A High Density Integrated Genetic Linkage Map of Soybean and the Development of a 1536 Universal Soy Linkage Panel for Quantitative Trait Locus Mapping

Single nucleotide polymorphisms (SNPs) are the marker of choice for many researchers due to their abundance and the high-throughput methods available for their multiplex analysis. Only recently have SNP markers been available to researchers in soybean [Glycine max (L.) Merr.] with the release of the third version of the consensus genetic linkage map that added 1141 SNP markers to the map. Our objectives were to add 2500 additional SNP markers to the soybean integrated map and select a set of 1536 SNPs to create a universal linkage panel for high-throughput soybean quantitative trait locus (QTL) mapping. The GoldenGate assay is one high-throughput analysis method capable of genotyping 1536 SNPs in 192 DNA samples over a 3-d period. We designed GoldenGate assays for 3456 SNPs (2956 new plus 500 previously mapped) which were used to screen three recombinant inbred line populations and diverse germplasm. A total of 3000 workable assays were obtained which added about 2500 new SNP markers to create a fourth version of the soybean integrated linkage map. To create a “Universal Soy Linkage Panel” (USLP 1.0) of 1536 SNP loci, SNPs were selected based on even distribution throughout each of the 20 consensus linkage groups and to have a broad range of allele frequencies in diverse germplasm. The 1536 USLP 1.0 will be able to quickly create a comprehensive genetic map in most QTL mapping populations and thus will serve as a useful tool for high-throughput QTL mapping

Mutational analysis of the major soybean UreF paralogue involved in urease activation

The soybean genome duplicated ∼14 and 45 million years ago and has many paralogous genes, including those in urease activation (emplacement of Ni and CO2 in the active site). Activation requires the UreD and UreF proteins, each encoded by two paralogues. UreG, a third essential activation protein, is encoded by the single-copy Eu3, and eu3 mutants lack activity of both urease isozymes. eu2 has the same urease-negative phenotype, consistent with Eu2 being a single-copy gene, possibly encoding a Ni carrier. Unexpectedly, two eu2 alleles co-segregated with missense mutations in the chromosome 2 UreF paralogue (Ch02UreF), suggesting lack of expression/function of Ch14UreF. However, Ch02UreF and Ch14UreF transcripts accumulate at the same level. Further, it had been shown that expression of the Ch14UreF ORF complemented a fungal ureF mutant. A third, nonsense (Q2*) allelic mutant, eu2-c, exhibited 5- to 10-fold more residual urease activity than missense eu2-a or eu2-b, though eu2-c should lack all Ch02UreF protein. It is hypothesized that low-level activation by Ch14UreF is ‘spoiled’ by the altered missense Ch02UreF proteins (‘epistatic dominant-negative’). In agreement with active ‘spoiling’ by eu2-b-encoded Ch02UreF (G31D), eu2-b/eu2-c heterozygotes had less than half the urease activity of eu2-c/eu2-c siblings. Ch02UreF (G31D) could spoil activation by Chr14UreF because of higher affinity for the activation complex, or because Ch02UreF (G31D) is more abundant than Ch14UreF. Here, the latter is favoured, consistent with a reported in-frame AUG in the 5' leader of Chr14UreF transcript. Translational inhibition could represent a form of ‘functional divergence’ of duplicated genes

An EST-SSR Linkage Map of Raphanus sativus and Comparative Genomics of the Brassicaceae†

Raphanus sativus (2n = 2x = 18) is a widely cultivated member of the family Brassicaceae, for which genomic resources are available only to a limited extent in comparison to many other members of the family. To promote more genetic and genomic studies and to enhance breeding programmes of R. sativus, we have prepared genetic resources such as complementary DNA libraries, expressed sequences tags (ESTs), simple sequence repeat (SSR) markers and a genetic linkage map. A total of 26 606 ESTs have been collected from seedlings, roots, leaves, and flowers, and clustered into 10 381 unigenes. Similarities were observed between the expression patterns of transcripts from R. sativus and those from representative members of the genera Arabidopsis and Brassica, indicating their functional relatedness. The EST sequence data were used to design 3800 SSR markers and consequently 630 polymorphic SSR loci and 213 reported marker loci have been mapped onto nine linkage groups, covering 1129.2 cM with an average distance of 1.3 cM between loci. Comparison of the mapped EST-SSR marker positions in R. sativus with the genome sequence of A. thaliana indicated that the Brassicaceae members have evolved from a common ancestor. It appears that genomic fragments corresponding to those of A. thaliana have been doubled and tripled in R. sativus. The genetic map developed here is expected to provide a standard map for the genetics, genomics, and molecular breeding of R. sativus as well as of related species. The resources are available at http://marker.kazusa.or.jp/Daikon

