Craig E. Coleman

The Prolamins of Maize

Prolamins are the principal seed storage proteins of cereals. These proteins are soluble in alcohol and function in storage of nitrogen and amino acids for the germinating seedling (Osborne, 1908; Osborne and Mendel, 1914). Maize prolamins are called zeins, and they form accretions or protein bodies within the lumen of the rough endoplasmic reticulum (RER) of endosperm cells. Zeins are structurally distinct from the prolamins of the more distantly related cereals, wheat, barley, oats and rice (see chapters 3, 4 and 5), but they are very similar to the prolamins of the other panacoid cereals, e.g. sorghum, millet and Coix (see chapter 7).

A defective signal peptide tethers the floury-2 zein to the endoplasmic reticulum membrane.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD [alpha]-zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD [alpha]-zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this [alpha]-zein reveals a putative mutation alaine->valine (Ala->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 [alpha]-zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 [alpha]-zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 [alpha]-zein and the fl2 [alpha]-zein (Val->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 [alpha]-zein (Val->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 [alpha]-zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.

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.

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.

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.

Population genetic analysis of Bromus tectorum (Poaceae) indicates recent range expansion may be facilitated by specialist genotypes

PREMISE OF THE STUDY The mechanisms for range expansion in invasive species depend on how genetic variation is structured in the introduced range. This study examined neutral genetic variation in the invasive annual grass Bromus tectorum in the Intermountain Western United States. Patterns of microsatellite (SSR) genotype distribution in this highly inbreeding species were used to make inferences about the roles of adaptively significant genetic variation, broadly adapted generalist genotypes, and facultative outcrossing in the recent range expansion of B. tectorum in this region. METHODS We sampled 20 individuals from each of 96 B. tectorum populations from historically and recently invaded habitats throughout the region and used four polymorphic SSR markers to characterize each individual. KEY RESULTS We detected 131 four-locus SSR genotypes; however, the 14 most common genotypes collectively accounted for 79.2% of the individuals. Common SSR genotypes were not randomly distributed among habitats. Instead, characteristic genotypes sorted into specific recently invaded habitats, including xeric warm and salt desert as well as mesic high-elevation habitats. Other SSR genotypes were common across a range of historically invaded habitats. We observed very few heterozygous individuals (0.58%). CONCLUSIONS Broadly adapted, generalist genotypes appear to dominate historically invaded environments, while recently invaded salt and warm desert habitats are dominated by distinctive SSR genotypes that contain novel alleles. These specialist genotypes are not likely to have resulted from recombination; they probably represent more recent introductions from unknown source populations. We found little evidence that outcrossing plays a role in range expansion.

De novo genome assembly of the fungal plant pathogen Pyrenophora semeniperda.

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen that has a wide host range within the Poaceae. One of its hosts is cheatgrass (Bromus tectorum), a species exotic to the United States that has invaded natural ecosystems of the Intermountain West. As a natural pathogen of cheatgrass, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing seeds within the seed bank; however, few genetic resources exist for the fungus. Here, the genome of P. semeniperda isolate assembled from sequence reads of 454 pyrosequencing is presented. The total assembly is 32.5 Mb and includes 11,453 gene models encoding putative proteins larger than 24 amino acids. The models represent a variety of putative genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, extensive rearrangements, including inter- and intrachromosomal rearrangements, were found when the P. semeniperda genome was compared to P. tritici-repentis, a related fungal species.

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.

The Ghost of Outcrossing Past in Downy Brome, an Inbreeding Annual Grass

We investigated the frequency of outcrossing in downy brome (Bromus tectorum L.), a cleistogamous weedy annual grass, in both common garden and wild populations, using microsatellite and single nucleotide polymorphic (SNP) markers. In the common garden study, 25 lines with strongly contrasting genotypes were planted in close proximity. We fingerprinted 10 seed progeny from 8 individuals of each line and detected 15 first-generation heterozygotes for a t-value (corrected for cryptic crosses) of 0.0082. Different genotypes were significantly overrepresented as maternal versus paternal parents of heterozygotes, suggesting gender-function-dependent genetic control of outcrossing rates. In 4 wild populations (>300 individuals each), expected heterozygosity ranged from 0.149 to 0.336, whereas t-values ranged from 0.0027 to 0.0133, indicating high levels of both genetic diversity and inbreeding. Up to a third of the individuals in each population belonged to groups with identical or nearly identical SNP genotypes, whereas many of the remaining individuals were members of loose clusters of apparently related plants that probably represent descendants from past outcrossing events. Strict inbreeding in some lineages within a population with occasional outcrossing in others may be related to positive selection on adaptive syndromes associated with specific inbreeding lineages, or possibly to among-lineage differences in genetic regulation of outcrossing.

Lack of Host Specialization on Winter Annual Grasses in the Fungal Seed Bank Pathogen Pyrenophora semeniperda

Generalist plant pathogens may have wide host ranges, but many exhibit varying degrees of host specialization, with multiple pathogen races that have narrower host ranges. These races are often genetically distinct, with each race causing highest disease incidence on its host of origin. We examined host specialization in the seed pathogen Pyrenophora semeniperda by reciprocally inoculating pathogen strains from Bromus tectorum and from four other winter annual grass weeds (Bromus diandrus, Bromus rubens, Bromus arvensis and Taeniatherum caput-medusae) onto dormant seeds of B. tectorum and each alternate host. We found that host species varied in resistance and pathogen strains varied in aggressiveness, but there was no evidence for host specialization. Most variation in aggressiveness was among strains within populations and was expressed similarly on both hosts, resulting in a positive correlation between strain-level disease incidence on B. tectorum and on the alternate host. In spite of this lack of host specialization, we detected weak but significant population genetic structure as a function of host species using two neutral marker systems that yielded similar results. This genetic structure is most likely due to founder effects, as the pathogen is known to be dispersed with host seeds. All host species were highly susceptible to their own pathogen races. Tolerance to infection (i.e., the ability to germinate even when infected and thereby avoid seed mortality) increased as a function of seed germination rate, which in turn increased as dormancy was lost. Pyrenophora semeniperda apparently does not require host specialization to fully exploit these winter annual grass species, which share many life history features that make them ideal hosts for this pathogen.