Jose L. Gonzalez-Hernandez

A multiple species approach to biomass production from native herbaceous perennial feedstocks

Due to the rapid rate of worldwide consumption of nonrenewable fossil fuels, production of biofuels from cellulosic sources is receiving increased research emphasis. Here, we review the feasibility to produce lignocellulosic biomass on marginal lands that are not well-suited for conventional crop production. Large areas of these marginal lands are located in the central prairies of North America once dominated by tallgrass species. In this article, we review the existing literature, current work, and potential of two native species of the tallgrass prairie, prairie cordgrass (Spartina pectinata), and little bluestem (Schizachyrium scoparium) as candidates for commercial production of biofuel. Based on the existing literature, we discuss the need to accelerate research in the areas of agronomy, breeding, genetics, and potential pathogens. Cropping systems based on maintaining biodiversity across landscapes are essential for a sustainable production and to mitigate impact of pathogens and pests.

Morphology and biomass production of prairie cordgrass on marginal lands

Prairie cordgrass (Spartina pectinata Link.) is indigenous throughout most of the continental United States and Canada to 60°N latitude and is well suited to marginal land too wet for maize (Zea mays L.) and switchgrass (Panicum virgatum L.). Evaluations of prairie cordgrass in Europe and North America indicated it has high potential for biomass production, relative to switchgrass, in short‐season areas. Our objective was to describe morphology and biomass production and partitioning in mature stands of ‘Red River’ prairie cordgrass and determine biomass production of natural populations on marginal land. This study was conducted from 2000 to 2008 in eastern South Dakota. Mean biomass production of mature stands of Red River was 12.7 Mg ha−1. Leaves composed >88% of the biomass, and 60% of the tillers had no internodes. Belowground biomass to a depth of approximately 25 cm, not including roots, was 21 Mg ha−1. Tiller density ranged from 683 tillers m−2 for a 10‐year‐old stand to 1140 tillers m−2 for a 4‐year‐old stand. The proaxis was composed of about eight phytomers, with rhizomes originating at proximal nodes and erect tillers at distal nodes. Vegetative propagation was achieved by both phalanx and guerilla growth. Differences among natural populations for biomass were expressed on gravelly marginal land. However, production, averaged across populations, was low (1.37 Mg ha−1) and comparable to ‘Cave‐In‐Rock’ switchgrass (1.67 Mg ha−1) over a 4‐year period. The large carbon storage capacity of prairie cordgrass in proaxes and rhizomes makes it useful for carbon sequestration purposes. Prairie cordgrass should be compared with switchgrass and other C4 perennial grasses along environmental gradients to determine optimum landscape positions for each and to maximize bioenergy production and minimize inputs.

Family-based mapping of quantitative trait loci in plant breeding populations with resistance to Fusarium head blight in wheat as an illustration

Traditional quantitative trait loci (QTL) mapping approaches are typically based on early or advanced generation analysis of bi-parental populations. A limitation associated with this methodology is the fact that mapping populations rarely give rise to new cultivars. Additionally, markers linked to the QTL of interest are often not immediately available for use in breeding and they may not be useful within diverse genetic backgrounds. Use of breeding populations for simultaneous QTL mapping, marker validation, marker assisted selection (MAS), and cultivar release has recently caught the attention of plant breeders to circumvent the weaknesses of conventional QTL mapping. The first objective of this study was to test the feasibility of using family-pedigree based QTL mapping techniques generally used with humans and animals within plant breeding populations (PBPs). The second objective was to evaluate two methods (linkage and association) to detect marker-QTL associations. The techniques described in this study were applied to map the well characterized QTL, Fhb1 for Fusarium head blight resistance in wheat (Triticumaestivum L.). The experimental populations consisted of 82 families and 793 individuals. The QTL was mapped using both linkage (variance component and pedigree-wide regression) and association (using quantitative transmission disequilibrium test, QTDT) approaches developed for extended family-pedigrees. Each approach successfully identified the known QTL location with a high probability value. Markers linked to the QTL explained 40–50% of the phenotypic variation. These results show the usefulness of a human genetics approach to detect QTL in PBPs and subsequent use in MAS.

