Quantitative trait locus analysis and construction of consensus genetic map for foliar disease resistance based on two recombinant inbred line populations in cultivated groundnut (Arachis hypogaea L.).

Late leaf spot (LLS) and rust have the greatest impact on yield losses worldwide in groundnut (Arachis hypogaea L.). With the objective of identifying tightly linked markers to these diseases, a total of 3,097 simple sequence repeats (SSRs) were screened on the parents of two recombinant inbred line (RIL) populations, namely TAG 24 × GPBD 4 (RIL-4) and TG 26 × GPBD 4 (RIL-5), and segregation data were obtained for 209 marker loci for each of the mapping populations. Linkage map analysis of the 209 loci resulted in the mapping of 188 and 181 loci in RIL-4 and RIL-5 respectively. Using 143 markers common to the two maps, a consensus map with 225 SSR loci and total map distance of 1,152.9 cM was developed. Comprehensive quantitative trait locus (QTL) analysis detected a total of 28 QTL for LLS and 15 QTL for rust. A major QTL for LLS, namely QTL(LLS)01 (GM1573/GM1009-pPGPseq8D09), with 10.27-62.34% phenotypic variance explained (PVE) was detected in all the six environments in the RIL-4 population. In the case of rust resistance, in addition to marker IPAHM103 identified earlier, four new markers (GM2009, GM1536, GM2301 and GM2079) showed significant association with the major QTL (82.96% PVE). Localization of 42 QTL for LLS and rust on the consensus map identified two candidate genomic regions conferring resistance to LLS and rust. One region present on linkage group AhXV contained three QTL each for LLS (up to 67.98% PVE) and rust (up to 82.96% PVE). The second candidate genomic region contained the major QTL with up to 62.34% PVE for LLS. Molecular markers associated with the major QTL for resistance to LLS and rust can be deployed in molecular breeding for developing groundnut varieties with enhanced resistance to foliar diseases. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9661-z) contains supplementary material, which is available to authorized users.

Quantitative trait locus analysis and construction of consensus genetic map for drought tolerance traits based on three recombinant inbred line populations in cultivated groundnut (Arachis hypogaea L.).

Groundnut (Arachis hypogaea L.) is an important food and cash crop grown mainly in semi-arid tropics (SAT) regions of the world where drought is the major constraint on productivity. With the aim of understanding the genetic basis and identification of quantitative trait loci (QTL) for drought tolerance, two new recombinant inbred line (RIL) mapping populations, namely ICGS 76 × CSMG 84-1 (RIL-2) and ICGS 44 × ICGS 76 (RIL-3), were used. After screening of 3,215 simple sequence repeat (SSR) markers on the parental genotypes of these populations, two new genetic maps were developed with 119 (RIL-2) and 82 (RIL-3) SSR loci. Together with these maps and the reference map with 191 SSR loci based on TAG 24 × ICGV 86031 (RIL-1), a consensus map was constructed with 293 SSR loci distributed over 20 linkage groups, spanning 2,840.8 cM. As all these three populations segregate for drought-tolerance-related traits, a comprehensive QTL analysis identified 153 main effect QTL (M-QTL) and 25 epistatic QTL (E-QTL) for drought-tolerance-related traits. Localization of these QTL on the consensus map provided 16 genomic regions that contained 125 QTL. A few key genomic regions were selected on the basis of the QTL identified in each region, and their expected role in drought adaptation is also discussed. Given that no major QTL for drought adaptation were identified, novel breeding approaches such as marker-assisted recurrent selection (MARS) and genomic selection (GS) approaches are likely to be the preferred approaches for introgression of a larger number of QTL in order to breed drought-tolerant groundnut genotypes. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11032-011-9660-0) contains supplementary material, which is available to authorized users.

Identification of several small main-effect QTLs and a large number of epistatic QTLs for drought tolerance related traits in groundnut (Arachis hypogaea L.).

