Exploring new transgenes for pyramiding resistance to potato tuber moth in potato

Potato tuber moth, Phthorimaea operculella (Zeller) (PTM), is a major insect pest of potato crops. The larvae mine into foliage and exposed tubers, making them difficult to control with insecticide applications or cultural methods. This thesis describes strategies to genetically engineer potato plants resistant to PTM. It includes the construction of chimeric cry genes, the insertion of these genes into standard binary vectors, and the subsequent transformation of potato using Agrobacterium-mediated gene transfer. Multiplex-PCR was performed to confirm the presence of the selectable marker gene and specific cry gene(s) in all regenerated lines. Multiplex RT-PCR was performed to confirm the transgene expression in transgenic lines. Southern blot and ELISA analyses were performed for transgenic lines to reveal the gene copy number and Cry protein level respectively. In transgenic potato it is often desirable to couple high-level expression in foliage with no expression in the edible tubers, especially for resistance to pests that primarily infest foliage. To accomplish this, a light inducible Lhca3 promoter for transcriptional control of cry1Ac9 and cry9Aa2 genes for resistance to PTM was successfully used. Significantly inhibited larval growth of PTM on excised greenhouse-grown leaves was observed in 51 % of the cry1Ac9-trangenic lines and 84% of the cry9Aa2-transgenic lines. RT-PCR analysis identified several transgenic lines with high levels of cry gene mRNA in leaves and no to low levels in tubers. This thesis has also demonstrated the effectiveness of two further cry genes, cry1Ba1 and cry1Ca5, for PTM resistance in transgenic potato. This is important to assemble a suite of genes capable of effecting PTM control to allow future evaluation of different approaches toward deployment of PTM resistance management strategies. Over 90% of the cry1Ca5-transgenic lines gave 100 % larval mortality of PTM on excised greenhouse-grown leaves and tubers bioassays. 40-50% of the cry1Ba1-transgenic lines gave 50 to 100% of larval mortality of PTM on excised greenhouse-grown leaves and tubers bioassays. An experimental approach to simulate cry gene pyramiding in potatoes was designed and used to assess the interaction of three different cry genes, cry1Ac9, cry9Aa2 and cry1Ba1 genes, in pair wise combinations. It is important to investigate how the expression of two dissimilar Bt toxins interact to confer insect resistance in transgenic plants before developing pyramided plants. The results showed that all combinations of the three cry genes were largely consistent with additive impacts on PTM larvae, although results from the combination of the cry1Ac9 and cry9Aa2 genes were suggestive of slight synergistic effects. Potato lines transgenic for both cry1Ac9 gene and cry9Aa2 gene were developed to investigate the effects of gene pyramiding. The feasibility of two strategies for Agrobacterium-mediated trans gene pyramiding in potato were also evaluated. The results of this thesis demonstrated that potatoes transgenic for cry genes offer a valuable approach to develop elite plant material for potential use as an additional component in integrated pest management of PTM

Tuber shape and eye depth variation in a diploid family of Andean potatoes.

BACKGROUND: Tuber appearance is highly variable in the Andean cultivated potato germplasm. The diploid backcross mapping population ‘DMDD’ derived from the recently sequenced genome ‘DM’ represents a sample of the allelic variation for tuber shape and eye depth present in the Andean landraces. Here we evaluate the utility of morphological descriptors for tuber shape for identification of genetic loci responsible for the shape and eye depth variation. RESULTS: Subjective morphological descriptors and objective tuber length and width measurements were used for assessment of variation in tuber shape and eye depth. Phenotypic data obtained from three trials and male–female based genetic maps were used for quantitative trait locus (QTL) identification. Seven morphological tuber shapes were identified within the population. A continuous distribution of phenotypes was found using the ratio of tuber length to tuber width and a QTL was identified in the paternal map on chromosome 10. Using toPt-437059, the marker at the peak of this QTL, the seven tuber shapes were classified into two groups: cylindrical and non-cylindrical. In the first group, shapes classified as ‘compressed’, ‘round’, ‘oblong’, and ‘long-oblong’ mainly carried a marker allele originating from the male parent. The tubers in this group had deeper eyes, for which a strong QTL was found at the same location on chromosome 10 of the paternal map. The non-cylindrical tubers classified as ‘obovoid’, ‘elliptic’, and ‘elongated’ were in the second group, mostly lacking the marker allele originating from the male parent. The main QTL for shape and eye depth were located in the same genomic region as the previously mapped dominant genes for round tuber shape and eye depth. A number of candidate genes underlying the significant QTL markers for tuber shape and eye depth were identified. CONCLUSIONS: Utilization of a molecular marker at the shape and eye depth QTL enabled the reclassification of the variation in general tuber shape to two main groups. Quantitative measurement of the length and width at different parts of the tuber is recommended to accompany the morphological descriptor classification to correctly capture the shape variation. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12863-015-0213-0) contains supplementary material, which is available to authorized users

A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Most published genome sequences are drafts, and most are dominated by computational gene prediction. Draft genomes typically incorporate considerable sequence data that are not assigned to chromosomes, and predicted genes without quality confidence measures. The current Actinidia chinensis (kiwifruit) 'Hongyang' draft genome has 164\ua0Mb of sequences unassigned to pseudo-chromosomes, and omissions have been identified in the gene models

Additional file 3: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Comparison of predicted paired end distance to genome.Heatmaps of alignment distance scores for the alignment of the read pairs from the 9Kb long-insert mate-paired-end (LIMP) library to each of the 29 chromosomes within the Red5 whole genome assembly and. Individual chromosome plots were prepared using hagfish_blockplot from the software program ‘hagfish’ ( https://github.com/mfiers/hagfish/ ). Individual images were cropped for height (not length) then cut and pasted into a table format for easier viewing. Each image depicted the entire length of the chromosome but all images are of standard length irrespective of chromosome length. Green regions indicate mate pairs aligning to the whole genome sequence within the expected distance of the library. Black indicates regions without mate pair alignment. Pinkish-red indicates regions where the distance between mated paired end reads is shorter (assembly compression relative to physical genome) or longer (assembly expansion relative to physical genome). (PPTX 432 kb

Additional file 8: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Sequenced cDNA’s used to verify the gene models.Fasta formatted predicted protein sequences of 550 bidirectionally sequenced expressed sequence tag clones from A. chinensis var. chinensis used in evaluating manually annotated gene models of Red5. (FASTA 220 kb

Additional file 7: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

Details of sequenced cDNA’s generated. Fasta formatted sequences of 812 bidirectionally sequenced expressed sequence tag clones from A. chinensis var. chinensis used in evaluating manually annotated gene models of Red5. (FASTA 1204 kb

Additional file 10: of A manually annotated Actinidia chinensis var. chinensis (kiwifruit) genome highlights the challenges associated with draft genomes and gene prediction in plants

The manual annotation process.Flow diagram of manual annotation process. A. Timeline showing the manual annotation process. *see materials and methods. B. Annotation followed a 5 step process. The annotator training was completed in the form of both workshops and YouTube training videos. ** https://www.youtube.com/playlist?list=PLcBe8nhQVgUg1zqOsdeRuVq9QVsLfj_Y9 . (PPTX 47 kb