Ksenija Gasic

RNA Extraction From Different Apple Tissues Rich in Polyphenols and Polysaccharides for cDNA Library Construction

Recovering RNA of high quality and quantity is a prerequisite for ensuring representation of all expressed genes in a cDNA library. An efficient procedure for isolating RNA from bud, internodal shoot, flower, and fruit tissues of apple has been developed. This protocol does not involve the use of phenol, lyophilization, or ultracentrifugation. In addition, this protocol overcomes problems of both RNA degradation and low yield attributed to oxidation by polyphenolic compounds and coprecipitation with polysaccharides, both abundant components in apple fruit and flower tissues. Isolated RNA is of high quality and is undegraded as assessed by spectrophotometric readings and electrophoresis in denaturing agarose gels. RNA quality is further assessed following its use in reverse transcription and cDNA library construction, and it can be used for a number of downstream analyses, including Northern blot hybridization and reverse transcription-polymerase chain reaction (RT-PCR). With this modified protocol, 25–900 μg of total RNA is routinely obtained from 1 g of fresh material. This method is of low cost and easy to perform.

Development of a set of SNP markers present in expressed genes of the apple

Molecular markers associated with gene coding regions are useful tools for bridging functional and structural genomics. Due to their high abundance in plant genomes, single nucleotide polymorphisms (SNPs) are present within virtually all genomic regions, including most coding sequences. The objective of this study was to develop a set of SNPs for the apple by taking advantage of the wealth of genomics resources available for the apple, including a large collection of expressed sequenced tags (ESTs). Using bioinformatics tools, a search for SNPs within an EST database of approximately 350,000 sequences developed from a variety of apple accessions was conducted. This resulted in the identification of a total of 71,482 putative SNPs. As the apple genome is reported to be an ancient polyploid, attempts were made to verify whether those SNPs detected in silico were attributable either to allelic polymorphisms or to gene duplication or paralogous or homeologous sequence variations. To this end, a set of 464 PCR primer pairs was designed, PCR was amplified using two subsets of plants, and the PCR products were sequenced. The SNPs retrieved from these sequences were then mapped onto apple genetic maps, including a newly constructed map of a Royal Gala x A689-24 cross and a Malling 9 x Robusta 5, map using a bin mapping strategy. The SNP genotyping was performed using the high-resolution melting (HRM) technique. A total of 93 new markers containing 210 coding SNPs were successfully mapped. This new set of SNP markers for the apple offers new opportunities for understanding the genetic control of important horticultural traits using quantitative trait loci (QTL) or linkage disequilibrium analysis. These also serve as useful markers for aligning physical and genetic maps, and as potential transferable markers across the Rosaceae family.

Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance.

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an ArabidopsisthalianaAtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.

Expression of Arabidopsis phytochelatin synthase in Indian mustard (Brassica juncea) plants enhances tolerance for Cd and Zn

Phytochelatins (PCs) are heavy metal binding peptides that play an important role in sequestration and detoxification of heavy metals in plants. In this study, our goal was to develop transgenic plants with increased tolerance for and accumulation of heavy metals from soil by expressing an ArabidopsisthalianaAtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A 35S promoter fused to a FLAG–tagged AtPCS1 cDNA was expressed in Indian mustard, and transgenic lines, designated pc lines, were evaluated for tolerance to and accumulation of Cd and Zn. Transgenic plants with moderate AtPCS1 expression levels showed significantly higher tolerance to Cd and Zn stress, but accumulated significantly less Cd and Zn than wild type plants in both shoot and root tissues. However, transgenic plants with highest expression of the transgene did not exhibit enhanced Cd and Zn tolerance. Shoots of Cd-treated pc plants had significantly higher levels of phytochelatins and thiols than wild-type plants. Significantly lower concentrations of gluthatione in Cd-treated shoot and root tissues of transgenic plants were observed. Moderate expression levels of phytochelatin synthase improved the ability of Indian mustard to tolerate certain levels of heavy metals, but at the same time did not increase the accumulation potential for Cd and Zn.