Cultivated groundnut or peanut (Arachis hypogaea L.), an allotetraploid (2n = 4x = 40), is a self pollinated and widely grown crop in the semi-arid regions of the world. Improvement of drought tolerance is an important area of research for groundnut breeding programmes. Therefore, for the identification of candidate QTLs for drought tolerance, a comprehensive and refined genetic map containing 191 SSR loci based on a single mapping population (TAG 24 x ICGV 86031), segregating for drought and surrogate traits was developed. Genotyping data and phenotyping data collected for more than ten drought related traits in 2-3 seasons were analyzed in detail for identification of main effect QTLs (M-QTLs) and epistatic QTLs (E-QTLs) using QTL Cartographer, QTLNetwork and Genotype Matrix Mapping (GMM) programmes. A total of 105 M-QTLs with 3.48-33.36% phenotypic variation explained (PVE) were identified using QTL Cartographer, while only 65 M-QTLs with 1.3-15.01% PVE were identified using QTLNetwork. A total of 53 M-QTLs were such which were identified using both programmes. On the other hand, GMM identified 186 (8.54-44.72% PVE) and 63 (7.11-21.13% PVE), three and two loci interactions, whereas only 8 E-QTL interactions with 1.7-8.34% PVE were identified through QTLNetwork. Interestingly a number of co-localized QTLs controlling 2-9 traits were also identified. The identification of few major, many minor M-QTLs and QTL × QTL interactions during the present study confirmed the complex and quantitative nature of drought tolerance in groundnut. This study suggests deployment of modern approaches like marker-assisted recurrent selection or genomic selection instead of marker-assisted backcrossing approach for breeding for drought tolerance in groundnut.

Fine mapping of the sunflower resistance locus Pl(ARG) introduced from the wild species Helianthus argophyllus.

Downy mildew, caused by Plasmopara halstedii, is one of the most destructive diseases in cultivated sunflower (Helianthus annuus L.). The dominant resistance locus Pl(ARG) originates from silverleaf sunflower (H. argophyllus Torrey and Gray) and confers resistance to all known races of P. halstedii. We mapped Pl(ARG) on linkage group (LG) 1 of (cms)HA342 × ARG1575-2, a population consisting of 2,145 F(2) individuals. Further, we identified resistance gene candidates (RGCs) that cosegregated with Pl(ARG) as well as closely linked flanking markers. Markers from the target region were mapped with higher resolution in NDBLOS(sel) × KWS04, a population consisting of 2,780 F(2) individuals that does not segregate for Pl(ARG). A large-insert sunflower bacterial artificial chromosome (BAC) library was screened with overgo probes designed for markers RGC52 and RGC151, which cosegregated with Pl(ARG). Two RGC-containing BAC contigs were anchored to the Pl(ARG) region on LG 1.

Selection for low erucic acid and genetic mapping of loci affecting the accumulation of very long-chain fatty acids in meadowfoam seed storage lipids.

Erucic acid (22:1(13)) has been identified as an anti-nutritional compound in meadowfoam (Limnanthes alba) and other oilseeds in the Brassicales, a classification which has necessitated the development of low erucic acid cultivars for human consumption. The erucic acid concentrations of meadowfoam wild types (8%-24%) surpass industry standards for human consumption (

The first SSR-based genetic linkage map for cultivated groundnut (Arachis hypogaea L.).

Molecular markers and genetic linkage maps are pre-requisites for molecular breeding in any crop species. In case of peanut or groundnut (Arachis hypogaea L.), an amphidiploid (4X) species, not a single genetic map is, however, available based on a mapping population derived from cultivated genotypes. In order to develop a genetic linkage map for tetraploid cultivated groundnut, a total of 1,145 microsatellite or simple sequence repeat (SSR) markers available in public domain as well as unpublished markers from several sources were screened on two genotypes, TAG 24 and ICGV 86031 that are parents of a recombinant inbred line mapping population. As a result, 144 (12.6%) polymorphic markers were identified and these amplified a total of 150 loci. A total of 135 SSR loci could be mapped into 22 linkage groups (LGs). While six LGs had only two SSR loci, the other LGs contained 3 (LG_AhXV) to 15 (LG_AhVIII) loci. As the mapping population used for developing the genetic map segregates for drought tolerance traits, phenotyping data obtained for transpiration, transpiration efficiency, specific leaf area and SPAD chlorophyll meter reading (SCMR) for 2 years were analyzed together with genotyping data. Although, 2-5 QTLs for each trait mentioned above were identified, the phenotypic variation explained by these QTLs was in the range of 3.5-14.1%. In addition, alignment of two linkage groups (LGs) (LG_AhIII and LG_AhVI) of the developed genetic map was shown with available genetic maps of AA diploid genome of groundnut and Lotus and Medicago. The present study reports the construction of the first genetic map for cultivated groundnut and demonstrates its utility for molecular mapping of QTLs controlling drought tolerance related traits as well as establishing relationships with diploid AA genome of groundnut and model legume genome species. Therefore, the map should be useful for the community for a variety of applications.

