Comparative Analysis and Functional Annotation of a Large Expressed Sequence Tag Collection of Apple

Abstract

A total of 34 apple (Malus × domestica Borkh.) cDNA libraries were constructed from root, leaf, bud, shoot, fl ower, 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 5ʹ end and 42,619 clones were sequenced from the 3ʹ 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.

Gene Expression is Highly Regulated in Early Developing Fruit of Apple

“…Geneva 3041×Malus sieversii. Tissues used for library construction were collected from either trees grown in an experimental orchard (University of Illinois, Urbana, IL) or from greenhouse-grown plants subjected to various biotic (Cornell University and USDA-ARS, Geneva, NY) or abiotic (USDA-ARS, Kearneysville, WV) stresses, as previously described (Gasic et al 2009). …”

“…cDNAs from replicate extractions were pooled in equal amounts before adaptor ligation. cDNA libraries were constructed following procedures described in Bonaldo et al (1996) and Soares et al (1994) and as reported by Gasic et al (2009). Briefly, double-stranded cDNAs were sizeselected to enrich for molecules >500 bp, adaptored with EcoRI adapters (Promega, Madison, WI) at both ends, and then digested with NotI.…”

“…Clone preparation and sequencing were as described by Gasic et al (2009). All sequences were deposited in Genbank, and contig assembly was done on "clean" sequences having minimum lengths of 100 nucleotides and minimum quality scores of 20, following vector trimming and discarding of low-quality sequences.…”

dHPLC efficiency for semi-automated cDNA-AFLP analyses and fragment collection in the apple scab-resistance gene model

“…The second most upregulated TDF in this category is similar to an Arabidopsis transcript encoding for a LRPK protein and to an apple EST from cv. GoldRush leaf tissue challenged with V. inaequalis (Gasic et al 2009). The third up-regulated TDF aligns with an apple EST from root tissues challenged with Phytophtora cactorum (this EST is also similar to an Arabidopsis transcript for a transducin/WD40-repeat containing protein) and its expression levels were significantly up-regulated at 24 and 48 hpi.…”

Ectopic Expression of Apple F3′H Genes Contributes to Anthocyanin Accumulation in the Arabidopsis tt7 Mutant Grown Under Nitrogen Stress

“…However, genes encoding F3#H have not yet been reported in apple, although they play important roles in both flower and fruit coloration. Recently, a large number of EST sequences from apple have been developed in our laboratory and deposited in the GenBank/EMBL/DDBJ databases (Gasic et al, 2009). These EST sequences together with our previously constructed bacterial artificial chromosome (BAC) libraries (Xu et al, 2001(Xu et al, , 2002 provide us with a unique opportunity to investigate genes involved in flavonoid biosynthesis in apple.…”