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Diaporthe gulyae Shivas et al. was initially described as causing Phomopsis stem canker of sunflower (Helianthus annuus L.) in the United States in 2010. In this study, the impact of growth stage on disease severity associated with D. gulyae in sunflower accessions was investigated in the greenhouse. One hundred thirty-nine accessions were inoculated with a single isolate of D. gulyae using the mycelium contact inoculation method at V4 to V6 (four to six true leaves) and R4 to R6 (inflorescence to ray flower wilting) growth stages. The experiment was conducted in a completely randomized design with six replications and repeated once. Disease severity was assessed at 14 days postinoculation using a 0-to-5 rating scale. A significant interaction between growth stage and accessions (P < 0.0001) was observed for disease severity (relative treatment effect [RTE]) caused by D. gulyae. Among the 139 accessions, 24, three, and two accessions had significantly lower RTE when inoculated at V4 to V6, R4 to R6, and both growth stages, respectively, upon comparing with the inbred susceptible check ‘HA 288’. This study suggests that accessions differ in their susceptibility to D. gulyae, possibly because of changes in resistance response during host development. Future studies on sunflower-D. gulyae interaction are warranted for developing commercial hybrids with resistance to the causal fungus.

Delayed planting is recommended to reduce damage from sunflower insect pests in the United States, including the sunflower moth, Homoeosoma electellum (Hulst) and banded sunflower moth, Cochylis hospes Walsingham. However, in some locations, planting earlier or growing later-maturing hybrids could improve yield or oil content of sunflowers which would partially offset any added costs from insect pests or their management. Because the abundance and distribution of some sunflower insects have changed since recommendations for delayed planting were developed, experimental plots were grown in 2012 and 2013 at sites in North Dakota, Nebraska, Iowa, and Illinois. Sunflowers were planted two to four weeks earlier than normal, including hybrids that flower two to three weeks later than elite commercial hybrids. The sum of seed damaged by sunflower moth, banded sunflower moth, and red sunflower seed weevil, Smicronyx fulvus LeConte, (i. e., total percentage) was influenced by location, but not the relative maturity of tested entries. However, when damage attributed solely to the red sunflower seed weevil was analyzed, more damaged seed were found for late-maturing entries in North Dakota and Nebraska. In addition to the trial data, current pest populations are lower than when delayed planting was first recommended and insecticide use during sunflower bloom is both common and effective. Together, these observations suggest factoring insect pests into planting time decisions may be unnecessary, except for areas with a history of problems with severe pests that cannot be managed using insecticides (e. g., sunflower midge, Contarinia schulzi Gagné).

The pages of this extensive book document the potential of a great many North American plants to enhance the productivity, sustainability, and nutritional quality of crops or to be further developed into important cultivated species in their own right. But this potential can only be realized if the plants are adequately conserved to ensure their survival and availability for research, invested in to promote their development, and marketed so as to be attractive to producers and consumers. We outline some of the key steps needed to boost the conservation and use of our regional cornucopia. In situ and ex situ conservation of North America’s useful plants are being accomplished by a variety of institutions with different mandates, but habitat destruction and other threats to wild populations continue to negatively impact many species. Information sharing, coordinating efforts, filling research gaps for wild plants, and increasing support for conservation will be necessary to more comprehensively safeguard these plants and to make them available for use. Technologies enabling more efficient exploration of the diversity within these species are rapidly advancing and offer the potential to contribute to quick advances in improvement of cultivars, but considerable further research and partnerships are needed to generate and share the results widely. Marketing of new crops can take advantage of the increasing public interest in diverse and nutritious foods, learning from successful collaborations between producers, researchers, and consumers. As a whole, North America already possesses a strong foundation from which the conservation and use of its flora can be enhanced. This includes many protected areas, strong conservation institutions, innovative research, and the willingness to collaborate across fields, institutions, and borders. There are still many silos that need to be broken down and reorganized through innovative partnerships to better conserve and benefit from the North American cornucopia. But given the incredible diversity of interesting and useful plants in the region, the remarkable efforts for many decades by many organizations to care for these plants and share them with humanity, and the increasing public interest in more diverse, healthy, and resilient food and agricultural systems, there is reason for hope.

