Poonam Chandel

Allele Mining in Solanum Germplasm: Cloning and Characterization of RB-Homologous Gene Fragments from Late Blight Resistant Wild Potato Species

The late blight disease can be managed by introduction of resistance (R) genes from the wild Solanum species into the cultivated potato. The R genes are mostly comprised of the nucleotide binding site-leucine rich repeat (NBS-LRR) domains and share nucleotide sequence homology in the crop species. In this study, we used potato R gene-specific primers to amplify homologous genes from wild species. A total of 39 wild species were tested for late blight resistance by challenge inoculation of Phytophthora infestans under controlled conditions. Of these, only 15 species were highly resistant (HR) and these were PCR (polymerase chain reaction) amplified by 53 primers representing 21 R genes of potato. Further, only single, distinct, and reproducible gene fragments were cloned and sequenced. Following sequence processing and analysis, 17 non-redundant sequences of RB-homologous genes were identified with uninterrupted open reading frames (ORFs) and nucleotide sequence homologies to known late blight R genes. Finally, 17 RB-homologous gene fragments amplified by the primers of the RB gene were isolated from 11 wild species. The isolation and characterization of 17 RB-homologous gene fragments from wild potato species may serve as an important genomic resource for the novel gene discovery in late blight resistance breeding programs.

Organelle Genome Analysis in Somatic Hybrids Between Solanum tuberosum and S. pinnatisectum Revealed Diverse Cytoplasm Type in Potato

Organelle genome diversity was analysed in interspecific potato somatic hybrids using chloroplast (cp)- and mitochondrial (mt)-specific molecular markers. Out of total 25 markers (15 cpDNA and 10 mtDNA) tested in total 16 samples, only four mtDNA primers (rpS14/cob, Nsm2, ALM4/ALM5 and ALM6/ALM7) detected polymorphism, whereas other primers were monomorphic. Cluster analysis showed higher genetic diversity among the genotypes by mtDNA profiles than that by cpDNA. Ten haplotypes were grouped by cluster analysis comprised of maximum seven genotypes in haplotype no. 3. Monomorphic markers did not reveal variability in our samples and suggest highly conserved organelle genomic regions. New genomic arrangements were observed in the somatic hybrids for mt polymorphic loci. Our study suggests that somatic hybrids are comprised of diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type chloroplast, and α-, β- and γ-type mitochondrial genome, and have unique potential to widen the cultivated potato gene pool by breeding methods.

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.