Investigation of the Transcriptome of Prairie Cord Grass, a New Cellulosic Biomass Crop

Prairie cordgrass (Spartina pectinata Bosc ex Link) is being developed as a cellulosic biomass crop. Development of this species will require numerous steps, including breeding, agronomy, and characterization of the species genome. The research in this paper describes the first investigation of the transcriptome of prairie cordgrass via Next Generation Sequencing Technology, 454 GS FLX. A total of 556,198 expressed sequence tags (ESTs) were produced from four prairie cordgrass tissues: roots, rhizomes, immature inflorescence, and hooks. These ESTs were assembled into 26,302 contigs and 71,103 singletons. From these data were identified, EST–SSR (simple sequence repeat) regions and cell wall biosynthetic pathway genes suitable for the development of molecular markers which can aid the breeding process of prairie cordgrass by means of marker assisted selection.

Validating DNA Polymorphisms Using KASP Assay in Prairie Cordgrass (Spartina pectinata Link) Populations in the U.S.

Single nucleotide polymorphisms (SNPs) are one of the most abundant DNA variants found in plant genomes and are highly efficient when comparing genome and transcriptome sequences. SNP marker analysis can be used to analyze genetic diversity, create genetic maps, and utilize marker-assisted selection breeding in many crop species. In order to utilize these technologies, one must first identify and validate putative SNPs. In this study, 121 putative SNPs, developed from a nuclear transcriptome of prairie cordgrass (Spartina pectinata Link), were analyzed using KASP technology in order to validate the SNPs. Fifty-nine SNPs were validated using a core collection of 38 natural populations and a phylogenetic tree was created with one main clade. Samples from the same population tended to cluster in the same location on the tree. Polymorphisms were identified within 52.6% of the populations, split evenly between the tetraploid and octoploid cytotypes. Twelve selected SNP markers were used to assess the fidelity of tetraploid crosses of prairie cordgrass and their resulting F2population. These markers were able to distinguish true crosses and selfs. This study provides insight into the genomic structure of prairie cordgrass, but further analysis must be done on other cytotypes to fully understand the structure of this species. This study validates putative SNPs and confirms the potential usefulness of SNP marker technology in future breeding programs of this species.

Mapping of two loci conferring resistance to wheat stem rust pathogen races TTKSK (Ug99) and TRTTF in the elite hard red spring wheat line SD4279

Since its identification in the late 1990s, the stem rust pathogen (Puccinia graminis. f. sp. tritici (Pgt)) strain Ug99 (race TTKSK) has represented a worldwide wheat production threat due to its ability to overcome most of the resistance genes present in commercial cultivars. In order to address this challenge, resistance genes in wheat cultivars as well as in wild relatives have been identified. However, stem rust resistance breeding is facing a new challenge with the recent discovery in Ethiopia of a new race of Pgt (TRTTF) capable of defeating Sr13, SrTmp, and Sr1RAmigo genes that conferred resistance to the Ug99 race group. As part of an ongoing screening process at USDA-ARS Cereal Disease Laboratory, SD4279, an elite line from the hard red spring wheat breeding program at South Dakota State University, was found to be resistant to both races TTKSK and TRTTF. The objectives posed in this research were (1) to characterize the genetics of resistance to stem rust in SD4279 and (2) to identify molecular markers linked to race TTKSK (Ug99) and TRTTF resistance in SD4279. A mapping population composed of 92 F2:3 families was evaluated for resistance to TTKSK and TRTTF. A single-gene conferring resistance to TTKSK, likely Sr9h, was mapped on chromosome arm 2BL. Also, a single gene was located on chromosome arm 6AS conferring resistance to TRTTF. Based on the type of reaction and map location, we postulate that the 6AS resistance gene is Sr8a which has not been mapped previously using DNA markers.