Cultivated sunflower, with seeds valued for oil, snack consumption and bird feed, and flowers popular in gardens and in the ornamental trade, is native to North America, although its development into a global oilseed crop, briefly described here, depended on the international agricultural community. Sunflower crop wild relatives (CWR), all native to North America, are a rich source of genetic diversity for crop improvement and have been used extensively throughout of the history of sunflower breeding. Traits from Helianthus wild species have been used to increase disease resistance and abiotic stress tolerance and create a reliable hybrid breeding system; examples are described in this chapter. Despite widespread use of sunflower CWR, there is a critical need to fill significant geographic gaps in ex situ collections and to increase conservation of wild sunflower species in situ. Recent genomic developments have made the use of wild species more feasible in shorter timelines emphasizing the potential value of increased conservation efforts.

Successful collection of wild plant germplasm requires careful preparation. Based on the author’s experiences in the United States, this chapter describes the process of preparing and executing explorations to collect wild plant germplasm in North America. An outline is provided, followed by more detailed descriptions of the steps of setting up an exploration framework, obtaining permits and permissions, and developing trip logistics. Establishing an exploration framework involves locating populations of the target species through compilation of herbarium voucher data and additional resources and through contacts with plant experts and other knowledgeable people. Most places in North America require permission or permits for any vegetation disturbance activities. Neither the United States nor Canada has an overarching national level permit; every landowner needs to be contacted. Mexico requires a national collection permit although the national permit does not grant access; therefore, every landowner needs to be contacted. The process of obtaining permits can take several months or longer. Travel logistics involve coordination and development of an efficient route based on population locations, the preparation of suitable maps, and the acquisition of appropriate devices and support equipment.

Brassica carinata is an emerging oilseed crop in the United States, and root diseases caused by Fusarium have the potential to cause yield losses in production. In this study, B. carinata plants were randomly sampled at vegetative and seed development plant stages from South Dakota State University experimental plots. Reddish-brown lesions were observed on roots of sampled plants from which F. acuminatum, F. oxysporum, F. solani, and F. sporotrichioides were recovered. The Fusarium species were identified based on morphology and phylogenetic analyses of the translation elongation factor 1-α gene region. Pathogenicity of the four Fusarium species was evaluated on five B. carinata accessions using a modified inoculum layer method in the greenhouse. At 21 days after inoculation, root rot severity caused by Fusarium on the B. carinata accessions was assessed on a rating scale of 0 to 4 and evaluated using relative treatment effects (RTEs). The F. oxysporum isolate caused significant differences in RTE (P = 0.01) among the B. carinata accessions. However, there were no significant differences in RTE among the B. carinata accessions in response to F. acuminatum (P = 0.82), F. solani (P = 0.76), and F. sporotrichioides (P = 0.47) isolates.

Katelyn (Reg. No. GP-35, PI 673443) pennycress (Thlaspi arvense L.) germplasm was publicly released by the USDA–ARS in 2014 as part of a new crop improvement program. Katelyn was developed by two generations of mass selection based on the germination response of freshly harvested pennycress seeds. The original seed source was the wild population Beecher (PI 672505) collected from a winter fallow cornfield 3.2 km north of Hanna City, IL. The germination rate of the original parent seed under conditions of 12-h light/dark cycles at 27.5°C/11.5°C was 0%. Beecher seed kept in the dark under otherwise identical conditions germinated at 7% compared with Katelyn S2 seed, which had an immediate post-harvest germination rate of 91% for seed kept in the dark and 81% under the 12-h light/dark 27.5°C/11.5°C conditions.

