J. Mitchell McGrath

Construction of a sugar beet BAC library from a hybrid with diverse traits

A bacterial artificial chromosome (BAC) library of the 750-Mbp sugar beet genome represented in hybrid US H20 was constructed fromHind III-digested DNA, with an average insert size of 120 kbp. US H20 is a variety grown in the eastern United States. It exhibits heterosis for emergence and yield, presumably because of its hybridity between eastern and western US germplasm sources. Filter arrays were used to assess the abundance and distribution of particular nucleotide sequences. An rRNA gene probe found that 1.2% of the library carried sequences similar to these highly repetitive and conserved sequences. A simple sequence repeat element (CA)8 thought to be predominantly distributed throughout centromere regions of all chromosomes was present in 1.7% of clones. For more than half of the 28 randomly chosen expressed sequence tags (ESTs) used as probes, a higher-than-expected number of single-copy hybridization signals was observed. Assuming 6× genome coverage, this suggests that many duplicate genes exist in the beet genome.

Cultivar-specific seedling vigor and expression of a putative oxalate oxidase germin-like protein in sugar beet (Beta vulgaris L.)

For genetic screening and breeding purposes, an in vitro germination system that reflects relative field emergence potential was used to screen for germination-enhancing and stress-induced genes from germinating seedlings from two varieties of sugar beet. Three full-length germin-like protein (GLP) gene classes were recovered from stress-germinated seedlings of a superior emerging variety. GLP gene expression, oxalate oxidase protein activity, the H2O2 content of stressed seedlings, but not catalase activity, were induced by stress germination conditions (e.g. excess water, NaCl, mannitol, or oxalate) in a good emerging hybrid and were not induced in a poor emerging variety. Only one of the three germin-like protein genes (BvGer165) was differentially regulated, and was induced only in the good emerger. Hydrogen peroxide promoted germination and partially compensated solute-depressed germination percentages. Unlike other solute recovery by hydrogen peroxide regimes, recovery in oxalic acid plus H2O2 was cultivar-independent. A block in oxalate metabolism is postulated to contribute to lower germination under stress in the lower emerging variety. Selection for stress-induced germin expression, or for down-stream targets, presents the first direct target to enable breeding for improved field emergence of sugar beet.

Introgression and stabilization oferwinia tuber soft rot resistance into potato after somatic hybridization ofsolanum tuberosum ands. brevidens

Resistance to potato tuber soft rot caused byErwinia carotovora was transferred fromSolanum brevidens to the cultivated potato over the course of four backcross generations originating from a somatic hybrid. Soft rot reactions were determined via a tuber plug inoculation method developed during the course of these experiments. Soft rot resistance was highest in the somatic hybrid (only ca. 20% of tubers and plugs showed evidence of severe rotting) and lowest among progeny of control potato x potato crosses (ca. 80% of tuber plugs showed severe rot). Backcross generations involving somatic hybrids were intermediate in their reaction, and resistance stabilized to about 60% of tuber plugs showing severe rot in the BC2 through the BC4. Reciprocal crosses showed no difference in the inheritance of soft rot resistance, indicating that neitherS. brevidens norS. tuberosum donor cytoplasm had a significant effect on the expression of resistance. Crosses between BC3 siblings where noS. brevidens genetic markers were detected but resistance was segregating demonstrated a dosage effect for soft rot resistance. We conclude that introgression of soft rot resistance has occurred and that at least one locus responsible for resistance inS. brevidens now resides in theS. tuberosum genome.