Identifying quantitative trait loci for resistance to Sclerotinia head rot in two USDA sunflower germplasms.

Sclerotinia head rot is a major disease of sunflower in the world, and quantitative trait loci (QTL) mapping could facilitate understanding of the genetic basis of head rot resistance and breeding in sunflower. One hundred twenty-three F2:3 and F2:4 families from a cross between HA 441 and RHA 439 were studied. The mapping population was evaluated for disease resistance in three field experiments in a randomized complete block design with two replicates. Disease incidence (DI) and disease severity (DS) were assessed. A genetic map with 180 target region amplification polymorphism, 32 simple sequence repeats, 11 insertion-deletion, and 2 morphological markers was constructed. Nine DI and seven DS QTL were identified with each QTL explaining 8.4 to 34.5% of phenotypic variance, suggesting the polygenic basis of the resistance to head rot. Five of these QTL were identified in more than one experiment, and each QTL explained more than 12.9% of phenotypic variance. These QTL could be useful in sunflower breeding. Although a positive correlation existed between the two disease indices, most of the respective QTL were located in different chromosomal regions, suggesting a different genetic basis for the two indices.

Identification and mapping of SNPs from ESTs in sunflower.

More than 67,000 expressed sequence tags (ESTs) have recently been generated for sunflower (Helianthus), including 44,000 from cultivated confectionery (RHA280) and oilseed (RHA801) lines of Helianthus annuus and 23,000 from drought- and salt-tolerant wild sunflowers, H. argophyllus and H. paradoxus, respectively. To create a transcript map for sunflower, we identified 605 ESTs that displayed small insertion-deletion polymorphism (SNP) variation in silico, had apparent tissue-specific expression patterns, and/or were ESTs with candidate functions in traits such as development, cell transport, metabolism, plant defense, and tolerance to abiotic stress. Primer pairs for 535 of the loci were designed from the ESTs and screened for polymorphism in recombinant inbred lines derived from a cross between the same cultivars (RHA280 x RHA801) employed for sequencing. In total, 273 of the loci amplified polymorphic products, of which 243 mapped to the 17 linkage groups previously identified for sunflower. Comparisons with previously mapped QTL revealed some cases where ESTs with putatively related functions mapped near QTLs identified in other crosses for salt tolerance and for domestication traits such as stem diameter, shattering, flowering time, and achene size.

Identification and validation of QTL for Sclerotinia midstalk rot resistance in sunflower by selective genotyping.

Midstalk rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is an important cause of yield loss in sunflower (Helianthus annuus L.). Objectives of this study were to: (1) estimate the number, genomic positions and genetic effects of quantitative trait loci (QTL) for resistance to midstalk rot in line TUB-5-3234, derived from an interspecific cross; (2) determine congruency of QTL between this line and other sources of resistance; and (3) make inferences about the efficiency of selective genotyping (SG) in detecting QTL conferring midstalk rot resistance in sunflower. Phenotypic data for three resistance (stem lesion, leaf lesion and speed of fungal growth) and two morphological (leaf length and leaf length with petiole) traits were obtained from 434 F3 families from cross CM625 (susceptible) x TUB-5-3234 (resistant) under artificial infection in field experiments across two environments. The SG was applied by choosing the 60 most resistant and the 60 most susceptible F3 families for stem lesion. For genotyping of the respective F2 plants, 78 simple sequence repeat markers were used. Genotypic variances were highly significant for all traits. Heritabilities and genotypic correlations between reMidstalk rot, caused by Sclerotinia sclerotiorum (Lib.) de Bary, is an important cause of yield loss in sunflower (Helianthus annuus L.). Objectives of this study were to: (1) estimate the number, genomic positions and genetic effects of quantitative trait loci (QTL) for resistance to midstalk rot in line TUB-5-3234, derived from an interspecific cross; (2) determine congruency of QTL between this line and other sources of resistance; and (3) make inferences about the efficiency of selective genotyping (SG) in detecting QTL conferring midstalk rot resistance in sunflower. Phenotypic data for three resistance (stem lesion, leaf lesion and speed of fungal growth) and two morphological (leaf length and leaf length with petiole) traits were obtained from 434 F3 families from cross CM625 (susceptible) x TUB-5-3234 (resistant) under artificial infection in field experiments across two environments. The SG was applied by choosing the 60 most resistant and the 60 most susceptible F3 families for stem lesion. For genotyping of the respective F2 plants, 78 simple sequence repeat markers were used. Genotypic variances were highly significant for all traits. Heritabilities and genotypic correlations between resistance traits were moderate to high. Three to four putative QTL were detected for each resistance trait explaining between 40.8% and 72.7% of the genotypic variance (PTS). Two QTL for stem lesion showed large genetic effects and corroborated earlier findings from the cross NDBLOSsel (resistant) x CM625 (susceptible). Our results suggest that SG can be efficiently used for QTL detection and the analysis of congruency for resistance genes across populations.