Basal stalk rot (BSR) and head rot (HR) caused by Sclerotinia sclerotiorum (Lib.) de Bary are traditionally major diseases of sunflower (Helianthus annuus L.) in the United States, while Phomopsis stem canker (PSC) caused by Phomopsis helianthi Munt.-Cvet. et al. has become increasingly damaging in recent years. Host resistance is the most effective approach to managing these diseases. Here we report new sources of resistance to these diseases identified in the USDA sunflower Plant Introduction (PI) collection. A total of 260 PIs, including USDA-ARS–developed inbred lines, were tested in multiple years and locations of North Dakota, South Dakota, and Minnesota. The effects of genotype (G), environment (E), and G × E interaction were significant. Broad sense heritability (H²) for BSR, HR, and PSC were estimated as 0.70, 0.79, and 0.83, respectively, reflecting a large genetic variation in the panel for these diseases. A total of 6.2, 29.6, and 36.9% of the genotypes showed significantly higher resistance than checks against BSR, HR, and PSC, respectively. Two PIs, 531389 and 531366, were significantly more resistant than checks against all three diseases. Head rot disease incidence was positively correlated with PSC (r = 0.52, p < 0.0001), while it was negatively correlated with BSR (r = −0.16, p < 0.05). A total of 13 PIs, mostly from Hungary, exhibited dual resistance against HR and PSC. Overall, sufficient variation exists among germplasm sources to facilitate further resistance breeding of sunflower against BSR, HR, and PSC diseases.

Four accessions of cultivated sunflower (Helianthus annuus) and silverleaf sunflower (Helianthus argophyllus), were each grown in three locations (Georgia, British Columbia, and Iowa) at different planting densities and phenotyped for biomass-related traits and wood biochemistry. In most environments, H. argophyllus produced significantly more biomass than H. annuus. Cell wall chemistry for a subset of plants grown in Georgia and Iowa was assessed using analytical wet chemistry methods to measure lignin and sugar content/composition. The analysis of lignin and the S/G-lignin ratios for a larger number of samples (n > 250) was also assessed by high-throughput pyrolysis Molecular Beam Mass Spectrometry. Average pyMBMS estimated lignin content (i.e., dry weight fraction) for 60 °C dried basal stem samples of H. annuus and H. argophyllus was 29.6% (range, 24.0%–34.6%) and 28.6% (range, 24.6%–33.3%), respectively when averaged across all environments. The average S/G lignin mass ratio was 1.5 (range, 1.0–2.0) for H. annuus and 1.7 (range, 1.0–2.4) in H. argophyllus. Stem samples from these two species only differed statistically for a few cell wall chemistry traits; however, accession level differences within each species were apparent. Cell wall chemistry in both species was significantly affected by both location and planting density, thus demonstrating the need to select for these traits in the environment for which the crop will be produced. Overall, these results show that cultivated sunflower and silverleaf sunflower both possess the necessary phenotypic diversity to facilitate the development of a hybrid sunflower with improved lignocellulosic biofuels traits, namely increased biomass, decreased lignin, and increased glucan.

Gold-of-pleasure or false flax (Camelina sativa L. Crantz) is being developed as an alternative oil crop for biodiesel and for food use. The seed meal, which contains three relatively unique glucosinolates, is being evaluated for approval for use as an ingredient in animal feeds and for other uses. The objective of this research was to develop reproducible methods for the isolation of large quantities of pure camelina glucosinolates (glucoarabin, glucocamelinin, and 11-(methylsulfinyl)-undecylglucosinolate) and develop efficient methods for quantifying these compounds. The separation and purification of the camelina glucosinolates were achieved using a combination of reverse phase chromatography, counter-current chromatography and ion exchange chromatography. An efficient reverse phase HPLC separation method was used to quantitate the glucosinolate content in camelina seed and plant extracts. The quantitation methodology was used to measure glucosinolate levels in seeds from 30 cultivars grown in a U.S. field trial and measure glucosinolates levels in sprouted camelina seeds.

Crop germplasm collections are valuable resources for ongoing plant breeding efforts. To fully utilize such collections, however, researchers need detailed information about the amount and distribution of genetic diversity present within collections. Here, we report the results of a population genetic analysis of the primary gene pool of sunflower (Helianthus annuus L.) based on a broad sampling of 433 cultivated accessions from North America and Europe, as well as a range-wide collection of 24 wild sunflower populations. Gene diversity across the cultivars was 0.47, as compared with 0.70 in the wilds, indicating that cultivated sunflower harbors roughly two-thirds of the total genetic diversity present in wild sunflower. Population structure analyses revealed that wild sunflower can be subdivided into four genetically distinct population clusters throughout its North American range, whereas the cultivated sunflower gene pool could be split into two main clusters separating restorer lines from the balance of the gene pool. Use of a maximum likelihood method to estimate the contribution of the wild gene pool to the cultivated sunflower germplasm revealed that the bulk of the cultivar diversity is derived from two wild sunflower population genetic clusters that are primarily composed of individuals from the east-central United States, the same general region in which sunflower domestication is believed to have occurred. We also identified a nested subset of accessions that capture as much of the allelic diversity present within the sampled cultivated sunflower germplasm collection as possible. At the high end, a core set of 288 captured nearly 90% of the alleles present in the full set of 433, whereas a core set of just 12 accessions was sufficient to capture nearly 50% of the total allelic diversity present within this sample of cultivated sunflower.

