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The harvesting of sweet cherry (Prunus avium L.) fruit is a labor-intensive process. The mechanical harvesting of sweet cherry fruit is feasible; however, it is dependent on the formation of an abscission zone at the fruit–pedicel junction. The natural propensity for pedicel-–fruit abscission zone (PFAZ) activation varies by cultivar, and the general molecular basis for PFAZ activation is not well characterized. In this study, ethylene-inducible change in pedicel fruit retention force (PFRF) was recorded in a developmental time-course with a concomitant analysis of the PFAZ transcriptome from three sweet cherry cultivars. In ‘Skeena’, mean PFRF for both control and treatment fruit dropped below the 0.40 kg-force (3.92 N) threshold for mechanical harvesting, indicating the activation of a discrete PFAZ. In ‘Bing’, mean PFRF for both control and treatment groups decreased over time. However, a mean PFRF conducive to mechanical harvesting was achieved only in the ethylene-treated fruit. While in ‘Chelan’ the mean PFRF of the control and treatment groups did not meet the threshold required for efficient mechanical harvesting. Transcriptome analysis of the PFAZ region followed by the functional annotation, differential expression analysis, and gene ontology (GO) enrichment analyses of the data facilitated the identification of phytohormone-responsive and abscission-related transcripts, as well as processes that exhibited differential expression and enrichment in a cultivar-dependent manner over the developmental time-course. Additionally, read alignment-based variant calling revealed several short variants in differentially expressed genes, associated with enriched gene ontologies and associated metabolic processes, lending potential insight into the genetic basis for different abscission responses between the cultivars. These results provide genetic targets for the induction or inhibition of PFAZ activation, depending on the desire to harvest the fruit with or without the stem attached. Understanding the genetic mechanisms underlying the development of the PFAZ will inform future cultivar development while laying a foundation for mechanized sweet cherry harvest.

The ancient plant production practice of grafting which instantly imparts new physiological properties to the desirable scion still remains shrouded in mystery. Yet, grafting remains a widely used technique in the production of several horticultural species. In a composite grafted plant, rootstocks control many aspects of scion growth and physiology including yield and quality attributes as well as biotic and abiotic stress tolerance. Broadly, physical, physiological, biochemical and molecular mechanisms have been reviewed to develop an integrated understanding of this enigmatic process that challenges existing genetic paradigms. This review summarizes the reported mechanisms underlying some of the economically important traits and identifies several key points to consider when conducting rootstock scion interaction experiments. Study of the somatogenetic interactions between rootstock and scion is a field that is ripe for discovery and vast improvements in the coming decade. Further, utilization of rootstocks based on a better understanding of the somatogenetic interactions is highly relevant in the current agricultural environment where there is a need for sustainable production practices. Rootstocks may offer a non-transgenic approach to rapidly respond to the changing environment and expand agricultural production of annual and perennial crops where grafting is feasible in order to meet the global food, fiber and fuel demands of the future.

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3'UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 ( hcef2 ) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force ( pmf ), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2 , which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

Background Sweet cherry ( Prunus avium L.), a non-model crop with narrow genetic diversity, is an important member of sub-family Amygdoloideae within Rosaceae. Compared to other important members like peach and apple, sweet cherry lacks in genetic and genomic information, impeding understanding of important biological processes and development of efficient breeding approaches. Availability of single nucleotide polymorphism (SNP)-based molecular markers can greatly benefit breeding efforts in such non-model species. RNA-seq approaches employing second generation sequencing platforms offer a unique avenue to rapidly identify gene-based SNPs. Additionally, haplotype markers can be rapidly generated from transcript-based SNPs since they have been found to be extremely utile in identification of genetic variants related to health, disease and response to environment as highlighted by the human HapMap project. Results RNA-seq was performed on two sweet cherry cultivars, Bing and Rainier using a 3' untranslated region (UTR) sequencing method yielding 43,396 assembled contigs. In order to test our approach of rapid identification of SNPs without any reference genome information, over 25% (10,100) of the contigs were screened for the SNPs. A total of 207 contigs from this set were identified to contain high quality SNPs. A set of 223 primer pairs were designed to amplify SNP containing regions from these contigs and high resolution melting (HRM) analysis was performed with eight important parental sweet cherry cultivars. Six of the parent cultivars were distantly related to Bing and Rainier, the cultivars used for initial SNP discovery. Further, HRM analysis was also performed on 13 seedlings derived from a cross between two of the parents. Our analysis resulted in the identification of 84 (38.7%) primer sets that demonstrated variation among the tested germplasm. Reassembly of the raw 3'UTR sequences using upgraded transcriptome assembly software yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs. Conclusions This approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB), genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

Most previous studies on genetic fingerprinting and cultivar relatedness in sweet cherry were based on isoenzyme, RAPD, and simple sequence repeat (SSR) markers. This study was carried out to assess the utility of single nucleotide polymorphism (SNP) markers generated from 3' untranslated regions (UTR) for genetic fingerprinting in sweet cherry. A total of 114 sweet cherry germplasm representing advanced selections, commercial cultivars, and old cultivars imported from different parts of the world were screened with seven SSR markers developed from other Prunus species and with 40 SNPs obtained from 3' UTR sequences of Rainier and Bing sweet cherry cultivars. Both types of marker study had 99 accessions in common. The SSR data was used to validate the SNP results. Results showed that the average number of alleles per locus, mean observed heterozygosity, expected heterozygosity, and polymorphic information content values were higher in SSRs than in SNPs although both set of markers were similar in their grouping of the sweet cherry accessions as shown in the dendrogram. SNPs were able to distinguish sport mutants from their wild type germplasm. For example, \"Stella\" was separated from \"Compact Stella.\" This demonstrates the greater power of SNPs for discriminating mutants from their original parents than SSRs. In addition, SNP markers confirmed parentage and also determined relationships of the accessions in a manner consistent with their pedigree relationships. We would recommend the use of 3' UTR SNPs for genetic fingerprinting, parentage verification, gene mapping, and study of genetic diversity in sweet cherry.