Functional differentiation of the sugar beet root system as indicator of developmental phase change

Developmental phase transitions in the plant root system have not been well characterized. In this study we compared the dynamics of sucrose accumulation with changes in gene expression analyzed with cDNA-amplified fragment length polymorphism (AFLP) in the developing tap root of sugar beet (Beta vulgaris, L.) during the first 9 weeks after emergence (WAE). Although differences between lines were evident as soon as 9 WAE, sucrose showed a marked increase in the rate of accumulation between 4 and 6 WAE and a remarkable shift in gene expression was observed between 5 and 6 WAE. These changes were evident in two unrelated genetic backgrounds and suggest that physiological and gene expression changes represent a functional differentiation of the tap root. These changes were considered as indicators of a developmental change in the sugar beet root system. To identify genes and metabolic pathways involved in this developmental shift, a root cDNA library was hybridized with probes enriched for 3- and 7-WAE transcripts and differentially expressed transcripts were analyzed by cDNA microarray. Several genes involved in the regulation of tissue development were found to be differentially regulated. Genes involved in protein metabolism, disease-related and secretory system were upregulated before the functional differentiation transition, while genes under hormonal control were upregulated after the functional differentiation transition. This developmental phase change of the root system is important to understand plant developmental regulation at the whole-plant level and will likely be useful as early selection parameter in breeding programs.

Evaluation of genetic diversity and root traits of sea beet accessions of the Adriatic Sea coast

Thirty-nine sea beet [Beta vulgaris L. ssp. maritima (L.) Arcang.] accessions of the Adriatic coast were screened genetically and for their adaptive morpho-functional root traits in order to identify new sources of abiotic resistances for sugar beet breeding programs. Genetic diversity was evaluated with 21 microsatellites markers that identified 44 polymorphic alleles. Sea beets grouped into two main clusters: the West and the East Adriatic coast groups, with the latter showing higher genetic diversity. Among sea beet accessions with desirable root traits, four accessions have proved to be interesting for sugar beet [B. vulgaris (L.) ssp. vulgaris] breeding aimed to improve tolerance to nutritional stresses. Lastovo (ID 29) and Zut (ID 34) accessions were characterized by the highest values of RER, TRL, FRL and RSA still maintaining a high value of RTD, while Grado (ID 21) an Portic (ID 23) accessions were characterized by the highest RTD, but with low values of RER, TRL, FRL and RSA parameters.

Assisted Breeding in Sugar Beets

Molecular insight and methods applied to plant breeding and germplasm enhancement is the goal of assisted breeding, also known as marker assisted breeding, marker assisted selection, molecular plant breeding, or genome-wide selection, among others. The basic idea is that most, if not all, heritable components of agronomic performance can be assayed with an unbiased, uniform, and comparatively inexpensive set of measures that are highly or completely correlated with phenotypic values. Typically such heritable components are genetically controlled, thus the panoply of DNA-based correlative methods should provide the requisite tools for assisted breeding to be successful. Whether DNA-based methods are successful in predicting agronomic performance depends on a host of considerations, many of which remain to be completely addressed for sugar beet, and thus assisted breeding remains somewhat empirical in practice, at least with respect to the specific traits of interest. Most sugar beet breeding and improvement programs have initiated assisted breeding efforts, primarily as a first step to discover marker associations with traits. These initial activities serve a number of important functions such as determining the complexity of trait inheritance, narrowing the possible range of genes and loci involved in phenotypic expression in different populations, and following the progress of sugar beet improvement following hybridization with wild and unadapted germplasm. It is unlikely that breeding will or should ever rely completely on assisted methods, however selection of the most promising germplasm can be greatly accelerated using molecular insights and knowledge.Molecular insight and methods applied to plant breeding and germplasm enhancement is the goal of assisted breeding, also known as marker assisted breeding, marker assisted selection, molecular plant breeding, or genome-wide selection, among others. The basic idea is that most, if not all, heritable components of agronomic performance can be assayed with an unbiased, uniform, and comparatively inexpensive set of measures that are highly or completely correlated with phenotypic values. Typically such heritable components are genetically controlled, thus the panoply of DNA-based correlative methods should provide the requisite tools for assisted breeding to be successful. Whether DNA-based methods are successful in predicting agronomic performance depends on a host of considerations, many of which remain to be completely addressed for sugar beet, and thus assisted breeding remains somewhat empirical in practice, at least with respect to the specific traits of interest. Most sugar beet breeding and improvement programs have initiated assisted breeding efforts, primarily as a first step to discover marker associations with traits. These initial activities serve a number of important functions such as determining the complexity of trait inheritance, narrowing the possible range of genes and loci involved in phenotypic expression in different populations, and following the progress of sugar beet improvement following hybridization with wild and unadapted germplasm. It is unlikely that breeding will or should ever rely completely on assisted methods, however selection of the most promising germplasm can be greatly accelerated using molecular insights and knowledge.

