John E. Erpelding

Genetic diversity and population structure analysis of accessions in the US historic sweet sorghum collection

Sweet sorghum has the potential to become a versatile feedstock for large-scale bioenergy production given its sugar from stem juice, cellulose/hemicellulose from stalks, and starch from grain. However, for researchers to maximize its feedstock potential a first step includes additional evaluations of the 2,180 accessions with varied origins in the US historic sweet sorghum collection. To assess genetic diversity of this collection for bioenergy breeding and population structure for association mapping, we selected 96 accessions and genotyped them with 95 simple sequence repeat markers. Subsequent genetic diversity and population structure analysis methods identified four subpopulations in this panel, which correlated well with the geographic locations where these accessions originated or were collected. Model comparisons for three quantitative traits revealed different levels of population structure effects on flowering time, plant height, and brix. Our results suggest that diverse germplasm accessions curated from different geographical regions should be considered for plant breeding programs to develop sweet sorghum cultivars or hybrids, and that this sweet sorghum panel can be further explored for association mapping.

An annotated genetic map of loblolly pine based on microsatellite and cDNA markers

BackgroundPrevious loblolly pine (Pinus taeda L.) genetic linkage maps have been based on a variety of DNA polymorphisms, such as AFLPs, RAPDs, RFLPs, and ESTPs, but only a few SSRs (simple sequence repeats), also known as simple tandem repeats or microsatellites, have been mapped in P. taeda. The objective of this study was to integrate a large set of SSR markers from a variety of sources and published cDNA markers into a composite P. taeda genetic map constructed from two reference mapping pedigrees. A dense genetic map that incorporates SSR loci will benefit complete pine genome sequencing, pine population genetics studies, and pine breeding programs. Careful marker annotation using a variety of references further enhances the utility of the integrated SSR map.ResultsThe updated P. taeda genetic map, with an estimated genome coverage of 1,515 cM(Kosambi) across 12 linkage groups, incorporated 170 new SSR markers and 290 previously reported SSR, RFLP, and ESTP markers. The average marker interval was 3.1 cM. Of 233 mapped SSR loci, 84 were from cDNA-derived sequences (EST-SSRs) and 149 were from non-transcribed genomic sequences (genomic-SSRs). Of all 311 mapped cDNA-derived markers, 77% were associated with NCBI Pta UniGene clusters, 67% with RefSeq proteins, and 62% with functional Gene Ontology (GO) terms. Duplicate (i.e., redundant accessory) and paralogous markers were tentatively identified by evaluating marker sequences by their UniGene cluster IDs, clone IDs, and relative map positions. The average gene diversity, He , among polymorphic SSR loci, including those that were not mapped, was 0.43 for 94 EST-SSRs and 0.72 for 83 genomic-SSRs. The genetic map can be viewed and queried at http://www.conifergdb.org/pinemap.ConclusionsMany polymorphic and genetically mapped SSR markers are now available for use in P. taeda population genetics, studies of adaptive traits, and various germplasm management applications. Annotating mapped genes with UniGene clusters and GO terms allowed assessment of redundant and paralogous EST markers and further improved the quality and utility of the genetic map for P. taeda.

Molecular genetic evaluation of sorghum germplasm differing in response to fungal diseases: Rust (Puccinia purpurea) and anthracnose (Collectotrichum graminicola)

To evaluate genetic diversity in relation to rust and anthracnose disease response, ninety-six accessions were randomly selected from the core collection database of the Germplasm Research Information Network (GRIN) and characterized by a set of 40 SSR markers. The mean value of polymorphism information content (PIC) was 0.8228. Two dendrograms were generated from the molecular genetic data and field morphological data, respectively. The genetic dendrogram demonstrates that the accessions can be classified into three main clades and nine subgroups. The branched subgroups correlated very well with the locations where the accessions were collected. Geographical origin of accessions had significant influences on genetic similarity of sorghum germplasm. Out of 96 accessions, only eight accessions were highly resistant to both rust and anthracnose. All the accessions from South Africa and Mali were highly resistant to anthracnose. The information from genetic classification would be useful for choosing parents to make crosses in sorghum breeding programs and classifying sorghum accessions in germplasm management.

