Genetic mapping of QTLs conditioning soybean sprout yield and quality

Soybean sprouts have been used as a food in the Orient since ancient times. In this study, 92 restriction fragment length polymorphism (RFLP) loci and two morphological markers (W1 and T) were used to identify quantitative trait loci (QTLs) associated with soybean sprout-related traits in 100 F₂-derived lines from the cross of ’Pureunkong’×’Jinpumkong 2’. The genetic map consisted of 76 loci which covered about 756 cM and converged into 20 linkage groups. Eighteen markers remained unlinked. Phenotypic data were collected in 1996 and 1997 for hypocotyl length, percentage of abnormal seedlings, and sprout yield 6 days after germination at 20°C. Hypocotyl length was determined as the average length from the point of initiation of the first secondary root to the point of attachment of the cotyledons. The number of decayed seeds and seedlings, plus the number of stunted seedlings (less than 2-cm growth), was recorded a s abnormal seedlings. Seed weight was determined based on the 50-seed sample. Sprout yield was recorded as the total fresh weight of soybean sprouts produced from the 50-seed sample divided by the dry weight of the 50-seed sample. Four QTLs were associated with sprout yield in the combined analysis across 2 years. For the QTL linked to L154 on the Linkage Group (LG) G the positive allele was derived from Pureunkong (R ² = 0.19), whereas at the other three QTLs (A089 on LG B1, A668n on LG K and B046 on LG L) the positive alleles were from Jinpumkong 2. QTLs conditioning seed weight were linked to markers A802n (LG B1), A069 (LG E), Cr321 (LG F) and A235 (LG G). At these four markers, the Jinpumkong allele increased seed weight. Markers K011n on LG B1, W1 on LG F and A757 on LG L were linked to QTLs conditioning hypocotyl length; and Bng119, K455n and K418n to QTLs conditioning the abnormal seedlings. The QTLs conditioning sprout yield were in the same genomic locations as the QTLs for seed weight identified in this population or from previously published research, indicating that QTLs for sprout yield are genetically linked to seed-weight QTLs or else that seed-weight QTLs pleiotropically condition sprout yield. These data demonstrate that effective marker-assisted selection may be feasible for enhancing sprout yield in a soybean. The transgressive segregation of sprout yield, as well as the existence of two QTLs conditioning greater than 10% of the phenotypic variation in sprout yields provides an opportunity to select for progeny lines with a greater sprout yield than currently preferred cultivars such as Pureunkong. Received: 23 August 2000 / Accepted: 23 January 2001     

Combining cry1Ac with QTL alleles from PI 229358 to improve soybean resistance to lepidopteran pests

A QTL conditioning corn earworm resistance in soybean PI 229358 and asynthetic Bacillus thuringiensis cry1Ac transgene from therecurrent parent Jack-Bt were pyramided intoBC₂F₃ plants by marker-assisted selection. Segregatingindividuals were genotyped at SSR markers linked to an anitbiosis/antixenosisQTL on linkage group M, and were tested for the presence ofcry1Ac. Marker-assisted selection was used during andafter the two backcrosses to develop a series of BC₂F₃plants with or without the crylAc transgene and the QTLconditioning for resistance BC₂F₃ plants that werehomozygous for parental alleles at markers on LG M, and whicheither had or lacked cry1Ac, were assigned to one of fourpossible genotype classes. These plants were used in no-choice, detached leaffeeding bioassays with corn earworm and soybean looper larvae (Lepidoptera:Noctuidae) to evaluate the relative antibiosis in the different genotypeclasses. Resistance was measured as larval weight gain and degree of foliageconsumption. Few larvae of either species survived on leaves expressing theCry1Ac protein. Though not as great as the effect of Cry1Ac, the PI229358-derived LG M QTL also had a detrimental effect on larval weights of bothpest species, and on defoliation by corn earworm, but did not reduce defoliation bysoybean looper. Weights of soybean looper larvae fed foliage from transgenicplants with the PI-derived QTL were lower than those of larvae fed transgenictissue with the corresponding Jack chromosomal segment. This work demonstratesthe usefulness of SSRs for marker-assisted selection in soybean, and shows thatcombining transgene-and QTL-mediated resistance to lepidopteran pests may be aviable strategy for insect control.     