Methylation of somatic and sperm DNA in the homosporous fern Ceratopteris richardii

Plants, in general, have a high proportion of their CpG and CpNpG nucleotide motifs modified with 5-methylcytosine (5mC). Developmental changes in the proportion of 5mC are evident in mammals, particularly during gametogenesis and embryogenesis, but little information is available from flowering plants due to the intimate association of gametes with sporophytic tissues. In ferns, sperm are uninucleate and free-swimming and thus are easily isolated. We have examined 5mC in DNA isolated from fern sperm and other tissues with methylation-sensitive and -insensitive restriction enzyme isoschizomers, Southern blots probed with chloroplast and nuclear ribosomal RNA genes and end-labeled restriction fragments. We conclude that fern sperm DNA is methylated to a similar or greater degree than DNA isolated from either sporophytes or gametophytes.

Sugar Beet, Energy Beet, and Industrial Beet

Sugar beet (Beta vulgaris) is a temperate root crop grown primarily as a source of sucrose for human diets. Breeding has focused on sucrose yield, which is simply the product of total root yield times the proportion of sucrose in the harvested roots, minus loss of sucrose in molasses due to impurities. Molasses is a source of betaine, which is recovered as a feed supplement. The non-sucrose dry matter (marc), mostly cell wall material, is used primarily for fodder. Beet juice, molasses, or sucrose solutions are easily fermented into ethanol, while whole beets or pulp is being used for biogas production. Beets have potential as a bio-resource for additional industrial and chemical feedstocks. Sugar beet vinasse is rich in glutamate that may be economically converted and substituted for some high-value petrochemicals. Cell wall material is low in lignin and thus is readily saccharified and fermented and may also serve as an economical resource for monosaccharides for which novel polymers may be developed. Procedures for sugar beet breeding are directly applicable to breed beets for alternative and novel uses.

Discovery of resistance to seedling disease caused by Rhizoctonia solani AG2-2, description of the host-pathogen interaction, and development of a seedling disease screening nursery

Sugarbeet seedling mortality caused by the damping-off pathogen Rhizoctonia solani AG2-2 is perhaps the most serious biotic cause of stand failure in the Michigan growing regions, and is likely important worldwide. Resistance to the seedling disease has not been available. Resistance would be beneficial in establishing uniform stands of beets, and the resulting improved harvest quality of similarly sized beets delivered to the factory. An initial set of experiments to examine disease progression in susceptible hosts was conducted with high and low virulence isolates in order to identify targets of opportunity for biotechnology manipulation, and during this work, the crown-and-root-rot (CRR) resistant release EL51 was demonstrated to survive early challenge by the highly virulent isolate Rl. Subsequently, the host-pathogen interaction was examined in detail using light and fluorescence microscopy. The resistance reaction was characterized by the failure of the pathogen to ramify the water-conducting stele tissues of the young hypocotyl, with an apparent barrier at the narrow endodermis. Field experiments were initiated to determine if resistance was expressed under agronomic conditions by a simple modification of traditional CRR screening to that of inoculating 3-week-old seedlings. Full stands of EL51 were present at the end of the season, and stands of the susceptible hybrid USH20 were decimated. In 2006, the entire East Lansing CRR nursery was inoculated at the seedling stage, and clear germplasm differences in disease reaction were seen. Interestingly, the disease continued to develop throughout the growing season, suggesting both seedling and CRR resistance can be selected simultaneously with a simple modification in the timing of inoculation. Abbreviations: CRR Crown and Root Rot; DI Disease index; DPI Days post inoculation; RSD Rhizoctonia seedling disease; WPI Weeks post inoculation.