Mikel R. Stevens

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.

Developmental contributions to phenotypic variation in functional leaf traits within quaking aspen clones

Phenotypic variation in plant traits is strongly influenced by genetic and environmental factors. Over the life span of trees, developmental factors may also strongly influence leaf phenotypes. The objective of this study was to fill gaps in our understanding of developmental influences on patterns of phenotypic trait variation among different-aged ramets within quaking aspen (Populus tremuloides Michx.) clones. We hypothesized that phenotypic variation in leaf functional traits is strongly influenced by developmental cues as trees age. We surveyed eight aspen clones, each with eight distinct age classes ranging from 1 to 160 years in age, and selected three ramets per age class for sample collection. Leaf traits measured included photosynthesis, stomatal conductance, water use efficiency, specific leaf area, and concentrations of N, phosphorus, sucrose, starch, condensed tannins and phenolic glycosides. Using regression analysis, we examined the relationships between ramet age and expression of leaf functional traits. The data showed significant correlations between ramet age and 10 of the 12 phenotypic traits measured. Eight of the phenotypic traits demonstrated a non-linear relationship in which large changes in phenotype occurred in the early stages of ramet development and stabilized thereafter. Water relations, nutrient concentration, leaf gas exchange and phenolic glycosides tended to decrease from early to late development, whereas sucrose, condensed tannin concentrations and water use efficiency increased with ramet age. We hypothesize that ontogenetically derived phenotypic variation leads to fitness differentials among different-aged ramets, which may have important implications for clone fitness. Age-related increases in phenotypic diversity may partially underlie aspens ability to tolerate the large environmental gradients that span its broad geographical range.

Assessment of genetic diversity in the USDA and CIP-FAO international nursery collections of quinoa (Chenopodium quinoa Willd.) using microsatellite markers

Quinoa ( Chenopodium quinoa Willd.) is a staple food crop for millions of impoverished rural inhabitants of Andean South America where it has been cultivated for millennia. Interest in quinoa, due largely to its superior nutritional characteristics, is fuelling a growing export market and has led to an increased focus on genetic research and the development of quinoa breeding programmes throughout South America. The success of these breeding programmes will rely heavily on the development of core germplasm collections and germplasm conservation. We report the development of a set of fluorescence-tagged microsatellite molecular markers that can be used to characterize genetic diversity within quinoa germplasm and we use this set of 36 microsatellites markers to genetically characterize the diversity of 121 accessions of C. quinoa held in the USDA germplasm bank, 22 accessions from the CIP-FAO international nursery collection and eight accessions representing parents from genetic mapping populations. A total of 420 alleles were detected among the quinoa accessions with an average of 11 alleles detected per microsatellite locus. Genetic heterogeneity was observed in 32% of the quinoa accessions at a given locus and suggests that many of these accessions represent heterogeneous seed lots or landraces. Both unweighted pair-group method with arithmetic averages (UPGMA) and principle components analysis (PCA) analyses partitioned the quinoa accessions into two main clusters. The first major cluster consisted of accessions from the Andean highlands of Peru, Bolivia, Ecuador, Argentina and extreme northeastern Chile. The other main cluster contained accessions from both the lowlands of Chile and a set of USDA accessions with no known passport data, collected by Emigdio Ballon. Using the patterns of genetic diversity detected within the C. quinoa accessions we discuss hypotheses regarding quinoas centre of diversity, including highland and lowland ecotype clustering patterns, origin of lowland varieties, origin of domestication, and diversity levels in the USDA and CIP-FAO collections.

Combining transgenic and natural resistance to obtain broad resistance to tospovirus infection in tomato (Lycopersicon esculentum mill)

This study was undertaken to develop tomato plants with broad resistanceto tospoviruses which are a major limiting factor to tomato productionworldwide. A nontransgenic tomato line Stevens-Rodale (S-R), six transgenictomato lines expressing the nucleocapsid (N) protein gene of the lettuceisolate of tomato spotted wilt virus (TSWV-BL), and progeny of the crosses between S-Rand three of the transgenic lines homozygous for the N gene were evaluated fortheir resistance to tospovirus infection in greenhouse inoculation tests. S-Rhas the Sw-5 gene that confers resistance to several TSWVisolates. The six transgenic lines showed high levels of resistance wheninoculated with either TSWV-BL or a tomato isolate from Hawaii (TSWV-H).However, these same plants were highly susceptible to the Brazilian isolate ofgroundnut ringspot virus (GRSV-BR). Plants with the Sw-5gene were resistant to TSWV-BL and GRSV-BR, but were susceptible to TSWV-H.When inoculated with any of the three viruses, the F1 progeny of thecrosses exhibited a susceptible, tolerant, or resistant phenotype with a higherproportion of the plants being either tolerant or resistant. When F2progeny from F1 resistant plants of each cross were inoculated withany of the three viruses, a higher proportion of tolerant and resistant plantswas observed compared to the F1 progeny. Our results show thepotential to obtain broad resistance to tospoviruses by combining transgenicand natural resistance in a single plant.