QTL mapping of Sclerotinia midstalk-rot resistance in sunflower.

In many sunflower-growing regions of the world, Sclerotinia sclerotiorum (Lib.) de Bary is the major disease of sunflower (Helianthus annuus L.). In this study, we mapped and characterized quantitative trait loci (QTL) involved in resistance to S. sclerotiorum midstalk rot and two morphological traits. A total of 351 F3 families developed from a cross between a resistant inbred line from the germplasm pool NDBLOS and the susceptible line CM625 were assayed for their parental F2 genotype at 117 codominant simple sequence repeat markers. Disease resistance of the F3 families was screened under artificial infection in field experiments across two sowing times in 1999. For the three resistance traits (leaf lesion, stem lesion, and speed of fungal growth) and the two morphological traits, genotypic variances were highly significant. Heritabilities were moderate to high (h2=0.55-0.89). Genotypic correlations between resistance traits were highly significant (P<0.01) but moderate. QTL were detected for all three resistance traits, but estimated effects at most QTL were small. Simultaneously, they explained between 24.4% and 33.7% of the genotypic variance for resistance against S. sclerotiorum. Five of the 15 genomic regions carrying a QTL for either of the three resistance traits also carried a QTL for one of the two morphological traits. The prospects of marker-assisted selection (MAS) for resistance to S. sclerotiorum are limited due to the complex genetic architecture of the trait. MAS can be superior to classical phenotypic selection only with low marker costs and fast selection cycles.

PlArg from Helianthus argophyllus is unlinked to other known downy mildew resistance genes in sunflower.

The PlArg locus in the sunflower (Helianthus annuus L.) inbred line Arg1575-2 conferring resistance to at least four tested races (300, 700, 730, 770) of downy mildew (Plasmopara halstedii) was localized by the use of simple sequence repeat (SSR) markers. Bulked segregant analysis (BSA) was conducted on 126 individuals of an F2 progeny from a cross between a downy mildew susceptible line, CmsHA342, and Arg1575-2. Twelve SSR markers linked to the PlArg locus were identified. All markers were located proximal to PlArg on linkage group LG1 based on the map of Yu et al. (2003) in a window of 9.3 cM. Since PlArg was mapped to a linkage group different from all other Pl genes previously mapped with SSRs, it can be concluded that PlArg provides a new source of resistance against P. halstedii in sunflower.

Quantitative trait loci for broomrape (Orobanche cumana Wallr.) resistance in sunflower.

Broomrape (Orobanche cumana Wallr.) is a root parasite of sunflower that is regarded as one of the most important constraints of sunflower production in the Mediterranean region. Breeding for resistance is the most effective method of control. P-96 is a sunflower line which shows dominant resistance to broomrape race E and recessive resistance to the very new race F. The objective of this study was to map and characterize quantitative trait loci (QTL) for resistance to race E and to race F of broomrape in P-96. A population from a cross between P-96 and the susceptible line P-21 was phenotyped for broomrape resistance in four experiments, two for race E and two for race F, by measuring different resistance parameters (resistance or susceptibility, number of broomrape per plant, and proportion of resistant plants per F(3) family). This population was also genotyped with microsatellite and RFLP markers. A linkage map comprising 103 marker loci distributed on 17 linkage groups was developed, and composite interval mapping analyses were performed. In total, five QTL ( or1.1, or3.1, or7.1 or13.1 and or13.2) for resistance to race E and six QTL ( or1.1, or4.1, or5.1, or13.1, or13.2 and or16.1) for resistance to race F of broomrape were detected on 7 of the 17 linkage groups. Phenotypic variance for race E resistance was mainly explained by the major QTL or3.1 associated to the resistance or susceptibility character ( R(2)=59%), while race F resistance was explained by QTL with a small to moderate effect ( R(2) from 15.0% to 38.7%), mainly associated with the number of broomrape per plant. Or3.1 was race E-specific, while or1.1, or13.1 and or13.2 of were non-race specific. Or13.1, and or13.2 were stable across the four experiments. Or3.1, and or7.1 were stable over the two race E experiments and or1.1 and or5.1 over the two race F experiments. The results from this study suggest that resistance to broomrape in sunflower is controlled by a combination of qualitative, race-specific resistance affecting the presence or absence of broomrape and a quantitative non-race specific resistance affecting their number.