Field evaluation of soybean engineered with a synthetic cry1Ac transgene for resistance to corn earworm, soybean looper, velvetbean caterpillar (Lepidoptera: Noctuidae), and lesser cornstalk borer (Lepidoptera: Pyralidae)

A transgenic line of the soybean 'Jack', Glycine max (L.) Merrill, expressing a synthetic cry1Ac gene from Bacillus thuringiensis variety kurstaki (Jack-Bt), was evaluated for resistance to four lepidopteran pests in the field. Jack-Bt and genotypes serving as susceptible and resistant controls were planted in field cages and artificially infested with larvae of corn earworm, Helicoverpa zea (Boddie), and velvetbean caterpillar, Anticarsia gemmatalis (Hübner), in 1996, 1997, and 1998, and also with soybean looper, Pseudoplusia includens (Walker), in 1996. Susceptible controls included Jack (1996-1998), 'Cobb' (1996), and Jack-HPH (1996). GatIR 81-296 was used as the resistant control in all 3 yr. Compared with untransformed Jack, Jack-Bt showed three to five times less defoliation from corn earworm and eight to nine times less damage from velvetbean caterpillar. Defoliation of GatIR 81-296 was intermediate between that of Jack and Jack-Bt for corn earworm, and similar to that of Jack for velveltbean caterpillar. Jack-Bt exhibited significant, but lower resistance to soybean looper. Jack-Bt also showed four times greater resistance than Jack to natural infestations of lesser cornstalk borer, Elasmopalpus lignosellus (Zeller), in conventional field plots at two locations in 1998. Data from these experiments suggest that expression of this cry1Ac construct in soybean should provide adequate levels of resistance to several lepidopteran pests under field conditions.     

Thrombomodulin Contributes to Gamma Tocotrienol-Mediated Lethality Protection and Hematopoietic Cell Recovery in Irradiated Mice

Systemic administration of recombinant thrombomodulin (TM) confers radiation protection partly by accelerating hematopoietic recovery. The uniquely potent radioprotector gamma tocotrienol (GT3), in addition to being a strong antioxidant, inhibits the enzyme hydroxy-methyl-glutaryl-coenzyme A reductase (HMGCR) and thereby likely modulates the expression of TM. We hypothesized that the mechanism underlying the exceptional radioprotective properties of GT3 partly depends on the presence of endothelial TM. In vitro studies confirmed that ionizing radiation suppresses endothelial TM (about 40% at 4 hr after 5 Gy γ-irradiation) and that GT3 induces TM expression (about 2 fold at the mRNA level after 5 μM GT3 treatment for 4 hr). In vivo survival studies showed that GT3 was significantly more effective as a radioprotector in TM wild type (TM^+/+) mice than in mice with low TM function (TM^Pro/-). After exposure to 9 Gy TBI, GT3 pre-treatment conferred 85% survival in TM^+/+ mice compared to only 50% in TM^Pro/-. Thus, GT3-mediated radiation lethality protection is partly dependent on endothelial TM. Significant post-TBI recovery of hematopoietic cells, particularly leukocytes, was observed in TM^+/+ mice (p = 0.003), but not in TM^Pro/- mice, despite the fact that GT3 induced higher levels of granulocyte colony stimulating factor (G-CSF) in TM^Pro/- mice (p = 0.0001). These data demonstrate a critical, G-CSF-independent, role for endothelial TM in GT3-mediated lethality protection and hematopoietic recovery after exposure to TBI and may point to new strategies to enhance the efficacy of current medical countermeasures in radiological/nuclear emergencies.     

Fine Mapping and Characterization of Candidate Genes that Control Resistance to Cercospora sojina K. Hara in Two Soybean Germplasm Accessions

Frogeye leaf spot (FLS), caused by the fungus Cercospora sojina K. Hara, may cause a significant yield loss to soybean growers in regions with a warm and humid climate. Two soybean accessions, PI 594891 and PI 594774, were identified to carry a high level of resistance similar to that conditioned by the Rcs3 gene in ''Davis''. Previously, we reported that the resistance to FLS in these two plant introductions (PIs) was controlled by a novel gene (s) on chromosome 13 that is different from Rcs3. To fine-map the novel FLS resistance gene(s) in these two PIs, F_{2: 3} seeds from the crosses between PI 594891 and PI 594774, and the FLS susceptible genotype ''Blackhawk'' were genotyped with SNP markers that were designed based on the SoySNP50k iSelect BeadChip data to identify recombinant events and locate candidate genes. Analysis of lines possessing key recombination events helped narrow down the FLS-resistance genomic region in PI 594891 from 3.3 Mb to a 72.6 kb region with five annotated genes. The resistance gene in PI 594774 was fine-mapped into a 540 kb region that encompasses the 72.6 kb region found in PI 594891. Sequencing five candidate genes in PI 594891 identified three genes that have several mutations in the promoter, intron, 5'', and 3'' UTR regions. qPCR analysis showed a difference in expression levels of these genes in both lines compared to Blackhawk in the presence of C. sojina. Based on phenotype, genotype and haplotype analysis results, these two soybean accessions might carry different resistance alleles of the same gene or two different gene(s). The identified SNPs were used to develop Kompetitive Allele Specific PCR (KASP) assays to detect the resistance alleles on chromosome 13 from the two PIs for marker-assisted selection.     