Proteomic Responses of Switchgrass and Prairie Cordgrass to Senescence

Senescence in biofuel grasses is a critical issue because early senescence decreases potential biomass production by limiting aerial growth and development. 2-Dimensional, differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometry of selected protein spots was used to evaluate differences between leaf proteomes of early (ES)- and late- senescing (LS) genotypes of Prairie cordgrass (ES/LS PCG) and switchgrass (ES/LS SG), just before and after senescence was initiated. Analysis of the manually filtered and statistically evaluated data indicated that 69 proteins were significantly differentially abundant across all comparisons, and a majority (41%) were associated with photosynthetic processes as determined by gene ontology analysis. Ten proteins were found in common between PCG and SG, and nine and 18 proteins were unique to PCG and SG respectively. Five of the 10 differentially abundant spots common to both species were increased in abundance, and five were decreased in abundance. Leaf proteomes of the LS genotypes of both grasses analyzed before senescence contained significantly higher abundances of a 14-3-3 like protein and a glutathione-S-transferase protein when compared to the ES genotypes, suggesting differential cellular metabolism in the LS vs. the ES genotypes. The higher abundance of 14-3-3 like proteins may be one factor that impacts the senescence process in both LS PCG and LS SG. Aconitase dehydratase was found in greater abundance in all four genotypes after the onset of senescence, consistent with literature reports from genetic and transcriptomic studies. A Rab protein of the Ras family of G proteins and an s-adenosylmethionine synthase were more abundant in ES PCG when compared with the LS PCG. In contrast, several proteins associated with photosynthesis and carbon assimilation were detected in greater abundance in LS PCG when compared to ES PCG, suggesting that a loss of these proteins potentially contributed to the ES phenotype in PCG. Overall, this study provides important data that can be utilized toward delaying senescence in both PCG and SG, and sets a foundational base for future improvement of perennial grass germplasm for greater aerial biomass productivity.

Advances towards a marker-assisted selection breeding program in prairie cordgrass, a biomass crop.

Prairie cordgrass (Spartina pectinata Bosc ex Link) is an indigenous, perennial grass of North America that is being developed into a cellulosic biomass crop suitable for biofuel production. Limited research has been performed into the breeding of prairie cordgrass; this research details an initial investigation into the development of a breeding program for this species. Genomic libraries enriched for four simple sequence repeat (SSR) motifs were developed, 25 clones from each library were sequenced, identifying 70 SSR regions, and primers were developed for these regions, 35 of which were amplified under standard PCR conditions. These SSR markers were used to validate the crossing methodology of prairie cordgrass and it was found that crosses between two plants occurred without the need for emasculation. The successful cross between two clones of prairie cordgrass indicates that this species is not self-incompatible. The results from this research will be used to instigate the production of a molecular map of prairie cordgrass which can be used to incorporate marker-assisted selection (MAS) protocols into a breeding program to improve this species for cellulosic biomass production.

Construction of dense linkage maps “on the fly” using early generation wheat breeding populations

In plant species, construction of framework linkage maps to facilitate quantitative trait loci mapping and molecular breeding has been confined to experimental mapping populations. However, development and evaluation of these populations is detached from breeding efforts for cultivar development. In this study, we demonstrate that dense and reliable linkage maps can be constructed using extant breeding populations derived from a large number of crosses, thus eliminating the need for extraneous population development. Using 565 segregating F1 progeny from 28 four-way cross breeding populations, a linkage map of the hexaploid wheat genome consisting of 3,785 single nucleotide polymorphism (SNP) loci and 22 simple sequence repeat loci was developed. Map estimation was facilitated by application of mapping algorithms for general pedigrees implemented in the software package CRI-MAP. The developed linkage maps showed high rank-order concordance with a SNP consensus map developed from seven mapping studies. Therefore, the linkage mapping methodology presented here represents a resource efficient approach for plant breeding programs that enables development of dense linkage maps “on the fly” to support molecular breeding efforts.

Genetics and Partitioning for Biomass of Prairie Cordgrass Compared to Switchgrass on Marginal Cropland

Prairie cordgrass (Spartina pectinata Link) has potential as a bioenergy crop in poorly drained cropland in much of temperate North America. Our objectives were (1) describe morphological differences between two prairie cordgrass populations and switchgrass for biomass production and pattern of biomass accumulation in leaf and stem tissue, (2) estimate genetic variation for biomass production and other agronomic traits in a population of prairie cordgrass from South Dakota, and (3) increase knowledge of the impact of insects on biomass production of prairie cordgrass. On poorly drained cropland in South Dakota, a natural population of prairie cordgrass (SD CG) from South Dakota produced 75% more biomass than a natural population from North Dakota, and a selected population of prairie cordgrass produced >2 times the biomass of switchgrass. However, when prairie cordgrass was fed on by larvae of the four-lined borer [(Resapamea stipata (Morr.)], yield of the two species was similar. Prairie cordgrass had a lower frequency of reproductive tillers (RTRs) and larger leaves than switchgrass. SD CG had high genetic variation for disease resistance, moderate genetic variation for biomass yield, and low genetic variation for inflorescences m−2. Seven out of 57 half-sib families from SD CG produced more biomass than “summer” switchgrass during a year with normal precipitation, whereas only one family did so during a drought year. RTR declined rapidly with stand age. The four-lined borer was identified as a serious threat to biomass production of prairie cordgrass in eastern South Dakota.