Virulence and Molecular Genotyping Studies of Sporisorium reilianum Isolates in Sorghum

Head smut, caused by the fungal pathogen Sporisorium reilianum, has been reported with increasing frequency in the grain sorghum growing areas of Texas. To facilitate analysis of changes in pathogen virulence, four inoculation techniques were examined: soil and teliospore mixture, seed coating, media placement, and syringe injection. Of the four, syringe injection was determined to be the most effective. Inoculations of sorghum host differentials BTx643, BTx7078, BTx635, SC170-6-17 (TAM2571), SA281 (Early Hegari), and Tx414 showed 23 of 32 Texas isolates were race 4. Two isolates from College Station, TX, were classified as race 1, but no race 2 or 3 isolates were found. New, virulent races 5 and 6 were identified among isolates from south Texas. Using 16 amplified fragment length polymorphism (AFLP) primer combinations, genetic diversity was assessed in DNA samples from 49 S. reilianum isolates, including 44 sorghum isolates from Texas, two from Uganda, and one from Mali; and two maize isolates from Mexico. Single-base extensions with EcoRI and MseI primers in the selective amplification increased the number of informative polymorphic bands. High genetic dissimilarity (50%) was observed between isolates originating from maize and those originating from sorghum. The resultant dendrogram, made using cluster analysis, grouped the Texas S. reilianum isolates into four small clusters with ≥82% similarity. Other than for two race 6 isolates from Weslaco, TX, no evidence for geographical or other restrictions on gene flow was evident.

A Pictorial Technique for Mass Screening of Sorghum Germplasm for Anthracnose (Colletotrichum sublineolum) Resistance

Globally, the foliar phase of anthracnose is one of the most destructive diseases of sorghum. In most cases, anthracnose resistance screening relies on the use of a spore suspension. This method is usually conducted after sundown and when there is the possibility of dew formation the following morning. Using a spore suspension for sorghum anthracnose field evaluation in College Station, Texas over five years (1996, 1997, 1999-2001) yielded inconsistent linkage results and failed to identify any closely linked molecular markers. For large scale screening of sorghum germplasm for anthracnose (Colletotrichum sublineolum) resistance, plants are inoculated in the field or in the green house at either 30 d after planting or at the 8-10 leaf-stage. In field inoculation, the use of C. sublineolum-colonized sorghum grains was shown to be the most efficient and effective in identifying resistant sources. For effective, efficient, fast and accurate infection, approximately 10-20 seeds are placed in each plant leaf whorl and it takes about 16.7 kg of colonized grains to cover a 0.4 ha area. In the greenhouse, though colonized grains are equally effective, spray inoculation is preferred for easy and uniform coverage. Using this method of inoculum preparation, spore suspension was extracted and sprayed (106 conidia·ml-1), followed by 10 hr/d misting for 30 sec at 30-45 min interval continuously for a period of one month resulted in effective infection

Genetic diversity analysis of Gossypium arboreum germplasm accessions using genotyping-by-sequencing.

The diploid cotton species Gossypium arboreum possesses many favorable agronomic traits such as drought tolerance and disease resistance, which can be utilized in the development of improved upland cotton cultivars. The USDA National Plant Germplasm System maintains more than 1600 G. arboreum accessions. Little information is available on the genetic diversity of the collection thereby limiting the utilization of this cotton species. The genetic diversity and population structure of the G. arboreum germplasm collection were assessed by genotyping-by-sequencing of 375 accessions. Using genome-wide single nucleotide polymorphism sequence data, two major clusters were inferred with 302 accessions in Cluster 1, 64 accessions in Cluster 2, and nine accessions unassigned due to their nearly equal membership to each cluster. These two clusters were further evaluated independently resulting in the identification of two sub-clusters for the 302 Cluster 1 accessions and three sub-clusters for the 64 Cluster 2 accessions. Low to moderate genetic diversity between clusters and sub-clusters were observed indicating a narrow genetic base. Cluster 2 accessions were more genetically diverse and the majority of the accessions in this cluster were landraces. In contrast, Cluster 1 is composed of varieties or breeding lines more recently added to the collection. The majority of the accessions had kinship values ranging from 0.6 to 0.8. Eight pairs of accessions were identified as potential redundancies due to their high kinship relatedness. The genetic diversity and genotype data from this study are essential to enhance germplasm utilization to identify genetically diverse accessions for the detection of quantitative trait loci associated with important traits that would benefit upland cotton improvement.