Conserved simple sequence repeats for the Limnanthaceae (Brassicales).

The Limnanthaceae (Order Brassicales) is a family of 18 taxa of Limnanthes (meadowfoam) native to California, Oregon, and British Columbia. Cultivated meadowfoam ( L. alba Benth.), a recently domesticated plant, has been the focus of research and development as an industrial oilseed for three decades. The goal of the present research was to develop several hundred simple sequence repeat (SSR) markers for genetic mapping, molecular breeding, and genomics research in wild and cultivated meadowfoam taxa. We developed 389 SSR markers for cultivated meadowfoam by isolating and sequencing 1,596 clones from L. alba genomic DNA libraries enriched for AG(n) or AC(n) repeats, identifying one or more unique SSRs in 696 clone sequences, and designing and testing primers for 624 unique SSRs. The SSR markers were screened for cross- taxa utility and polymorphisms among ten of 17 taxa in the Limnanthaceae; 373 of these markers were polymorphic and 106 amplified loci from every taxon. Cross-taxa amplification percentages ranged from 37.3% in L. douglasii ssp. rosea (145/389) to 85.6% in L. montana (333/389). The SSR markers amplified 4,160 unique bands from 14 genotypes sampled from ten taxa (10.7 unique bands per SSR marker), of which 972 were genotype-specific. Mean and maximum haplotype heterozygosities were 0.71 and 0.90, respectively, among six L. alba genotypes and 0.63 and 0.93, respectively, among 14 genotypes (ten taxa). The SSR markers supply a critical mass of high-throughput DNA markers for biological and agricultural research across the Limnanthaceae and open the way to the development of a genetic linkage map for meadowfoam ( x = 5).

The development of a genetic map for meadowfoam comprised of amplified fragment length polymorphisms.

Limnanthes alba Benth. (meadowfoam), a diploid ( x=5) winter annual, produces novel very long-chain seed oils (C(20) and C(22)) with less than 2% saturated fatty acids. The first genetic map of meadowfoam, a recently domesticated species, is described herein. Two phenotypically diverse inbred lines, OMF40-11 ( L. alba ssp. alba) and OMF64 ( L. alba ssp. versicolor), were screened for amplified fragment length polymorphisms (AFLPs) using 16 primer combinations. Twenty three percent of the AFLP bands (415 out of 1,801) were polymorphic between OMF40-11 and OMF64. One hundred (OMF40-11xOMF64)xOMF64 BC(1) progeny were genotyped for 107 polymorphic AFLP markers produced by nine AFLP primer combinations. One hundred and three AFLP loci amalgamated into five linkage groups with 14 to 28 loci per linkage group (four loci segregated independently). The map was 698.5-cM long with a mean interlocus spacing of 6.7 cM and no dense clustering of loci. The segregation ratios for 25 loci (23.2%) were significantly distorted. Twenty one of the distorted loci (84%) had an excess of L. alba ssp. versicolor (recurrent parent) alleles. The distorted loci, apart from one locus on linkage group 4, were distally clustered on both ends of linkage groups 1, 4 and 5. The development of the map was facilitated by the small chromosome number, an abundance of restriction site polymorphisms between the two subspecies (23%), and a high multiplex ratio of the AFLP markers (112 per primer combination).

Stearoyl-ACP and oleoyl-PC desaturase genes cosegregate with quantitative trait loci underlying high stearic and high oleic acid mutant phenotypes in sunflower.