Cuphea is a potential new oilseed crop rich in medium-chain fatty acids (C8:0 to C14:0) that may serve as a renewable, biodegradable source of oil for lubricants, motor oil, and aircraft fuel. Impacts of climate and soil environment on cuphea growth and development are not well understood. The objective of this study was to evaluate the influence of climate and soil on growth, seed yield, and seed oil characteristics of two semi-domesticated cuphea genotypes [PSR23 and HC-10 ( Cuphea viscosissima Jacq. × C. lanceolata W.T. Aiton)] and three wild species [ Cuphea wrightii , Cuphea lutea , and C. viscosissima (VS-6-CPR-1)] that show potential for domestication. The study was conducted in 2007 and 2008 at field sites in North Dakota (ND), Minnesota (MN), Iowa (IA), and Illinois (IL). Cuphea PSR23 and HC-10 were direct seeded in the field, while the three wild species were transplanted. The two plantings were treated as separate experiments. Plant growth, seed yield and oil content for the two direct-seeded lines tended to be distinctly greater in MN and ND than IL and IA, which was related more to growth temperature than soil environment. The three wild species generally performed similarly across the four different environments. C. wrightii had the greatest oil content, ranging from 320 to 360 g kg −1 , which was comprised of 59–64% lauric acid. For each genotype, the content of its most prominent saturated medium-chain fatty acid (e.g., C10:0 or C12:0) increased with decreasing latitude of field site. Seed yields for C. w rightii and C. lutea were as high as 1116 kg ha −1 . Combined with relatively high seed oil contents (280–350 g kg −1 ) these species may be good candidates for domestication. Results indicate that PSR23 and HC-10 are more regionally adapted than the wild species studied, which tended to exhibit a greater range of adaptability to climate and soil conditions.

Genetic resources are the biological basis of global food security. Collection and preservation of wild relatives of important crop species such as sunflower provide the basic foundation to improve and sustain the crop. Acquisition through exploration is the initial step in the germplasm conservation process. There are 53 species of wild

Crop wild relatives (CWR) are a rich source of genetic diversity for crop improvement. Combining ecogeographic and phylogenetic techniques can inform both conservation and breeding. Geographic occurrence, bioclimatic, and biophysical data were used to predict species distributions, range overlap and niche occupancy in 36 taxa closely related to sunflower (Helianthus annuus L.). Taxa lacking comprehensive ex situ conservation were identified. The predicted distributions for 36 Helianthus taxa identified substantial range overlap, range asymmetry and niche conservatism. Specific taxa (e.g., Helianthus deblis Nutt., Helianthus anomalus Blake, and Helianthus divaricatus L.) were identified as targets for traits of interest, particularly for abiotic stress tolerance, and adaptation to extreme soil properties. The combination of techniques demonstrates the potential for publicly available ecogeographic and phylogenetic data to facilitate the identification of possible sources of abiotic stress traits for plant breeding programs. Much of the primary genepool (wild H. annuus) occurs in extreme environments indicating that introgression of targeted traits may be relatively straightforward. Sister taxa in Helianthus have greater range overlap than more distantly related taxa within the genus. This adds to a growing body of literature suggesting that in plants (unlike some animal groups), geographic isolation may not be necessary for speciation.