Chloroplast ribosomal RNA genes in Euglena gracilis exist as three clustered tandem repeats

Chloroplast ribosomal DNA from Euglena gracilis was partially purified, digested with restriction endonucleases BamHI or EcoRI and cloned into bacterial plasmids. Plasmids containing the ribosomal DNA were identified by their ability to hybridize to chloroplast ribosomal RNA and were physically mapped using restriction endonucleases BamHI, EcoRI, HindIII and HpaI. The nucleotide sequences coding for the 16S and the 23S chloroplast ribosomal RNAs were located on these plasmids by hybridizing the individual RNAs to denatured restriction endonuclease DNA fragments immobilized on nitrocellulose filters. Restriction endonuclease fragments from chloroplast DNA were analyzed in a similar fashion. These data permitted the localization on a BamHI map of the chloroplast DNA three tandemly arranged chloroplast ribosomal RNA genes. Each ribosomal RNA gene consisted of a 4.6 kilobase pair region coding for the 16S and 23S ribosomal RNAs and a 0.8 kilobase pair spacer region. The chloroplast ribosomal DNA represented 12% of the chloroplast DNA and is G + C rich.     

Comparison of four flow cytometric SNP detection assays and their use in plant improvement

Single nucleotide polymorphisms (SNPs) are attractive DNA markers due to their abundance and potential for use in automated high-throughput genotyping. Numerous SNP genotyping assays have been developed, but it is unclear which assays are best suited and most efficient for various types of plant improvement research. The objective of this study was to compare the accuracy, efficiency, and cost of four SNP genotyping assays: single-base extension (SBE), allele-specific primer extension (ASPE), oligonucleotide ligation (OL), and direct hybridization (DH). All four assay methods used the same Luminex 100 flow cytometer platform. Fifty-eight F₂-derived soybean [Glycine max (L.) Merr.] lines from a cross between inbred lines G99-G725 and N00-3350 were genotyped at four SNPs. SBE and ASPE clearly differentiated between the two homozygotes and the heterozygote at each SNP. Results were in agreement with those identified using the SNaPshot minisequencing assay as a control. In contrast, the OL and DH assays were unable to differentiate between genotypes at some of the SNPs. However, when the cost per data point for the four different assays was compared, the cost of OL and DH was only about 70% of that for SBE, with DH requiring the least time of the four assays. On the basis of cost and labor, ASPE is more cost-effective and simpler than SBE, and would therefore be a good method for genetic mapping and diversity studies which require a large number of markers and a high level of multiplexing. DH appears to be the most economical assay for marker-assisted selection, though optimization for DH would be required for some SNP markers.     

Genome Duplication in Soybean (Glycine Subgenus Soja)

Restriction fragment length polymorphism mapping data from nine populations (Glycine max X G. soja and G. max X G. max) of the Glycine subgenus soja genome led to the identification of many duplicated segments of the genome. Linkage groups contained up to 33 markers that were duplicated on other linkage groups. The size of homoeologous regions ranged from 1.5 to 106.4 cM, with an average size of 45.3 cM. We observed segments in the soybean genome that were present in as many as six copies with an average of 2.55 duplications per segment. The presence of nested duplications suggests that at least one of the original genomes may have undergone an additional round of tetraploidization. Tetraploidization, along with large internal duplications, accounts for the highly duplicated nature of the genome of the subgenus. Quantitative trait loci for seed protein and oil showed correspondence across homoeologous regions, suggesting that the genes or gene families contributing to seed composition have retained similar functions throughout the evolution of the chromosomes.     

Over de verdeeling van de agglutininen over de serumeiwitten

Ohne Zusammenfassung Voordracht voor de Vergadering van de Ned. Ver. v. Microbiologie, gehouden te Utrecht op 12 November 1938. Uitvoerige publicatie met literatuur volgt in den vorm van een dissertatie.