Response of sorghum accessions from Chad and Uganda to natural infection by the downy mildew pathogen, Peronosclerospora sorghi in Mexico and the USA

In this study, 78 accessions from Chad, Central Africa and 20 photoperiod insensitive accessions from Uganda, East Africa were evaluated for downy mildew resistance in Ocotlan, Mexico in 2004 and 2005. Ninety-four of these accessions were also evaluated at two locations in Wharton County, Texas, USA, in 2005. Accessions were planted in a randomized complete block design with each sorghum accession replicated three times. Disease incidence was determined from natural infection. Disease incidence varied between locations, with the highest mean disease incidence observed for the Mexican evaluation. Germplasm from Chad also showed a higher mean disease incidence than germplasm from Uganda. Several sources of downy mildew resistance were identified. Three accessions PI282860, PI282864, and PI563505 from Chad were shown to possess high levels of downy mildew resistance in Mexico and Texas, whereas PI282843, PI282877, PI549196, and PI563438 also from Chad exhibited high levels of resistance to the disease in Texas. Accessions PI297210, PI576386 and PI576395 from Uganda also showed downy mildew resistance in Mexico and Texas. These sorghum accessions from Chad and Uganda can be utilized in breeding for downy mildew resistance in Mexico and Texas.ZusammenfassungIn dieser Untersuchung wurden 78 Sorghum-Akzessionen aus dem Tschad und 20 photoperiodisch neutral reagierende Sorghum-Akzessionen aus Uganda hinsichtlich ihrer Resistenz gegenüber dem Falschen Mehltau in den Jahren 2004 und 2005 in Ocotlan, Mexiko, überprüft. Vierundneunzig dieser Akzessionen wurden 2005 auch in Wharton County, Texas, getestet. Die Akzessionen wurden in einer vollständig randomisierten Blockanlage mit drei Wiederholungen pro Akzession gepflanzt. Die Befallshäufigkeit wurde nach natürlicher Infektion bestimmt. Die Befallshäufigkeit variierte zwischen den Standorten, wobei die höchsten mittleren Werte in Mexiko beobachtet wurden. Tschadische Herkünfte wiesen höhere Befallshäufigkeiten auf als ugandische. Mehrere Resistenzquellen gegen den Falschen Mehltau wurden identifiziert. Die drei tschadischen Akzessionen PI282860, PI282864 und PI563505 zeigten hohe Grade von Mehltauresistenz in Mexiko und Texas, während die ebenfalls aus dem Tschad stammenden Akzessionen PI282843, PI282877, PI549196 und PI563438 hohe Resistenzgrade nur in Texas aufwiesen. Die ugandischen Akzessionen PI297210, PI576386 und PI576395 waren gegenüber dem Falschen Mehltau in Mexiko und Texas resistent. Diese Sorghum-Akzessionen aus Uganda und dem Tschad können daher zur Züchtung mehltauresistenter Sorghum-Sorten sowohl in Mexico als auch in Texas verwendet werden.

Inoculation Techniques for Identifying Resistance in Sorghum Genotypes to Sorghum Ergot

Abstract Seven inoculation methods were tested at multiple field sites with different climates in Texas and Mexico to determine the technique most suitable for routine screening of sorghum (Sorghum bicolor (L.) Moench) germplasm for resistance to Claviceps africana (causal agent of sorghum ergot). A single spray or sponge inoculation before flower initiation followed by bagging of the panicles for seven days was the most reliable technique for identifying ergot-resistant sorghum genotypes. This method ensures that unfertilized ovules and infection sites were exposed to the pathogen at the time of greatest vulnerability, thereby reducing the influence of flowering characteristics. This method is repeatable and rapid, thus providing a useful procedure for germplasm evaluation. Disease symptoms on sorghum genotypes are expressed within a reasonably short period. Compared with the other methods, inoculated and bagged panicles resulted in a significantly higher percentage of ergot-infected florets in all environments. Across sorghum lines, no significant increases in ergot severity were observed between inoculated nonbagged, inoculated and misted, and water-sprayed control panicles. In all environments, percent ergot severity per panicle across treatments generally increased from 10 to 15 days after inoculation with negligible increases in disease severity after 15 days. This suggests that ergot assessments after inoculation can be concluded within 15 days. None of the genotypes tested was completely resistant to sorghum ergot. However, NC+8R18, NC+7W97, and the two sister-lines of IS8525 (IS8525J and IS8525D) exhibited more resistance to ergot than the other sorghum lines tested.