Flowering dates and life forms of all available Brassica napus accessions conserved at the North Central Regional Plant Introduction Station (NCRPIS) were characterized, and a survey of molecular variation was conducted by using simple sequence repeats (SSR) in order to support better management of accessions with diverse life forms. To characterize flowering phenology, 598 B. napus accessions from the NCRPIS collection were planted in Iowa and Kansas field sites together with a current commercial cultivar and observed for days to flowering (first, 50% and 100% flowering) in 2003. Days from planting to 50% flowering ranged from 34 to 83 in Iowa and from 53 to 89 in Kansas. The mean accumulated growing degree days (GDD) to 50% flowering were 1,997 in Iowa, and 2,106 in Kansas. Between locations, the correlation in flowering time (r = 0.42) and the correlation in computed GDD (r = 0.40) were both significant. Differences in flowering-time rank were observed for several accessions. Accessions that failed to flower in Iowa in a single growing season comprised 28.5% of the accessions; of the flowering accessions, 100% plant flowering was not always achieved. Accessions were grouped according to flowering time. A stratified sample of 50 accessions was selected from these groups, including 10 non-flowering and 40 flowering accessions of diverse geographic origins and phenological variation. The flowering time observed in the sampled accessions when grown in the greenhouse were found to be significantly correlated to the flowering time observed in the field locations in Iowa (r = 0.79) and Kansas (r = 0.49). Thirty SSR markers, selected across 18 Brassica linkage groups from BrassicaDB, and 3 derived from Brassica expressed sequence tags (ESTs) were scored in the stratified sample. An average of three bands per SSR primer pair was observed. Associations of SSR marker fragments with the life forms were determined. Analysis of molecular variation by using cluster analysis and ordination resulted in recognizable, distinct groups of annual and biennial life-form types, which may have direct applications for planning and management of future seed regenerations.

This study was conducted to determine if Brassica germplasm bulks created and main- tained by the USDA-ARS North Central Plant Introduction Station (NCRPIS) were made with genetically indistinguishable component acces- sions and to examine newly identified putative duplicate accessions to determine if they can be bulked. Using ten microsatellite primer pairs, we genotyped two bulks of B. rapa L. ssp. dichotoma (Roxb.) Hanelt comprising four accessions and three bulks of B. rapa L. ssp. trilocularis (Roxb.) Hanelt comprising fourteen accessions, as well as four pairs of putatively duplicate accessions of B. napus L. Assignment tests on ten individual plants per accession were conducted using a model-based clustering method to arrive at prob- abilities of likelihood of accession assignment. The assignment tests indicated that one of the two bulks of B. rapa ssp. dichotoma involves geneti- cally heterogeneous accessions. It was observed in the B. rapa ssp. trilocularis bulks that the compo- nent accessions could be differentiated into groups, with misassignments observed most fre- quent within groups. In B. napus, only one of the four pairs of putative duplicates showed significant genetic differentiation. The other three pairs of putative duplicates lack differences and support the creation of bulks. The results of the assignment tests were in agreement with cluster analyses and tests of population differentiation. Implications of these results in terms of germplasm management include the maintenance and/or re-creation of some Brassica germplasm bulks by excluding those accessions identified as being unique in this study.

The I locus in soybean (Glycine max) corresponds to a region of chalcone synthase (CHS) gene duplications affecting seed pigmentation. We sequenced and annotated BAC clone 104J7, which harbors a dominant iiallele from Glycine max 'Williams 82', to gain insight into the genetic structure of this multigenic region in addition to examining its flanking regions. The 103-kb BAC encompasses a gene-rich region with 11 putatively expressed genes. In addition to six copies of CHS, these genes include: a geranylgeranyltransferase type II β subunit (E.C.2.5.1.60), a β-galactosidase, a putative spermine and (or) spermidine synthase (E.C.2.5.1.16), and an unknown expressed gene. Strikingly, sequencing data revealed that the 10.91-kb CHS1, CHS3, CHS4 cluster is present as a perfect inverted repeat separated by 5.87 kb. Contiguous arrangement of CHS paralogs could lead to folding into multiple secondary structures, hypothesized to induce deletions that have previously been shown to effect CHS expression. BAC104J7 also contains several gene fragments representing a cation/hydrogen exchanger, a 40S ribosomal protein, a CBL-interacting protein kinase, and the amino terminus of a subtilisin. Chimeric ESTs were identified that may represent read-through transcription from a flanking truncated gene into a CHS cluster, generating aberrant CHS RNA molecules that could play a role in CHS gene silencing.Key words: chalcone synthase, gene duplication, gene silencing, gene-rich region, soybean, BAC.