Enhanced resistance to early blight in transgenic tomato lines expressing heterologous plant defense genes

Genes coding for an iris ribosomal-inactivating protein (I-RIP), a maize β-glucanase (M-GLU), and a Mirabilis jalapa antimicrobial peptide (Mj-AMP1) were separately introduced into tomato (Lycopersicon esculentum cv. Sweet Chelsea) cotyledons via Agrobacterium tumefaciens-mediated transformation. Transgenic lines carrying each of the transgenes were confirmed for integration into the tomato genome using Southern blot hybridization. Transcription of I-RIP, M-GLU, and Mj-AMP1 genes in various transgenic lines was determined using Northern blot analysis. Plants of selected transgenic lines were inoculated with a 2–3×104 conidial spores/ml suspension of the fungal pathogen Alternaria solani, the causal agent of tomato early blight. Compared to control (non-transformed) plants, two transgenic lines carrying either a M-GLU or Mj-AMP1 showed enhanced resistance to early blight disease. None of the four lines carrying the I-RIP transgene showed increased resistance to early blight.

Gene Expression is Highly Regulated in Early Developing Fruit of Apple

An oligonucleotide-based microarray for apple was developed consisting of ~40,000 sequences, along with positive and negative controls, obtained from 34 cDNA libraries constructed from both vegetative and reproductive tissues at different stages of development, varying genotypes, and under different biotic and abiotic stresses. This apple microarray was used to investigate global gene expression profiles in early developing fruit of three apple genotypes, including “Golden Delicious”, “Gala”, and “Fuji”. A set of 3,348 genes, exhibiting significant differential expression profiles among the three different genotypes, was identified. This set primarily included genes encoding enzymes involved in metabolism and genes related to cell cycle. Differentially expressed genes were grouped into 17 functional categories. Quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) confirmed differential expression for most genes detected in the microarray analysis, particularly those involved in cell division, cell expansion, and cell enlargement. Among those genes investigated, EF-1 alpha and PRP exhibited differential expression in different apple genotypes and demonstrated a regulatory role during early fruit development. Moreover, a total of 12 differentially expressed genes were identified during early fruit development in three genotypes of apple. These included genes encoding for aminotransferase family protein, DnaJ heat shock, histone, rubisco activase, and tetratricopeptide (TPR) repeat containing protein. This genome-wide analysis suggested that genes engaged in early fruit development among different genotypes of apple are highly regulated.

Genotyping by Sequencing for SNP-Based Linkage Map Construction and QTL Analysis of Chilling Requirement and Bloom Date in Peach [ Prunus persica (L.) Batsch]

Low-cost, high throughput genotyping methods are crucial to marker discovery and marker-assisted breeding efforts, but have not been available for many ‘specialty crops’ such as fruit and nut trees. Here we apply the Genotyping-By-Sequencing (GBS) method developed for cereals to the discovery of single nucleotide polymorphisms (SNPs) in a peach F2 mapping population. Peach is a genetic and genomic model within the Rosaceae and will provide a template for the use of this method with other members of this family. Our F2 mapping population of 57 genotypes segregates for bloom time (BD) and chilling requirement (CR) and we have extensively phenotyped this population. The population derives from a selfed F1 progeny of a cross between ‘Hakuho’ (high CR) and ‘UFGold’ (low CR). We were able to successfully employ GBS and the TASSEL GBS pipeline without modification of the original methodology using the ApeKI restriction enzyme and multiplexing at an equivalent of 96 samples per Illumina HiSeq 2000 lane. We obtained hundreds of SNP markers which were then used to construct a genetic linkage map and identify quantitative trait loci (QTL) for BD and CR.

