Mapping of quantitative trait loci for canopy-wilting trait in soybean (Glycine max L. Merr)

Drought stress adversely affects [Glycine max (L.) Merr] soybean at most developmental stages, which collectively results in yield reduction. Little information is available on relative contribution and chromosomal locations of quantitative trait loci (QTL) conditioning drought tolerance in soybean. A Japanese germplasm accession, PI 416937, was found to possess drought resistance. Under moisture-deficit conditions, PI 416937 wilted more slowly in the field than elite cultivars and has been used as a parent in breeding programs to improve soybean productivity. A recombinant inbred line (RIL) population was derived from a cross between PI 416937 and Benning, and the population was phenotyped for canopy wilting under rain-fed field conditions in five distinct environments to identify the QTL associated with the canopy-wilting trait. In a combined analysis over environments, seven QTL that explained 75?% of the variation in canopy-wilting trait were identified on different chromosomes, implying the complexity of this trait. Five QTL inherited their positive alleles from PI 416937. Surprisingly, the other two QTL inherited their positive alleles from Benning. These putative QTL were co-localized with other QTL previously identified as related to plant abiotic stresses in soybean, suggesting that canopy-wilting QTL may be associated with additional morpho-physiological traits in soybean. A locus on chromosome 12 (Gm12) from PI 416937 was detected in the combined analysis as well as in each individual environment, and explained 27?% of the variation in canopy-wilting. QTL identified in PI 416937 could provide an efficient means to augment field-oriented development of drought-tolerant soybean cultivars.     

Seed yield of near-isogenic soybean lines with introgressed quantitative trait loci conditioning resistance to corn earworm (Lepidoptera: Noctuidae) and soybean looper (Lepidoptera: Noctuidae) from PI 229358

The development of superior soybean, Glycine max (L.) Merr., cultivars exhibiting resistance to insects has been hindered due to linkage drag, a common phenomenon when introgressing alleles from exotic germplasm. Simple-sequence repeat (SSR) markers were used previously to map soybean insect resistance (SIR) quantitative trait loci (QTLs) in a'Cobb' X PI 229358 population, and subsequently used to create near-isogenic lines (NILs) with SIR QTL i n a 'Benning' genetic background. SIR QTLs were mapped on linkage groups (LGs) M (SIRQTL-M), G (SIRQTL-G), and H (SIRQTL-H). The objectives of this study were to 1) evaluate linkage drag for seed yield by using Benning-derived NILs selected for SIRQTL-M, SIRQTL-H, and SIRQTL-G; 2) assess the amount of PI 229358 genome surrounding the SIR QTL in each Benning NIL; and 3) evaluate the individual effects these three QTLs on antibiosis and antixenosis to corn earworm, Helicoverpa zea (Boddie), and soybean looper, Pseudoplusia includens (Walker). Yield data collected in five environments indicated that a significant yield reduction is associated with SIRQTL-G compared with NILs without SIR QTL. Overall, there was no yield reduction associated with SIRQTL-M or SIRQTL-H. A significant antixenosis and antibiosis effect was detected for SIRQTL-M in insect feeding assays, with no effect detected in antixenosis or antibiosis assays for SIRQTL-G or SIRQTL-H without the presence of PI 229358 alleles at SIRQTL-M. These results support recent findings concerning these loci.     

Glutathione-S-transferase pi as a model protein for the characterisation of chemically reactive metabolites

Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N-acetyl-p-benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents.     

Genotype effects on proliferative embryogenesis and plant regeneration of soybean

Summary Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of proliferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in liquid cultures, for yield of cotyledon-stage embryos on differentiation medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture phenotype consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifications.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Cellular Responses of Resistant and Susceptible Soybean Genotypes Infected with Meloidogyne arenaria Races 1 and 2

The cellular responses induced by Meloidogyne arenaria races 1 and 2 in three soybean genotypes, susceptible CNS, resistant Jackson, and resistant PI 200538, were examined by light microscopy 20 days after inoculation. Differences in giant-cell development were greater between races than among the soybean genotypes. M. arenaria race 1 stimulated small, poorly formed giant-cells in contrast with M. arenaria race 2, which induced well-developed, thick-walled, multinucleate giant-cells. The number of nuclei per giant-celt was variable, but fewer nuclei were usually present in giant-cells induced by race 1 (mean 16 nuclei) than in giant-cells induced by race 2 (mean 41 nuclei). Differences observed in giant-cell development were related to differences in growth and maturation of M. arenaria races 1 and 2 and host suitability of the soybean genotypes.     