The genetic control of the synthesis of stearic acid (C18:0) and oleic acid (C18:1) in the seed oil of sunflower was studied through candidate-gene and QTL analysis. Two F(2) mapping populations were developed using the high C18:0 mutant CAS-3 crossed to either HA-89 (standard, high linoleic fatty acid profile), or HAOL-9 (high C18:1 version of HA-89). A stearoyl-ACP desaturase locus (SAD17A), and an oleoyl-PC de-saturase locus (OLD7) were found to cosegregate with the previously described Es1 and Ol genes controlling the high C18:0 and the high C18:1 traits, respectively. Using linkage maps constructed from AFLP and RFLP markers, these loci mapped to LG1 (SAD17A) and to LG14 (OLD7) and were found to underlie the major QTLs affecting the concentrations of C18:0 and C18:1, explaining around 80% and 56% of the phenotypic variance of these fatty acids, respectively. These QTLs pleiotropically affected the levels of other primary fatty acids in the seed storage lipids. A minor QTL affecting both C18:0 and C18:1 levels was identified on LG8 in the HAOL-9xCAS-3 F(2). This QTL showed a significant epistatic interaction for C18:1 with the QTL at the OLD7 locus, and was hypothesized to be a modifier of Ol. Two additional minor C18:0 QTLs were also detected on LG7 and LG3 in the HA-89xCAS-3 and the HAOL-9xCAS-3 F(2) populations, respectively. No association between a mapped FatB thioesterase locus and fatty acid concentration was found. These results provide strong support about the role of fatty acid desaturase genes in determining fatty acid composition in the seed oil of sunflower.

Candidate disease resistance genes in sunflower cloned using conserved nucleotide-binding site motifs: genetic mapping and linkage to the downy mildew resistance gene Pl1.

Disease resistance gene candidates (RGCs) belonging to the nucleotide-binding site (NBS) superfamily have been cloned from numerous crop plants using highly conserved DNA sequence motifs. The aims of this research were to (i) isolate genomic DNA clones for RGCs in cultivated sunflower (Helianthus annuus L.) and (ii) map RGC markers and Pl1, a gene for resistance to downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni) race 1. Degenerate oligonucleotide primers targeted to conserved NBS DNA sequence motifs were used to amplify RGC fragments from sunflower genomic DNA. PCR products were cloned, sequenced, and assigned to 11 groups. RFLP analyses mapped six RGC loci to three linkage groups. One of the RGCs (Ha-4W2) was linked to Pl1, a downy mildew resistance gene. A cleaved amplified polymorphic sequence (CAPS) marker was developed for Ha-4W2 using gene-specific oligonucleotide primers. Downy mildew susceptible lines (HA89 and HA372) lacked a 276-bp Tsp5091 restriction fragment that was present in downy mildew resistant lines (HA370, 335, 336, 337, 338, and 339). HA370 x HA372 F2 progeny were genotyped for the Ha-4W2 CAPS marker and phenotyped for resistance to downy mildew race 1. The CAPS marker was linked to but did not completely cosegregate with Pl1 on linkage group 8. Ha-4W2 was found to comprise a gene family with at least five members. Although genetic markers for Ha-4W2 have utility for marker-assisted selection, the RGC detected by the CAPS marker has been ruled out as a candidate gene for Pl1. Three of the RGC probes were monomorphic between HA370 and HA372 and still need to be mapped and screened for linkage to disease resistance loci.

An integrated restriction fragment length polymorphism--amplified fragment length polymorphism linkage map for cultivated sunflower.

Restriction fragment length polymorphism (RFLP) maps have been constructed for cultivated sunflower (Helianthus annuus L.) using three independent sets of RFLP probes. The aim of this research was to integrate RFLP markers from two sets with RFLP markers for resistance gene candidate (RGC) and amplified fragment length polymorphism (AFLP) markers. Genomic DNA samples of HA370 and HA372, the parents of the F2 population used to build the map, were screened for AFLPs using 42 primer combinations and RFLPs using 136 cDNA probes (RFLP analyses were performed on DNA digested with EcoRI, HindIII, EcoRV, or DraI). The AFLP primers produced 446 polymorphic and 1101 monomorphic bands between HA370 and HA372. The integrated map was built by genotyping 296 AFLP and 104 RFLP markers on 180 HA370 x HA372 F2 progeny (the AFLP marker assays were performed using 18 primer combinations). The HA370 x HA372 map comprised 17 linkage groups, presumably corresponding to the 17 haploid chromosomes of sunflower, had a mean density of 3.3 cM, and was 1326 cM long. Six RGC RFLP loci were polymorphic and mapped to three linkage groups (LG8, LG13, and LG15). AFLP markers were densely clustered on several linkage groups, and presumably reside in centromeric regions where recombination is reduced and the ratio of genetic to physical distance is low. Strategies for targeting markers to euchromatic DNA need to be tested in sunflower. The HA370 x HA372 map integrated 14 of 17 linkage groups from two independent RFLP maps. Three linkage groups were devoid of RFLP markers from one of the two maps.

