Correction to "Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae)".

[This corrects the article DOI: 10.3732/apps.1300089.].

Whole genome shotgun sequences for microsatellite discovery and application in cultivated and wild Macadamia (Proteaceae).

PREMISE OF THE STUDY: Next-generation sequencing (NGS) data are widely used for single-nucleotide polymorphism discovery and genetic marker development in species with limited available genome information. We developed microsatellite primers for the Proteaceae nut crop species Macadamia integrifolia and assessed cross-species transferability in all congeners to investigate genetic identification of cultivars and gene flow. • METHODS AND RESULTS: Primers were designed from both raw and assembled Illumina NGS paired-end reads. The final 12 microsatellite markers selected were polymorphic among wild individuals of all four Macadamia species-M. integrifolia, M. tetraphylla, M. ternifolia, and M. jansenii-and in commercial macadamia cultivars including hybrids. • CONCLUSIONS: We demonstrate the utility of raw and assembled Illumina NGS reads from total genomic DNA for the rapid development of microsatellites in Macadamia. These primers will facilitate future studies of population structure, hybridization, parentage, and cultivar identification in cultivated and wild Macadamia populations.

High-throughput sequencing and mutagenesis to accelerate the domestication of Microlaena stipoides as a new food crop.

Global food demand, climatic variability and reduced land availability are driving the need for domestication of new crop species. The accelerated domestication of a rice-like Australian dryland polyploid grass, Microlaena stipoides (Poaceae), was targeted using chemical mutagenesis in conjunction with high throughput sequencing of genes for key domestication traits. While M. stipoides has previously been identified as having potential as a new grain crop for human consumption, only a limited understanding of its genetic diversity and breeding system was available to aid the domestication process. Next generation sequencing of deeply-pooled target amplicons estimated allelic diversity of a selected base population at 14.3 SNP/Mb and identified novel, putatively mutation-induced polymorphisms at about 2.4 mutations/Mb. A 97% lethal dose (LD97) of ethyl methanesulfonate treatment was applied without inducing sterility in this polyploid species. Forward and reverse genetic screens identified beneficial alleles for the domestication trait, seed-shattering. Unique phenotypes observed in the M2 population suggest the potential for rapid accumulation of beneficial traits without recourse to a traditional cross-breeding strategy. This approach may be applicable to other wild species, unlocking their potential as new food, fibre and fuel crops.

Molecular basis of seed lipoxygenase null traits in soybean line OX948.

The poor stability and off-flavors of soybean oil and protein products can be reduced by eliminating lipoxygenases from soybean seed. Mature seeds of OX948, a lipoxygenase triple null mutant line, do not contain lipoxygenase proteins. The objective of this study was to determine the molecular basis of the seed lipoxygenase null traits in OX948. Comparisons of the sequences for lipoxygenase 1 (Lx1) and lipoxygenase 2 (Lx2) genes in the mutant (OX948) with those in a line with normal lipoxygenase levels (RG10) showed that the mutations in these genes affected a highly conserved group of six histidines necessary for enzymatic activity. The OX948 mutation in Lx1 is a 74 bp deletion in exon 8, which introduces a stop codon that prematurely terminates translation. A single T-A substitution in Lx2 changes histidine H532 (one of the iron-binding ligands essential for L-2 activity) to glutamine. The mutation in the lipoxygenase 3 (Lx3) gene in OX948 is in the promoter region and represents two single base substitutions in a cis-acting AAATAC paired box. All three mutations would result in the loss of lipoxygenase activity in mature seed. The seed lipoxygenase gene mutation-based molecular markers could be used to accelerate and simplify breeding efforts for soybean cultivars with improved flavor.

Targeted single nucleotide polymorphism (SNP) discovery in a highly polyploid plant species using 454 sequencing.