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 ( ) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force ( ), activation of the photoprotective q response, and the accumulation of H O . Surprisingly, was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome complex, and the ATP synthase, suggests that inefficient translation of leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H O in , which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H O accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H O , which in turn activates CEF. This activation could be from either H O acting as a redox signal, or by a secondary effect from H O inducing a deficit in ATP.

Sweet cherry is an important horticultural crop in Washington. Sweet cherry trees are grown as composite plants where the scion is grafted onto a rootstock. Rootstock genotype influences tree yield potential (among other traits) largely by affecting floral bud numbers. A transcriptomics approach (differential display and 3'UTR sequencing) was used to examine the genetic mechanism of the rootstock's effect on floral bud initiation. The first section of this dissertation outlines the development of key genomic resources for sweet cherry. Utilizing both reference mapping and de novo genome assembly for the sweet cherry variety 'Stella', 1.5 million single nucleotide polymorphisms were identified and 180Mb of genome were assembled respectively. The identified polymorphisms were screened and roughly 300,000 are located in peach genes and could be used as gene-based markers for future research. From this set, several genes involved in fruit ripening were found to contain nonsense mutations and could be the underlying cause for non-climacteric ripening in sweet cherry. Examining gene expression in developing floral buds of the scion, rootstock effects were identified through differential display and 3'UTR analyses. Differential display identified 207 transcripts that were putatively controlled by the rootstock genotype while 3'UTR identified 115 differentially expressed gene fragments. Several transcription factors including some with domains previously documented to control flowering were identified in these datasets and their expression levels were examined via qRT-PCR for verification. These studies have created vast amounts of information from both the sweet cherry genome and transcriptome. Many single nucleotide polymorphisms were identified and leveraged as DNA markers for screening sweet cherry populations. This demonstrated the rapid application of a small subset to aid sweet cherry breeding and population screening. Additionally, a computational program was developed to assess restriction sites in large datasets and used to convert the polymorphism information into cleaved amplified polymorphic sequence markers. Overall, the work described in this dissertation has laid the foundation for sweet cherry genomics research and identified several directions for future studies.

Abstract Harvesting of sweet cherry (Prunus avium L.) fruit is a labor-intensive process. Mechanical harvesting of sweet cherry fruit is feasible; however, it is dependent on the formation of an abscission zone at the fruit-pedicel junction. The natural propensity for pedicel-fruit abscission zone (PFAZ) formation varies by cultivar, and the general molecular basis for PFAZ formation is not well characterized. In this study, ethylene-inducible change in pedicel fruit retention force (PFRF) was recorded in a developmental time course with a concomitant analysis of the PFAZ transcriptome from three sweet cherry cultivars. In ‘Skeena’, mean PFRF for both control and treatment fruit dropped below the 0.40kg-force (3.92N) threshold for mechanical harvesting and indicating the formation of a discrete PFAZ. In ‘Bing’, mean PFRF for both control and treatment groups decreased over time. However, a mean PFRF conducive to mechanical harvesting was achieved only in the ethylene-treated fruit. While in ‘Chelan’ the mean PFRF of the control and treatment groups did not meet the threshold required for efficient mechanical harvesting. Transcriptome analysis of the PFAZ followed by the functional annotation, differential expression analysis, and gene ontology (GO) enrichment analyses of the data facilitated the identification of phytohormone-responsive and abscission-related transcripts as well as processes that exhibited differential expression and enrichment in a cultivar-dependent manner over the developmental time-course. Additionally, read alignment-based variant calling revealed several short variants in differentially expressed genes, associated with enriched gene ontologies and associated metabolic processes, lending potential insight into the genetic basis for different abscission responses between the cultivars. These results provide genetic targets for induction or inhibition of PFAZ formation, depending on the desire to harvest the fruit with or without the stem attached. Understanding the genetic mechanisms underlying the development of the PFAZ will inform future cultivar development while laying a foundation for mechanized sweet cherry harvest. Competing Interest Statement The authors have declared no competing interest. Footnotes * Added analysis related to the 6-hour time point. Repeated the variant analysis. Rewrote several sections. Rearranged and redrew the figures.

Agricultural productivity across the globe is threatened by environmental fluctuations. Years of human agronomic activity has further confounded the problem by increasing abiotic stress factors in the environment. There is an impending need for designer crops that can withstand an ever‐increasing level and range of abiotic stresses to continue to support the food needs of a burgeoning population. Plants are also being explored as a source of fuel, which necessitates production of appropriate crops in nonarable lands. Thus, abiotic stress‐resistant crops are relevant for both food and fuel needs in this century and beyond. Unlike biotic stresses, which are mostly monogenic traits and thus amenable to molecular breeding, abiotic stress resistance involves adjustments in morphological and physiological parameters. The multigenic nature of the trait makes it difficult, if not impossible, to breed crops in a timely manner that will be able to withstand the environmental fluctuations. This is particularly relevant for perennial crops. While annual crops can be bred in a matter of years, perennial crops stand to gain from directed biotechnological approaches. Several studies over the years in different plant species have identified various mechanisms and associated genes underlying abiotic stress resistance or tolerance. The scientific literature is replete with instances where these mechanisms were successfully unraveled in a heterologous host, tobacco that has long‐served as surrogate for test of novel gene function. These studies underpin the fact that abiotic stress resistance mechanisms are shared within the plant kingdom. This chapter summarizes the current body of knowledge on our understanding of abiotic stress tolerance as deciphered from studies performed in tobacco.