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Key Message: We identified QTL associated with protein and amino acids in a soybean mapping population that was grown in five environments. These QTL could be used in MAS to improve these traits. Soybean, rather than nitrogen-containing forages, is the primary source of quality protein in feed formulations for domestic swine, poultry, and dairy industries. As a sole dietary source of protein, soybean is deficient in the amino acids lysine (Lys), threonine (Thr), methionine (Met), and cysteine (Cys). Increasing these amino acids would benefit the feed industry. The objective of the present study was to identify quantitative trait loci (QTL) associated with crude protein (cp) and amino acids in the 'Benning' × 'Danbaekkong' population. The population was grown in five southern USA environments. Amino acid concentrations as a fraction of cp (Lys/cp, Thr/cp, Met/cp, Cys/cp, and Met + Cys/cp) were determined by near-infrared reflectance spectroscopy. Four QTL associated with the variation in crude protein were detected on chromosomes (Chr) 14, 15, 17, and 20, of which, a QTL on Chr 20 explained 55 % of the phenotypic variation. In the same chromosomal region, QTL for Lys/cp, Thr/cp, Met/cp, Cys/cp and Met + Cys/cp were detected. At these QTL, the Danbaekkong allele resulted in reduced levels of these amino acids and increased protein concentration. Two additional QTL for Lys/cp were detected on Chr 08 and 20, and three QTL for Thr/cp on Chr 01, 09, and 17. Three QTL were identified on Chr 06, 09 and 10 for Met/cp, and one QTL was found for Cys/cp on Chr 10. The study provides information concerning the relationship between crude protein and levels of essential amino acids and may allow for the improvement of these traits in soybean using marker-assisted selection.

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.

The crystal proteins coded by transgenes from Bacillus thuringiensis (Bt) have shown considerable value in providing effective insect resistance in a number of crop species, including soybean, Glycine max (L.) Merr. Additional sources of soybean insect resistance would be desirable to manage the development of tolerance/resistance to crystal proteins by defoliating insects and to sustain the deployment of Bt crops. The objective of this study was to evaluate the effects and interactions of three insect resistance quantitative trait loci (QTLs; QTL-M, QTL-H, and QTL-G) originating from Japanese soybean PI 229358 and a cry1Ac gene in a "Benning" genetic background. A set of 16 BC(6)F(2)-derived near isogenic lines (NILs) was developed using marker-assisted backcrosses and evaluated for resistance to soybean looper [SBL, Pseudoplusia includens (Walker)] and corn earworm [CEW, Helicoverpa zea (Boddie)] in field cage, greenhouse, and detached leaf assays. Both Bt and QTL-M had significantly reduced defoliation by both SBL and CEW and reduced larval weight of CEW. The antibiosis QTL-G had a significant effect on reducing CEW larval weight and also a significant effect on reducing defoliation by SBL and CEW in some assays. The antixenosis QTL-H had no main effect, but it appeared to function through interaction with QTL-M and QTL-G. Adding QTL-H and QTL-G further enhanced the resistance of the Bt and QTL-M combination to CEW in the field cage assay. These results should help guide the development of strategies for effective management of insect pests and for sustainable deployment of Bt genes.

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 F(2)-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.

Deployment of salt tolerant cultivars is an effective approach to minimize yield loss in a saline soil. In soybean, Glycine max (L.) Merr., substantial genetic variation exists for salt response. However, breeding for salt tolerance is hampered because no economically viable screening method has been developed for practical breeding. To facilitate the development of an effective screening method for salt tolerance in soybean, the present study was conducted to determine the heritability of salt tolerance and to identify associated quantitative trait loci (QTL). F2:5 lines from the cross of 'S-100' (salt tolerant) x 'Tokyo' (salt sensitive) were evaluated in a saline field in Hyde County, N.C., USA, in 1999 and in a greenhouse located in Raleigh, N.C., USA, in 2001. S-100 and Tokyo are ancestors of popular soybean cultivars released for the southern USA. The visual salt tolerance ratings of the F2:5 lines ranged from 0 (complete death) to 5 (normal healthy appearance). The entry-mean heritability for salt tolerance was 0.85, 0.48, and 0.57 in the field (four replications), greenhouse (two replications), and combined environments, respectively. The genotypic correlation between field and greenhouse ratings was 0.55, indicating reasonably good agreement between the two screening environments. To identify QTL associated with salt tolerance, each line was characterized with RFLP markers and an initial QTL single-factor analysis was completed. These results were used to identify genomic regions associated with the trait and to saturate the selected genomic regions with SSR markers to improve mapping precision. Subsequently, a major QTL for salt tolerance was discovered near the Sat_091 SSR marker on linkage group (LG) N, accounting for 41, 60, and 79% of the total genetic variation for salt tolerance in the field, greenhouse, and combined environments, respectively. The QTL allele associated with tolerance was derived from S-100. Pedigree tracking was used to examine the association between the salt tolerance QTL and flanking SSR marker alleles in U.S. cultivars descended from S-100 or Tokyo through 60 years of breeding. The presence of alleles from S-100 at the Sat_091 and Satt237 marker loci was always associated with salt tolerance in descendants. Alleles from Tokyo for these same markers were generally associated with salt sensitivity in descendent cultivars. The strong relationship between the SSR marker alleles and salt tolerance suggests that these markers could be used for marker-assisted selection in commercial breeding.

The Rxp locus in soybean [Glycine max (L.) Merr.] that conditions reaction to bacterial pustule was mapped by simple sequence repeat (SSR) marker analysis. A population of 116 F4-derived lines from a cross between the resistant parent Young and the susceptible parent PI 416937 was used for mapping. The Rxp locus was mapped 3.9 cM from Satt372 and 12.4 cM from Satt014 on linkage group D2. Linkage associations were confirmed by identifying a close association between the SSR genotype at each locus identified as flanking Rxp and the bacterial pustule reaction of individual lines derived from a population different from the one used for mapping. A molecular pedigree analysis showed that bacterial pustule-resistant cultivars inherited the resistance gene rxp from the ancestral cultivar CNS based on their consistent genotypic pattern at flanking marker loci. Based on the results of the study, marker-assisted selection for rxp would be very effective.

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.

Somatic embryos of jack, a Glycine max (L.) Merrill cultivar, were transformed using microprojectile bombardment with a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt cryIAc) driven by the 35S promoter and linked to the HPH gene. Approximately 10 g of tissue was bombarded, and three transgenic lines were selected on hygromycin-containing media and converted into plants. The recovered lines contained the HPH gene, but the Bt gene was lost in one line. The plasmid was rearranged in the second line, and the third line had two copies, one of which was rear-ranged. The CryIAc protein accumulated up to 46 ng mg-1 extractable protein. In detached-leaf bioassays, plants with an intact copy of the Bt gene, and to a lesser extent those with the rearranged copy, were protected from damage from corn earworm (Helicoverpa zea), soybean looper (Pseudoplusia includens), tobacco budworm (Heliothis virescens), and velvetbean caterpillar (Anticarsia gemmatalis). Corn earworm produced less than 3% defoliation on transgenic plants, compared with 20% on the lepidopteran-resistant breeding line GatIR81-296, and more than 40% on susceptible cultivars. Unlike previous reports of soybean transformation using this technique, all plants were fertile. To our knowledge, this is the first report of a soybean transgenic for a highly expressed insecticidal gene.

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.