Considerations for ethical practice in managed care.

How does one maintain an ethical practice while facing the requirements and limits of a health care system that is dominated by managed care? Psychologists are increasingly raising such questions about ethical issues when working in or contracting with managed care organizations. The authors review the process involved in ethical decision making and problem solving and focus on 4 areas in which ethical dilemmas most commonly arise in a managed care context: informed consent, confidentiality, abandonment, and utilization management-utilization review. The need for sustained and organized advocacy efforts to ensure patient access to quality health care is discussed, as is the impact of managed care's competitive marketplace on professional relationships. Hypothetical examples of typical dilemmas psychologists face in the current practice environment are provided to illustrate systematic ethical decision making.

Condensing enzymes from Cuphea wrightii associated with medium chain fatty acid biosynthesis.

Seed oils of most Cuphea species contain > 90% medium chain (C8-C14) fatty acids. Thioesterases with specificity for these substrates are important determinants of the medium chain phenotype. The role of condensing enzymes, however, has not been investigated. cDNA clones encoding beta-ketoacyl-acyl carrier protein (ACP) synthase (KAS) were isolated from C. wrightii, a C10/C12-producing species. Deduced amino acid sequences of four unique clones were approximately 60% identical to plant KAS I sequences and approximately 75% identical to a distinct class of KAS sequences recently identified in castor and barley. A 46 kDa protein that was observed only in developing and mature seed was detected using antiserum directed against recombinant Cuphea KAS protein. The 46 kDa protein was abundant in developing seeds of six medium chain-producing Cuphea species but barely detected in one long chain-producing species. A 48 kDa protein identified immunologically as KAS I was expressed in both medium and long chain-producing Cuphea species and was detected in all tissues tested. In in vitro assays, extracts from C. wrightii and C. viscosissima developing embryos were unable to extend fatty acid chains beyond C10 following treatment with 10 microns cerulenin, a potent inhibitor of KAS I. However, a C. viscosissima mutant, cpr-1, whose seed oils are deficient in caprate relative to wild type, was impaired in extension of C8 to C10 in this assay and Western analysis revealed a specific deficiency in 46 kDa KAS in cpr-1 embryos. These results implicate cerulenin-resistant condensing activity in production of medium chain fatty acids in Cuphea.

A Cuphea beta-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases.

Acyl-acyl carrier protein (ACP) thioesterases with specificities on medium chain substrates (C8-C14) are requisite enzymes in plants that produce 8:0, 10:0, 12:0 and 14:0 seed oils, but they may not be the sole enzymatic determinants of chain length. The contribution to chain length regulation of a beta-ketoacyl-ACP synthase, Cw KAS A1, derived from Cuphea wrightii, a species that accumulates 30% 10:0 and 54% 12:0 in seed oils, was investigated. Expression of Cw KAS A1 in Arabidopsis seeds reduced 16:0 from 8.2 to 6.2 mol%, suggesting a KAS II-type activity. In the presence of the KAS I inhibitor cerulenin, however, transgenic seed extracts extended 6:0- and 8:0-ACP at a rate four- to fivefold greater than extracts from untransformed plants, whereas no difference was observed in extension of 14:0- and 16:0-ACP. The effect of KAS A1 on seed oils was tested by combining it with the C. wrightii medium chain-specific thioesterases, Cw FatB1 and Cw FatB2, in crosses of transformed plants. Fatty acid synthesis thesis shifted towards shorter chains in progeny expressing both classes of enzymes. KasA1/FatB1 homozygotes produced threefold more 12:0 than the FatB1 parent while 14:0 and 16:0 were reduced by one-third and one-half, respectively. F2 progeny expressing KasA1 and FatB2 produced twofold more 10:0 and 1.4-fold more 12:0 than the FatB2 parent, and the double-transgenic progeny produced one-quarter less 14:0 and one-half less 16:0 than the FatB2 parent. It is hypothesized that the shift towards production of shorter chains resulted from increased pools of medium chain acyl-ACP resulting from KAS A1 activity. The combined activities of KAS A1 and FatB thioesterases appear to determine the C. wrightii phenotype.