Surveying the soybean genome with 683 bacterial artificial chromosome (BAC) contiguous groups (contigs) anchored by restriction fragment length polymorphisms (RFLPs) enabled us to explore microsyntenic relationships among duplicated regions and also to examine the physical organization of hypomethylated (and presumably gene-rich) genomic regions. Numerous cases where nonhomologous RFLPs hybridized to common BAC clones indicated that RFLPs were physically clustered in soybean, apparently in less than 25% of the genome. By extension, we speculate that most of the genes are clustered in less than 275 M of the soybean genome. Approximately 40%–45% of this gene-rich portion is associated with the RFLP-anchored contigs described in this study. Similarities in genome organization among BAC contigs from duplicate genomic regions were also examined. Homoeologous BAC contigs often exhibited extensive microsynteny. Furthermore, paralogs recovered from duplicate contigs shared 86%–100% sequence identity.Key words: Glycine max, bacterial artifical chromosome, restriction fragment length polymorphism, genome duplication, gene distribution.

Simple sequence repeats (SSRs) are versatile DNA markers that are readily assayed and highly informative. Unfortunately, non-targeted approaches to SSR development often leave large genomic regions without SSR markers. In some cases these same genomic regions are already populated by other types of DNA markers, especially restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAPDs), and amplified fragment length polymorphisms (AFLPs). To identify SSR markers in such regions, bacterial artificial chromosome (BAC) clones can be used as intermediaries. First, one or more BAC clones in a region of interest are identified through the use of an existing DNA marker. BAC clones uncovered in this initial step are then used to create a small insert DNA library that can be screened for the presence of SSR-containing clones. Because BAC inserts are often 100-kb pairs or more in size, most contain one or more SSRs. This strategy was applied to two regions of the soybean genome near genes that condition resistance to the soybean cyst nematode on molecular linkage groups G and A2. This targeted approach to identifying new DNA markers can readily be extended to other types of DNA markers, including single nucleotide polymorphisms.

Three fields were planted in Ames in 2010, two association mapping fields, N3 and A, and a recombinant inbred line field, N13. Phenotype data and images were transferred to UGA to support genetic and genomic analyses of woody biomass-related traits.

Several changes occur in wild-type Chlamydomonas reinhardtii upon exposure to limiting CO2, including induction of several polypeptides. Polypeptide induction was previously shown to correlate with appearance of the active CO2-concentrating mechanism (CCM) of this alga. In this paper induction of polypeptides by limiting CO2 was investigated in mutants with lesions in the CCM. Mutants with lesions in the ca-1 and pmp-1 loci exhibited alterations in polypeptide induction, but it was concluded that the alterations probably do not represent their primary genetic lesions. Other changes that occur in this alga in response to limiting CO2 were also investigated. Based on a lack of significant change in the transcript abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunit genes in the wild type, it was concluded that the previously reported transient decline in synthesis of both subunits is controlled at the translational level. A transient increase in the activity of the photorespiratory enzyme phosphoglycolate phosphatase was observed in the wild type but not in a mutant, cia-5, that lacks induction of the CCM. In addition, changes in expression of genes encoding periplasmic carbonic anhydrase, a 36-kDa membrane-associated protein and a chlorophyll-binding protein occurred in the wild type but not in cia-5 in response to limiting CO2. The absence of these changes in cia-5 was attributed to a lack of either the signal itself or transduction of the signal responsible for adaptation to limiting CO2, which led to speculation that a larger range of responses are regulated by the same signal than was previously recognized. Key words: photosynthesis, photorespiration, algae, inorganic carbon transport, transcription, translation.

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO(2) concentration for photoautrophic growth in spite of the apparent induction of a functional CO(2) concentrating mechanism in air-adapted cells. In 2% O(2) the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O(2), photosynthetic O(2) evolution is severely inhibited in the mutant by preillumination in limiting CO(2), although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O(2) evolution measurements. Net CO(2) uptake is also inhibited when the cells are exposed to air (21% O(2), 0.035% CO(2), balance N(2)) for longer than a few minutes. [(14)C]Phosphoglycolate accumulates within 5 minutes of photosynthetic (14)CO(2) fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO(2) requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.