Simple sequence repeat marker development and genetic mapping in quinoa (Chenopodium quinoa Willd.)

Quinoa is a regionally important grain crop in the Andean region of South America. Recently quinoa has gained international attention for its high nutritional value and tolerances of extreme abiotic stresses. DNA markers and linkage maps are important tools for germplasm conservation and crop improvement programmes. Here we report the development of 216 new polymorphic SSR (simple sequence repeats) markers from libraries enriched for GA, CAA and AAT repeats, as well as 6 SSR markers developed from bacterial artificial chromosome-end sequences (BES-SSRs). Heterozygosity (H) values of the SSR markers ranges from 0.12 to 0.90, with an average value of 0.57. A linkage map was constructed for a newly developed recombinant inbred lines (RIL) population using these SSR markers. Additional markers, including amplified fragment length polymorphisms (AFLPs), two 11S seed storage protein loci, and the nucleolar organizing region (NOR), were also placed on the linkage map. The linkage map presented here is the first SSR-based map in quinoa and contains 275 markers, including 200 SSR. The map consists of 38 linkage groups (LGs) covering 913 cM. Segregation distortion was observed in the mapping population for several marker loci, indicating possible chromosomal regions associated with selection or gametophytic lethality. As this map is based primarily on simple and easily-transferable SSR markers, it will be particularly valuable for research in laboratories in Andean regions of South America.

Identification of RAPD markers linked to fusarium crown and root rot resistance (Frl) in tomato

In tomato ( Lycopersicon esculentum Mill.) a single dominant gene ( Frl) on chromosome 9 confers resistance to fusarium crown and root rot (crown rot) incited by Fusarium oxysporum f. sp. radicis-lycopersici. To identify randomly amplified polymorphic DNA (RAPD) markers linked to Frl, crown rot susceptible and resistant tomato lines were screened for polymorphisms using 1000 random 10-mer primers and three reliable RAPD markers were found linked to Frl (UBC #s 116, 194, and 655). A codominant polymorphic PCR marker of TG101, a restriction fragment length polymorphic (RFLP) marker linked to Frl, was developed to facilitate the linkage studies. Using TG101 and the four RAPD markers, on a Frl segregating backcross population of 950 plants indicated that all belong to the same linkage group. The polymorphic allele order was found to be TG101 – 655 – 116 – 194 – Frl. UBC 194 was found to be 5.1 cM from Frl in this population. Furthermore, it was the only marker found in the resistant genotypes ‘Mocis’ and Fla 7226, whereas resistant genotypes ‘Momor’, Ohio 89-1, and Fla 7464 all had UBC 194 and UBC #s 116, 194, and 655.

Screening Two Lycopersicon peruvianum Collections for Resistance to Tomato spotted wilt virus

The United States Department of Agriculture (USDA) Research Service and the Tomato Genetics Resource Center (TGRC) Lycopersicon peruvianum germplasm collections (16,335 plants from 285 accessions) were screened with the Tomato spotted wilt virus (TSWV) isolates TSWV6 from Hawaii, and Anwa-1 from Western Australia. Using TSWV6 to screen for resistance, 10,634 L. peruvianum plants from 280 accessions were screened for resistance, resulting in 168 (60%) accessions with 1,437 (14%) plants indicating resistance, with all 1,404 89S (Sw-5+/Sw-5+) and 1,456 89R (Sw-5/Sw-5) controls infected. When using Anwa-1 for screening, 864 (15%) of 5,701 L. peruvianum plants were uninfected from 106 of the 181 accessions tested, and 472 (95%) of the 495 89S and 421 (73%) of the 574 89R controls were infected. Of the 172 accessions tested with both isolates, 54 were resistant to one isolate but not the other. Additionally, more accessions from the USDA than from the TGRC collection indicated resistance. TSWV-resistant accessions were somewhat equally distributed throughout the L. peruvianum geographic range, with an observation that northern Chile and southern Peru seemed to have an unusually high portion of accession indicating resistance. The value of Sw-5 is discussed in relationship to potential additional sources of TSWV resistance.

