Improvements in semi-arid agriculture: Sorghum bicolor sub-population molecular marker development and mild salinity stress in Vitis

Abstract

A multidisciplinary approach was taken to investigate two crops used in semi-aridagriculture (sorghum and grapevine). This dissertation is the result of two distinct projects. The first focused on sub-population specific molecular markers in sorghum and the second on salinity tolerance in grapevines. The work on sorghum tested the hypothesis: due to the genetic distinctions among sorghum sub-populations, molecular markers can be designed to differentiate and screen them in a high-throughput manner using next-generation sequencing. The work on grapevine tested the hypothesis: due to the previously reported correlation between photosynthetic decline and increased foliar Cl- concentrations in Vitis, foliar Cl- concentration increase is the cause of photosynthetic decline in Vitis. For the investigation of sub-population specific molecular marker development in sorghum, bioinformatic and plant breeding approaches were taken. For the investigation of grapevine salinity tolerance, plant physiology and bioinformatics techniques were employed. Recent plant breeding studies of several species have demonstrated the utility of combining molecular assessments of genetic distance with trait-linked SNP genotyping during the development of parent lines to maximize yield gains due to heterosis. SSRs (Short Sequence Repeat markers; also known as microsatellite markers) are the molecular marker of choice to determine genetic diversity, but the methods historically used to sequence them have been burdensome. The ability to analyze SSRs in a higher-throughput manner independent of laboratory conditions would increase their utility in molecular ecology, germplasm curation, and plant breeding programs worldwide. This project reports simple bioinformatics methods that can be used to generate genome-wide de novo SSRs in silico followed by targeted Next Generation Sequencing (NGS) validation of those that provide the most information about sub-population identity of a breeding line, which influences heterotic group selection. While these methods were optimized in sorghum [Sorghum bicolor (L.) Moench], they were developed to be applied to any species with a reference genome and high-coverage whole-genome sequencing data of individuals from the sub-populations to be characterized. An analysis of published sorghum genomes selected to represent its five main races (bicolor, caudatum, durra, kafir, and guinea; 75 samples total) identified 130,120 SSR motifs. Average lengths were 23.8 bp and 95% were between 10 and 92 bp, making them suitable for NGS. Validation through targeted sequencing amplified 188 of 192 assayed SSR loci. Results highlighted the distinctness of accessions from the guinea sub-group margaritiferum from all other sorghum accessions, consistent with previous studies of nuclear and mitochondrial DNA. SSRs that efficiently fingerprinted margaritiferum individuals (Xgma1 – Xgma6) are presented. Developing similar fingerprints of other sub-populations (Xunr1 – Xunr182) was not possible due to the extensive admixture between them in the data set analyzed. In summary, these methods were able to fingerprint specific sub-populations when rates of admixture between them are low. Moderate levels of Cl- have been associated with grapevine salt tolerance. Photosynthesis in grapevine is negatively correlated with foliar Cl- concentration. To further test this hypothesis, multiple mild salinity experiments on four different Vitis genotypes (Cabernet Sauvignon, Riparia Gloire, Ramsey, and SC2) were conducted and physiology, ion concentrations and gene expression were assessed. The salt tolerant rootstock Ramsey had greater Cl- exclusion capabilities than V. vinifera cultivars both during rooted cutting greenhouse experiments and three years of field-grafted experiments; SC2 also excluded Cl-. Differential gene expression indicated that salinity affected transcript abundance more in salt sensitive genotypes, especially chloroplast related transcripts. The transcript abundances of known anion transporters were determined and a family of putative B transporters were associated with the Cl- exclusion phenotype. Photosynthesis and growth were maintained in Ramsey and SC2 under mild salinity. However, photosynthesis declined in Cabernet Sauvignon with isosmotic 20 mM salt concentrations of NaCl, KCl, or NaNO3, independent of the salt type. While foliar Clconcentrations did correlate with salt tolerance during control and saline conditions, it was not found to be the cause of photosynthetic decline in Vitis during mild salinity.