Fifteen Rps genes confer resistance against the oomycete pathogen Phytophthora sojae, which causes root and stem rot disease in soybean. We have isolated a disease resistance gene-like sequence from the genomic region containing Rps1-k. Four classes of cDNA of the sequence were isolated from etiolated hypocotyl tissues that express the Rps1-k-encoded Phytophthora resistance. Sequence analyses of a cDNA clone showed that the sequence is a member of the coiled coil-nucleotide binding site–leucine rich repeat (CC-NBS–LRR)-type of disease resistance genes. It showed 36% identity to the recently cloned soybean resistance gene Rpg1-b, which confers resistance against Pseudomonas syringae pv. glycinea, and 56% and 38% sequence identity to putative resistance gene sequences from lotus and Medicago truncatula, respectively. The soybean genome contains about 38 copies of the sequence. Most of these copies are clustered in approximately 600 kb of contiguous DNA of the Rps1-k region. We have identified a recombinant that carries both rps1-k- and Rps1-k-haplotype-specific allelomorphs of two Rps1-k-linked molecular markers. An unequal crossover event presumably led to duplication of alleles for these two physically linked molecular markers. We hypothesize that the unequal crossing over was one of the mechanisms involved in tandem duplication of CC-NBS–LRR sequences in the Rps1-k region.

A total of 391 simple sequence repeat (SSR) markers designed from genomic DNA libraries, 24 derived from existing GenBank genes or ESTs, and five derived from bacterial artificial chromosome (BAC) end sequences were developed. In contrast to SSRs derived from EST sequences, those derived from genomic libraries were a superior source of polymorphic markers, given that the mean number of tandem repeats in the former was significantly less than that of the latter (P

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.

Soybean ( Glycine max L. Merrill) linkage group A2 contains a major resistance gene to the soybean cyst nematode ( Heterodera glycines Ichinohe) at the Rhg (4) locus near a gene encoding aspartokinase homoserine dehydrogenase (AK-HSDH) and also near the I locus affecting seed coat color. To identify clones related to this region of the genome, we used a PCR assay using primers designed from a gene encoding AK-HSDH to screen approximately 40,000 clones from a bacterial artificial chromosome (BAC) library constructed from genomic DNA of the susceptible cv. Williams 82. The identified BACs were screened with a second PCR assay using primers designed from DNA sequence associated with the I locus to confirm the location of the BACs. Only BAC Gm_ISb001_056_G02 (56G2) was positive for both assays. BAC 56G2 contains several genes previously associated with stress or defense response including genes with high sequence similarity to those encoding chalcone synthase, glucosyl-transferase, a heat-shock transcription factor, a membrane-associated salt-inducible protein, adenosyl homocysteinase, a protein kinase, and a G10-like protein. The map contributes to our understanding of the organization of the soybean genome and to the completion of a physical map of the soybean genome. In addition, the genes identified provide landmarks to identify BAC clones near the Rhg (4) locus in resistant soybean genomic libraries and provide a foundation for comparison of soybean cyst nematode resistant and -susceptible DNA sequences in this region.

PCR amplification was previously used to identify a cluster of resistance gene analogues (RGAs) on soybean linkage group J. Resistance to powdery mildew (Rmd-c), Phytophthora stem and root rot (Rps2), and an ineffective nodulation gene (Rj2) map within this cluster. BAC fingerprinting and RGA-specific primers were used to develop a contig of BAC clones spanning this region in cultivar “Williams 82” [rps2, Rmd (adult onset), rj2]. Two cDNAs with homology to the TIR/NBD/LRR family of R-genes have also been mapped to opposite ends of a BAC in the contig Gm_Isb001_091F11 (BAC 91F11). Sequence analyses of BAC 91F11 identified 16 different resistance-like gene (RLG) sequences with homology to the TIR/NBD/LRR family of disease resistance genes. Four of these RLGs represent two potentially novel classes of disease resistance genes: TIR/NBD domains fused inframe to a putative defense-related protein (NtPRp27-like) and TIR domains fused inframe to soybean calmodulin Ca2+-binding domains. RT-PCR analyses using gene-specific primers allowed us to monitor the expression of individual genes in different tissues and developmental stages. Three genes appeared to be constitutively expressed, while three were differentially expressed. Analyses of the R-genes within this BAC suggest that R-gene evolution in soybean is a complex and dynamic process.