Genetic Variation in Ustilago bullata: Molecular Genetic Markers and Virulence on Bromus tectorum Host Lines

The pathogen Ustilago bullata often causes epidemic levels of head smut disease in Intermountain populations of the inbreeding annual grass Bromus tectorum. We examined patterns of genetic variation for virulence and for amplified fragment length polymorphism (AFLP) markers in four U. bullata populations on B. tectorum in northern Utah and southern Nevada. Patterns of disease incidence generally supported a gene‐for‐gene model for virulence and resistance in this pathosystem. Most host lines were susceptible to most or all paired isolates included in cross‐inoculation tests, but we found evidence for the existence of four avirulence genes. Host lines with alleles conferring resistance were usually resistant to races not found in co‐occurring pathogen populations. The exception was the population at Potosi Pass, Nevada, where the prevalent host line possessed an allele conferring resistance to one of two co‐occurring races. The two pathogen races at Potosi Pass were also strongly genetically differentiated in terms of their AFLP genotypes, whereas for the other three populations, there was no relationship between virulence phenotype and AFLP genotype. There was little evidence for high levels of inbreeding in the northern pathogen populations, which showed no clear groupings of isolates within or among populations. The correlation of the virulence phenotype with the AFLP genotype at Potosi Pass was associated with the presence of a mating locus–linked haplolethal trait in the race pathogenic on the prevalent host line. The presence of this trait is coincident with a high level of intratetrad selfing in this race, thus isolating it from the co‐occurring race and leading to fixation of most AFLP markers. The Potosi Pass population was also clearly differentiated from the northern populations, which were genetically similar to each other, a pattern also seen in the co‐occurring host populations, providing evidence for the idea of pathogen dispersal through seedborne inoculum.

Expression and Evolutionary Relationships of the Chenopodium quinoa 11S Seed Storage Protein Gene

Quinoa (Chenopodium quinoa Willd.) is a food crop cultivated by subsistence farmers and commercial growers on the high Andean plateau, primarily in Bolivia, Peru, and Chile. Present interest in quinoa is due to its tolerance of harsh environments and its nutritional value. It is thought that the seed storage proteins of quinoa, particularly the 11S globulins and 2S albumins, are responsible for the relatively high protein content and ideal amino acid balance of the quinoa seed. Here we report the genomic and cDNA sequences for two 11S genes representing two orthologous loci from the quinoa genome. Important features of the genes and the proteins they encode are described on the basis of a comparison with homologous 11S sequences from other plant species. Gene expression and protein accumulation determined via reverse transcriptase real‐time PCR and SDS‐PAGE analyses are described. Additionally, we report the phylogenetic relationships between quinoa and 49 other species by using the coding DNA sequence for the well‐conserved 11S basic subunit.

Development and use of microsatellite markers for genetic diversity analysis of cañahua (Chenopodium pallidicaule Aellen)

Cañahua (Chenopodium pallidicaule Aellen) is a poorly studied, annual subsistence crop of the high Andes of South America. Its nutritional value (high in protein and mineral content) and ability to thrive in harsh climates make it an important regional food crop throughout the Andean region. The objectives of this study were to develop genetic markers and to quantify genetic diversity within cañahua. A set of 43 wild and cultivated cañahua genotypes and two related species (Chenopodium quinoa Willd. and Chenopodium petiolare Kunth) were evaluated for polymorphism using 192 microsatellite markers derived from random genomic cañahua sequences produced by 454 pyrosequencing of cañahua genomic DNA. Another 424 microsatellite markers from C. quinoa were also evaluated for cross-species amplification and polymorphism in cañahua. A total of 34 polymorphic microsatellite marker loci were identified which detected a total of 154 alleles with an average of 4.5 alleles per marker locus and an average heterozygosity value of 0.49. A cluster analysis, based on Nei genetic distance, clearly separated from wild cañahua genotypes from the cultivated genotypes. Within the cultivated genotypes, subclades were partitioned by AMOVA analysis into six model-based clusters, including a subclade consisting sole of erect morphotypes. The isolation by distance test displayed no significant correlation between geographic collection origin and genotypic data, suggesting that cañahua populations have moved extensively, presumably via ancient food exchange strategies among native peoples of the Andean region. The molecular markers reported here are a significant resource for ongoing efforts to characterize the extensive Bolivian and Peruvian cañahua germplasm banks, including the development of core germplasm collections needed to support emerging breeding programs.