QTL for seed protein and amino acids in the Benning × Danbaekkong soybean population

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

High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence

Background: The Soybean Consensus Map 4.0 facilitated the anchoring of 95.6% of the soybean whole genome sequence developed by the Joint Genome Institute, Department of Energy, but its marker density was only sufficient to properly orient 66% of the sequence scaffolds. The discovery and genetic mapping of more single nucleotide polymorphism (SNP) markers were needed to anchor and orient the remaining genome sequence. To that end, next generation sequencing and high-throughput genotyping were combined to obtain a much higher resolution genetic map that could be used to anchor and orient most of the remaining sequence and to help validate the integrity of the existing scaffold builds. Results: A total of 7,108 to 25,047 predicted SNPs were discovered using a reduced representation library that was subsequently sequenced by the Illumina sequence-by-synthesis method on the clonal single molecule array platform. Using multiple SNP prediction methods, the validation rate of these SNPs ranged from 79% to 92.5%. A high resolution genetic map using 444 recombinant inbred lines was created with 1,790 SNP markers. Of the 1,790 mapped SNP markers, 1,240 markers had been selectively chosen to target existing un-anchored or un-oriented sequence scaffolds, thereby increasing the amount of anchored sequence to 97%. Conclusion: We have demonstrated how next generation sequencing was combined with high-throughput SNP detection assays to quickly discover large numbers of SNPs. Those SNPs were then used to create a high resolution genetic map that assisted in the assembly of scaffolds from the 8× whole genome shotgun sequences into pseudomolecules corresponding to chromosomes of the organism

SNP Discovery and Linkage Map Construction in Cultivated Tomato

Few intraspecific genetic linkage maps have been reported for cultivated tomato, mainly because genetic diversity within Solanum lycopersicum is much less than that between tomato species. Single nucleotide polymorphisms (SNPs), the most abundant source of genomic variation, are the most promising source of polymorphisms for the construction of linkage maps for closely related intraspecific lines. In this study, we developed SNP markers based on expressed sequence tags for the construction of intraspecific linkage maps in tomato. Out of the 5607 SNP positions detected through in silico analysis, 1536 were selected for high-throughput genotyping of two mapping populations derived from crosses between ‘Micro-Tom’ and either ‘Ailsa Craig’ or ‘M82’. A total of 1137 markers, including 793 out of the 1338 successfully genotyped SNPs, along with 344 simple sequence repeat and intronic polymorphism markers, were mapped onto two linkage maps, which covered 1467.8 and 1422.7 cM, respectively. The SNP markers developed were then screened against cultivated tomato lines in order to estimate the transferability of these SNPs to other breeding materials. The molecular markers and linkage maps represent a milestone in the genomics and genetics, and are the first step toward molecular breeding of cultivated tomato. Information on the DNA markers, linkage maps, and SNP genotypes for these tomato lines is available at http://www.kazusa.or.jp/tomato/

EFFECTS OF GOAL SETTING, E-MAIL FEEDBACK AND GRAPHIC FEEDBACK ON THE PRODUCTIVITY OF PUBLIC SCHOOL ATTENDANCE CLERKS APPROVED

A package intervention, consisting of daily-adjusted goal setting, e-mail feedback, and graphic feedback, was used in a public school attendance office to increase the efficiency with which 3 attendance clerks documented student attendance. During the intervention phase, the attendance secretary set a daily goal for each attendance clerk. This goal was a percentage of student absences to be coded and entered in the school computer program. After establishing a daily goal, the attendance secretary provided daily feedback, in the form of a written e-mail response and graphed feedback to each clerk. If the subjects had attained their daily goal, the attendance secretary also delivered a praise statement along with the e-mail feedback. Results indicated that the intervention package was ineffective in producing change in the attendance clerks' absence coding behavior. 2 ii ACKNOWLEDGEMENT