James H. Oard

High transgene expression levels in sugarcane (Saccharum officinarum L.) driven by the rice ubiquitin promoter RUBQ2

Abstract Discovery and evaluation of new regulatory elements are urgently needed for future improvements in transgene expression in sugarcane. The rice polyubiquitin promoter RUBQ2 was fused to the β-glucuronidase (GUS) reporter gene and expression levels were monitored after particle bombardment and Agrobacterium-mediated transformation. DNA constructs containing RUBQ2 promoter produced higher levels of transient GUS expression by particle bombardment in calli and leaves compared with control constructs containing the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter or the maize polyubiquitin Ubi-1 promoter. Similarly, the RUBQ2 promoter produced higher levels of transient GUS expression in calli than the Ubi-1 promoter via Agrobacterium-mediated transformation using strains LABA4404 and AGL1. Use of Agrobacterium strain LBA4404 resulted in a 7-fold increase in transient gene expression over strain AGL1. Stably transformed plants were produced via particle bombardment, and stable GUS expression levels by RUBQ2 were increased 1.6-fold over those by Ubi-1, while no GUS expression was detected in the transgenic plants with CaMV 35S promoter. Southern blotting analysis revealed stable, multi-copy GUS gene integration into the sugarcane genome via particle bombardment. Results from this study indicate that RUBQ2 can serve as a new regulatory element to provide high levels of transgene expression in sugarcane.

Whole genome sequencing of elite rice cultivars as a comprehensive information resource for marker assisted selection.

Current advances in sequencing technologies and bioinformatics revealed the genomic background of rice, a staple food for the poor people, and provided the basis to develop large genomic variation databases for thousands of cultivars. Proper analysis of this massive resource is expected to give novel insights into the structure, function, and evolution of the rice genome, and to aid the development of rice varieties through marker assisted selection or genomic selection. In this work we present sequencing and bioinformatics analyses of 104 rice varieties belonging to the major subspecies of Oryza sativa. We identified repetitive elements and recurrent copy number variation covering about 200 Mbp of the rice genome. Genotyping of over 18 million polymorphic locations within O. sativa allowed us to reconstruct the individual haplotype patterns shaping the genomic background of elite varieties used by farmers throughout the Americas. Based on a reconstruction of the alleles for the gene GBSSI, we could identify novel genetic markers for selection of varieties with high amylose content. We expect that both the analysis methods and the genomic information described here would be of great use for the rice research community and for other groups carrying on similar sequencing efforts in other crops.

Molecular marker variability for southern root-knot nematode resistance in sweetpotato

Amplified fragment length polymorphism (AFLP) marker profiles for individuals in two F1 populations of sweetpotato [Ipomoea batatas (L.) Lam] were used in association studies to identify AFLP markers suitable for identification of plants possessing a resistant reaction to southern root-knot nematode race 3 [Meloidogyne incognita (Kofoid and White) Chitwood]. Population one consisted of 48 half-sib genotypes developed at the Louisiana State University (LSU) AgCenter. The second population consisted of 54 full-sibs developed by the East African and International Potato Center (CIP) sweetpotato breeding programs. Results for plant nematode resistance indicate a bimodal distribution among the genotypes for the LSU population and a normal distribution for the CIP population. Using analysis of molecular variance (AMOVA) at P < 0.001 and two multivariate analysis techniques i.e logistic regression and discriminant analysis, 5 and 4 AFLP markers that had a strong and significant association with respect to the resistance trait were selected for the LSU and CIP populations, respectively. A comparative analysis of the power of discriminant analysis models for southern root-knot nematode resistance class prediction achieved 88.78% (LSU) and 88.04% (CIP) classification efficiencies.

Confirming QTLs and Finding Additional Loci Responsible for Resistance to Rice Sheath Blight Disease

Rice sheath blight disease, caused by Rhizoctonia solani AG1-1A, is one of the most destructive rice diseases worldwide. Utilization of host resistance is the most economical and environmentally sound strategy in managing sheath blight (ShB). Ten ShB quantitative trait loci (QTLs) were previously mapped in a Lemont × Jasmine 85 recombinant inbred line (LJRIL) population using greenhouse inoculation methods at an early vegetative stage. However, confirmation of ShB-resistant QTLs under field conditions is critical for their utilization in marker-assisted selection (MAS) for improving ShB resistance in new cultivars. In the present study, we evaluated ShB resistance using 216 LJRILs under field conditions in Arkansas, Texas, and Louisiana during 2008 and 2009. We confirmed the presence of the major ShB-QTL qShB9-2 based on the field data and also identified one new ShB-QTL between markers RM221 and RM112 on chromosome 2 across all three locations. Based on the field verification of ShB evaluations, the microchamber and mist-chamber assays were simple, effective, and reliable methods to identify major ShB-QTLs like qShB9-2 in the greenhouse at early vegetative stages. The markers RM215 and RM245 were found to be closely linked to qShB9-2 in greenhouse and field assays, indicating that they will be useful for improving ShB resistance in rice breeding programs using MAS.