Construction of High Density Sweet Cherry (Prunus avium L.) Linkage Maps Using Microsatellite Markers and SNPs Detected by Genotyping-by-Sequencing (GBS)

Linkage maps are valuable tools in genetic and genomic studies. For sweet cherry, linkage maps have been constructed using mainly microsatellite markers (SSRs) and, recently, using single nucleotide polymorphism markers (SNPs) from a cherry 6K SNP array. Genotyping-by-sequencing (GBS), a new methodology based on high-throughput sequencing, holds great promise for identification of high number of SNPs and construction of high density linkage maps. In this study, GBS was used to identify SNPs from an intra-specific sweet cherry cross. A total of 8,476 high quality SNPs were selected for mapping. The physical position for each SNP was determined using the peach genome, Peach v1.0, as reference, and a homogeneous distribution of markers along the eight peach scaffolds was obtained. On average, 65.6% of the SNPs were present in genic regions and 49.8% were located in exonic regions. In addition to the SNPs, a group of SSRs was also used for construction of linkage maps. Parental and consensus high density maps were constructed by genotyping 166 siblings from a ‘Rainier’ x ‘Rivedel’ (Ra x Ri) cross. Using Ra x Ri population, 462, 489 and 985 markers were mapped into eight linkage groups in ‘Rainier’, ‘Rivedel’ and the Ra x Ri map, respectively, with 80% of mapped SNPs located in genic regions. Obtained maps spanned 549.5, 582.6 and 731.3 cM for ‘Rainier’, ‘Rivedel’ and consensus maps, respectively, with an average distance of 1.2 cM between adjacent markers for both ‘Rainier’ and ‘Rivedel’ maps and of 0.7 cM for Ra x Ri map. High synteny and co-linearity was observed between obtained maps and with Peach v1.0. These new high density linkage maps provide valuable information on the sweet cherry genome, and serve as the basis for identification of QTLs and genes relevant for the breeding of the species.

BAC-end sequence-based SNPs and Bin mapping for rapid integration of physical and genetic maps in apple.

A genome-wide BAC physical map of the apple, Malus x domestica Borkh., has been recently developed. Here, we report on integrating the physical and genetic maps of the apple using a SNP-based approach in conjunction with bin mapping. Briefly, BAC clones located at ends of BAC contigs were selected, and sequenced at both ends. The BAC end sequences (BESs) were used to identify candidate SNPs. Subsequently, these candidate SNPs were genetically mapped using a bin mapping strategy for the purpose of mapping the physical onto the genetic map. Using this approach, 52 (23%) out of 228 BESs tested were successfully exploited to develop SNPs. These SNPs anchored 51 contigs, spanning approximately 37 Mb in cumulative physical length, onto 14 linkage groups. The reliability of the integration of the physical and genetic maps using this SNP-based strategy is described, and the results confirm the feasibility of this approach to construct an integrated physical and genetic maps for apple.

Comparative analysis and functional annotation of a large expressed sequence tag collection of apple.

A total of 34 apple (Malus × domestica Borkh.) cDNA libraries were constructed from root, leaf, bud, shoot, flower, and fruit tissues, at various developmental stages and/or under biotic or abiotic stress conditions, and of several genotypes. From these libraries, 190,425 clones were partially sequenced from the 5′ end and 42,619 clones were sequenced from the 3′ end, and a total of 182,241 high‐quality expressed sequence tags (ESTs) were obtained. These coalesced into 23,442 tentative contigs and 9843 singletons, for a total of 33,825 apple unigenes. Functional annotation of this unigene set revealed an even distribution of apple sequences among the three main gene ontology categories. Of ∼33,000 apple unigenes, 8437 (25%) had no detectable homologs (E >0.1) in the Arabidopsis genome. When the entire apple unigene set was compared with the entire citrus [Citrus sinensis (L.) Osbeck] unigene set and the poplar (Populus trichocarpa Torr. & Gray) predicted proteome, both members of the core eudicot and rosids clade, 13,521 of apple unigenes matched one or more sequences in citrus, while 25,817 had counterparts in the poplar protein database. Apple–Arabidopsis–citrus–poplar comparisons revealed closer evolutionary relationships between apple and poplar than with the other two species. Genes involved in basic metabolic pathways appear to be largely conserved among apple, citrus, poplar, and Arabidopsis.