Clusters of Resistance-like genes (RLGs) have been identified from a variety of plant species. In soybean, RLG-specific primers and BAC-fingerprinting were used to develop a contig of overlapping BACs for a cluster of RLGs on soybean linkage group J. The resistance genes Rps2 (Phytophthora stem and root rot) and Rmd-c (powdery mildew) and the ineffective nodulation gene Rj2 were previously mapped to this region of linkage group J. PCR hybridization was used to place two TIR/NBD/LRR cDNAs on overlapping BACs from this contig. Both of the cDNAs were present on BAC 34P7. Fingerprinting of this BAC suggested as many as twelve different RLGs were present. Given the high nucleotide identity shared between cDNAs LM6 and MG13 (>90%), direct sequencing of this region would be difficult. More sequence information was needed about the RLGs within this region before sequencing could be undertaken. By comparing the genomic sequences of cDNAs LM6 and MG13 we identified conserved regions from which oligonucleotide primers specific to BAC 34P7 RLGs could be designed. The nine primer pairs spanned the genomic sequence of LM6 and produced overlapping RLG products upon amplification of BAC 34P7. Amplification products from 12 different RLGs were identified. On average, nucleotide identity between RLG sequences was greater than 95%. Examination of RLG sequences also revealed evidence of additions, deletions and duplications within targeted regions of these genes. Using previously mapped cDNAs we were able to quickly and inexpensively access multiple RLGs within a single specific cluster.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes. Key words: expression, R-genes, contig, RGAs, soybean.

Various growth and physiological parameters were measured in germinating, presenescent, and senescing soybean (Glycine max [L.] Merr.) cotyledons and in cotyledons rejuvenated by epicotyl removal 18 days after planting. The maximal measured carbon dioxide exchange rates (CER) in the cotyledons were in the range of those reported for field-grown soybean leaves. Rejuvenated cotyledons accumulated total chlorophyll in excess of the maximum observed in presenescent cotyledons. When photosynthetic rates were expressed per cotyledon, the CER in rejuvenated tissue recovered to the maximal rates observed in presenescent cotyledons. Ribulose-1,5-bisphosphate carboxylase/oxygenase in rejuvenated cotyledons also recovered to the maximal amount seen in presenescent cotyledons so that CER appeared to be a function of ribulose-1,5-bisphosphate carboxylase/oxygenase content during most of the period studied. Observations of the postillumination outburst of CO(2) and (14)C label in glycine indicated that photorespiration was occurring in the cotyledons and that photorespiration relative to photosynthesis was different in rejuvenated compared with presenescent cotyledons.

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO(2)-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO(2). Adaptation to air levels of CO(2) by Chlamydomonas involves induction of a CO(2)-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO(2)-enriched cells. Unlike WT, the high-CO(2)-requiring mutant, cia-5, does not respond to limiting CO(2) conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO(2) which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.

High-CO2 requiring mutants of Chlamydomonas reinhardtii have been isolated and used to study the CO2 concentrating mechanism which is induced in this unicellular green alga during growth under air levels of CO2 (1). One of the high-CO2 requiring mutants, 18–7F, has shown some characteristics that indicate it is not a mutant of some component of the CO2-concentrating mechanism but a mutant deficient in phosphoglycolate phosphatase, the first enzyme specific to the photorespiratory metabolic pathway.

The rate of respiration in sectioned leaves of Bryophyllum tubiflorum Harv. increases with decreasing section thickness. The rates of uninhibited respiration in 2- and 8-millimeter-thick sections are 74 and 46 microliters of O(2) per gram fresh weight of unruptured tissue per hour at 20 C, whereas the rate in the presence of cyanide is 31 microliters of O(2) in each case. The rates are unaffected by salicylhydroxamic acid, but cyanide and salicylhydroxamic acid together completely eliminate O(2) uptake. The capacity of the alternative respiratory pathway is thus initially high (estimated at 84% of the uninhibited respiratory rate in whole leaves) and remains constant but probably unexpressed subsequent to the rapid induction of wound respiration.