43% of SNPs among emmer accessions with 19% of SNPs affected by climatic factors. The top three bioclimatic factors driving SNP distribution were temperature seasonality, precipitation seasonality, and isothermality. Association mapping approaches revealed 57 SNPs associated with these bio-climatic variables. Out of 21 unique genomic regions controlling heading date variation, 10 (~50%) overlapped with SNPs showing significant association with at least one of the three bioclimatic variables. This result suggests that a substantial part of the genomic variation associated with local adaptation in wild emmer is driven by selection acting on loci regulating flowering. Conclusions: Wild emmer can serve as a good source for novel APR and seedling R genes for stripe rust resistance. APR for stripe rust is a complex trait conferred by several loci that may have an additive effect. GWAS is feasible in the wild emmer population, however, its detection power is limited. A panel of wild emmer tagged with more than 150K SNP is available for further GWAS of important traits. The insights gained by the bioclimatic-gentic associations should be taken into consideration when planning conservation strategies.

Oroginal Objectives: (i) identify DNA markers linked to the avirulence (Avr) locus and locate the Avr locus through genetic mapping with an inter-race Orobanche cumana population; (ii) develop high-throughput fingerprint DNA markers for genotypingO. cumana races; (iii) identify nucleotide binding domain leucine rich repeat (NB-LRR) genes encoding R proteins conferring resistance to O. cumana in sunflower; (iv) increase the resolution of the chromosomal segment harboring Or₅ and related R genes through genetic and physical mapping in previously and newly developed mapping populations of sunflower; and (v) develop high-throughput DNA markers for rapidly and efficiently identifying and transferring sunflower R genes through marker-assisted selection. Revisions made during the course of project: Following changes in O. cumana race distribution in Israel, the newly arrived virulent race H was chosen for further analysis. HA412-HO, which was primarily chosen as a susceptible sunflower cultivar, was more resistant to the new parasite populations than var. Shemesh, thus we shifted sunflower research into analyzing the resistance of HA412-HO. We exceeded the deliverables for Objectives #3-5 by securing funding for complete physical and high-density genetic mapping of the sunflower genome, in addition to producing a complete draft sequence of the sunflower genome. We discovered limited diversity between the parents of the O. cumana population developed for the mapping study. Hence, the developed DNA marker resources were insufficient to support genetic map construction. This objective was beyond the scale and scope of the funding. This objective is challenging enough to be the entire focus of follow up studies. Background to the topic: O. cumana, an obligate parasitic weed, is one of the most economically important and damaging diseases of sunflower, causes significant yield losses in susceptible genotypes, and threatens production in Israel and many other countries. Breeding for resistance has been crucial for protecting sunflower from O. cumana, and problematic because new races of the pathogen continually emerge, necessitating discovery and deployment of new R genes. The process is challenging because of the uncertainty in identifying races in a genetically diverse parasite. Major conclusions, solutions, achievements: We developed a small collection of SSR markers for genetic mapping in O. cumana and completed a diversity study to lay the ground for objective #1. Because DNA sequencing and SNPgenotyping technology dramatically advanced during the course of the study, we recommend shifting future work to SNP discovery and mapping using array-based approaches, instead of SSR markers. We completed a pilot study using a 96-SNP array, but it was not large enough to support genetic mapping in O.cumana. The development of further SNPs was beyond the scope of the grant. However, the collection of SSR markers was ideal for genetic diversity analysis, which indicated that O. cumanapopulations in Israel considerably differ frompopulations in other Mediterranean countries. We supplied physical and genetic mapping resources for identifying R-genes in sunflower responsible for resistance to O. cumana. Several thousand mapped SNP markers and a complete draft of the sunflower genome sequence are powerful tools for identifying additional candidate genes and understanding the genomic architecture of O. cumana-resistanceanddisease-resistance genes. Implications: The OrobancheSSR markers have utility in sunflower breeding and genetics programs, as well as a tool for understanding the heterogeneity of races in the field and for geographically mapping of pathotypes.The segregating populations of both Orobanche and sunflower hybrids are now available for QTL analyses.

Pod-filling, an important yield-determining stage is strongly influenced by water stress. This is particularly true for peanut (Arachishypogaea), wherein pods are developed underground and are directly affected by the water condition. Pod-filling in peanut has a significant genetic component as well, since genotypes are considerably varied in their pod-fill (PF) and seed-fill (SF) potential. The goals of this research were to: Examine the effects of genotype, irrigation, and genotype X irrigation on PF and SF. Detect global changes in mRNA and metabolites levels that accompany PF and SF. Explore the response of the duplicate peanut pod transcriptome to drought stress. Study how entire duplicated PF regulatory processes are networked within a polyploid organism. Discover locus-specific SNP markers and map pod quality traits under different environments. The research included genotypes and segregating populations from Israel and US that are varied in PF, SF and their tolerance to water deficit. Initially, an extensive field trial was conducted to investigate the effects of genotype, irrigation, and genotype X irrigation on PF and SF. Significant irrigation and genotypic effect was observed for the two main PF related traits, "seed ratio" and "dead-end ratio", demonstrating that reduction in irrigation directly influences the developing pods as a result of low water potential. Although the Irrigation × Genotype interaction was not statistically significant, one genotype (line 53) was found to be more sensitive to low irrigation treatments. Two RNAseq studies were simultaneously conducted in IL and the USA to characterize expression changes that accompany shell ("source") and seed ("sink") biogenesis in peanut. Both studies showed that SF and PF processes are very dynamic and undergo very rapid change in the accumulation of RNA, nutrients, and oil. Some genotypes differ in transcript accumulation rates, which can explain their difference in SF and PF potential; like cvHanoch that was found to be more enriched than line 53 in processes involving the generation of metabolites and energy at the beginning of seed development. Interestingly, an opposite situation was found in pericarp development, wherein rapid cell wall maturation processes were up-regulated in line 53. Although no significant effect was found for the irrigation level on seed transcriptome in general, and particularly on subgenomic assignment (that was found almost comparable to a 1:1 for A- and B- subgenomes), more specific homoeologous expression changes associated with particular biosynthesis pathways were found. For example, some significant A- and B- biases were observed in particular parts of the oil related gene expression network and several candidate genes with potential influence on oil content and SF were further examined. Substation achievement of the current program was the development and application of new SNP detection and mapping methods for peanut. Two major efforts on this direction were performed. In IL, a GBS approach was developed to map pod quality traits on Hanoch X 53 F2/F3 generations. Although the GBS approach was found to be less effective for our genetic system, it still succeeded to find significant mapping locations for several traits like testa color (linkage A10), number of seeds/pods (A5) and pod wart resistance (B7). In the USA, a SNP array was developed and applied for peanut, which is based on whole genome re-sequencing of 20 genotypes. This chip was used to map pod quality related traits in a Tifrunner x NC3033 RIL population. It was phenotyped for three years, including a new x-ray method to phenotype seed-fill and seed density. The total map size was 1229.7 cM with 1320 markers assigned. Based on this linkage map, 21 QTLs were identified for the traits 16/64 weight, kernel percentage, seed and pod weight, double pod and pod area. Collectively, this research serves as the first fundamental effort in peanut for understanding the PF and SF components, as a whole, and as influenced by the irrigation level. Results of the proposed study will also generate information and materials that will benefit peanut breeding by facilitating selection for reduced linkage drag during introgression of disease resistance traits into elite cultivars. BARD Report - Project4540 Page 2 of 10