RFLP loci associated with soybean seed protein and oil content across populations and locations

Molecular markers provide the opportunity to identify marker-quantitative trait locus (QTL) associations in different environments and populations. Two soybean [Glycine max (L.) Merr.] populations, Young x PI 416 937 and PI 97100 x Coker 237, were evaluated with restriction fragment length polymorphism (RFLP) markers to identify additional QTLs related to seed protein and oil. For the Young x PI 416937 population, 120 F₄-derived lines were secored for segregation at 155 RFLP loci. The F₄-derived lines and two parents were grown at Plains, G.a., and Windblow and Plymouth, N.C. in 1994, and evaluated for seed protein and oil. For the PI 97100 x Coker 237 population, 111 F₂-derived lines were evaluated for segregation at 153 RFLP loci. Phenotypic data for seed protein and oil were obtained in two different locations (Athens, G.a., and Blackville, S.C.) in 1994. Based on single-factor analysis of variance (ANOVA) for the Young x PI 416937 population, five of seven independent markers associated with seed protein, and all four independent markers associated with seed oil in the combined analysis over locations were detected at all three locations. For the PI 97 100 x Coker 237 population, both single-factor ANOVA and interval mapping were used to detect QTLs. Using single-factor ANOVA, three of four independent markers for seed protein and two of three independent markers for seed oil were detected at both locations. In both populations, singlefactor ANOVA, revealed the consistency of QTLs across locations, which might be due to the high heritability and the relatively few QTLs with large effects conditioning these traits. However, interval mapping of the PI 97100 x Coker 237 population indicated that QTLs identified at Athens for seed protein and oil were different from those at Blackville. This might result from the power of QTL mapping being dependent on the level of saturation of the genetic map. Increased seed protein was associated with decreased seed oil in the PI 97100 x Coker 237 population (r = –0.61). There were various common markers (P0.05) on linkage groups (LG) E, G,H,K, and UNK2 identified for both seed protein and oil. One QTL on LG E was associated with seed protein in both populations. The other QTLs for protein and oil were population specific.     

Early events in the cellular formation of proparathyroid hormone

Early events in the cellular synthesis and subsequent transfer into membrane-limited compartments of pre-proparathyroid hormone (pre-proPTH) and proparathyroid hormone (proPTH) were investigated by electrophoretic analyses of newly synthesized proteins in subcellular fractions of parthyroid gland slices pulse-labeled for 0.5-5 min with [(35)S] methionine. During these short times of incubation, both pre-proPTH and proPTH were confined to the microsomal fraction. Labeled pre-proPTH and proPTH were detected in a 30-s interval between 0.5 and 1.0 min of incubation. The radioactivity in proPTH became relatively constant between 3 and 5 min, whereas the radioactivity in ProPTH increased markedly over this period. When corrected for the known content of methionine in the prohormone and the prohormone, we found four times as much radiolabeled prohormone as prehormone between 0.5 and 1.0 min of synthesis. Sequestration of labeled prohomrone into endoplasmic reticulum compartments was shown by treatment of the microsomal fraction with chymotrypsin and trypsin, which resulted in the degradation of the prehormone but not of the prohormones. Approximately 50 percent of pre-prohormone and 25 percent of prohormone were released from the microsomes by their extraction with 1.0 M KCl, whereas 80-90 percent of both was released by treatment with Triton X-100. These results in intact cells support the signal hypothesis proposed by Blobel and his co-workers in studies utilizing cell-free systems, inasmuch as the results indicate transfer of prohormone into the cisternal space of the rough endoplasmic reticulum concomitant with the growth of the nascent polypeptide chain. Appearance of membrane-sequestered proPTH takes place without entry of pre-proPTH into the cisternal space, suggesting that proteolytic removal of the leader peptide occurs during transfer of the polypeptide through the lipid bilayer. Further evidence in support of this process is that pre-proPTH is only partly extracted from the microsomes by treatment with 1.0 M KCl, suggesting that a substantial fraction of the nascent pre-proPTH is integrally inserted into the membranes before it is cleaved to form proPTH.     

Discovery and utilization of QTLs for insect resistance in soybean

Insect resistance in soybean has been an objective in numerous breeding programs, but efforts to develop high yielding cultivars with insect resistance have been unsuccessful. Three Japanese plant introductions, PIs 171451, 227687 and 229358, have been the primary sources of insect resistance alleles, but a combination of quantitative inheritance of resistance and poor agronomic performance has hindered progress. Linkage drag caused by co-introgression of undesirable agronomic trait alleles linked to the resistance quantitative trait loci (QTLs) is a persistent problem. Molecular marker studies have helped to elucidate the numbers, effects and interactions of insect resistance QTLs in the Japanese PIs, and markers are now being used in breeding programs to facilitate transfer of resistance alleles while minimizing linkage drag. Molecular markers also make it possible to evaluate QTLs independently and together in different genetic backgrounds, and in combination with transgenes from Bacillus thuringiensis.