Discovering single nucleotide polymorphisms (SNPs) in specific genes in a heterozygous polyploid plant species, such as sugarcane, is challenging because of the presence of a large number of homologues. To discover SNPs for mapping genes of interest, 454 sequencing of 307 polymerase chain reaction (PCR) amplicons (> 59 kb of sequence) was undertaken. One region of a four-gasket sequencing run, on a 454 Genome Sequencer FLX, was used for pooled PCR products amplified from each parent of a quantitative trait locus (QTL) mapping population (IJ76-514 x Q165). The sequencing yielded 96,755 (IJ76-514) and 86,241 (Q165) sequences with perfect matches to a PCR primer used in amplification, with an average sequence depth of approximately 300 and an average read length of 220 bases. Further analysis was carried out on amplicons whose sequences clustered into a single contig using an identity of 80% with the program cap3. In the more polymorphic sugarcane parent (Q165), 94% of amplicons (227/242) had evidence of a reliable SNP--an average of one every 35 bases. Significantly fewer SNPs were found in the pure Saccharum officinarum parent--with one SNP every 58 bases and SNPs in 86% (213/247) of amplicons. Using automatic SNP detection, 1632 SNPs were detected in Q165 sequences and 1013 in IJ76-514. From 225 candidate SNP sites tested, 209 (93%) were validated as polymorphic using the Sequenom MassARRAY system. Amplicon re-sequencing using the 454 system enables cost-effective SNP discovery that can be targeted to genes of interest and is able to perform in the highly challenging area of polyploid genomes.

Seed and agronomic QTL in low linolenic acid, lipoxygenase-free soybean (Glycine max (L.) Merrill) germplasm.

Linolenic acid and seed lipoxygenases are associated with off flavours in soybean products. F5 recombinant inbred lines (RILs) from a cross between a low linolenic acid line (RG10) and a seed lipoxygenase-free line (OX948) were genotyped for simple sequence repeats (SSR), random amplified polymorphic DNA (RAPD), sequence-tagged sites (STS), and cleaved amplified polymorphic sequence (CAPS) markers and evaluated for seed and agronomic traits at 3 Ontario locations in 2 years. One hundred twenty markers covering 1247.5 cM were mapped to 18 linkage groups (LGs) in the soybean composite genetic map. Seed lipoxygenases L-1 and L-2 mapped as single major genes to the same location on LG G13-F. L-3 mapped to LG G11-E. This is the first report of a map position for L-3. A major quantitative trait locus (QTL) associated with reduced linolenic acid content was identified on LG G3-B2. QTLs for 12 additional seed and agronomic traits were detected. Linolenic acid content, linoleic acid content, yield, seed mass, protein content, and plant height QTL were present in at least 4 of 6 environments. Three to 8 QTLs per trait were detected that accounted for up to 78% of total variation. Linolenic acid and lipoxygenase loci did not overlap yield QTL, suggesting that it should be possible to develop high-yielding lines resistant to oxidative degradation by marker-assisted selection (MAS).

Inheritance and Interaction of Low Palmitic and Low Linolenic Soybean.

Decreasing the palmitic and linolenic acid content of soybean [Glycine max (L.) Merrill] oil would help improve its nutritional quality and oxidative stability. The altered fatty acid profile in soybean germplasm lines with decreased levels of palmitic and linolenic acid have been developed at the University of Guelph, Canada, by combining different mutant alleles through hybridization. The objectives of this study were to determine the inheritance and interaction of palmitic and linolenic acid levels in RG3 and RG1, and the effects of these altered fatty acid levels on other fatty acids. RG3 and RG1 (low palmitic approximately 45 g kg(-1) and linolenic approximately 40 g kg(-1)) were crossed reciprocally to several soybean lines with altered or normal fatty acid profiles. Analysis of the reciprocal F(2) generations indicated no maternal or cytoplasmic effects for palmitic or linolenic acid content. Chi-square analyses of the F(2) generation demonstrated that RG3 and RG1 contained two alleles, fap1 and fapx, that controlled palmitic acid content. In addition, RG1 had a third allele, fan, which reduced its linolenic acid content. Calculation of gene substitution values indicated additive gene action at the fap1 and fan loci, whereas the fapx locus involved partial dominance. Correlation coefficients indicated no association between palmitic and linolenic acid. Decreases in palmitic and linolenic acid content were associated primarily with increases in linoleic acid content. Since fap1, fapx, and fan were inherited independently of each other and appeared to behave in an additive manner, RG3 and RG1 can be used in breeding programs as additional valuable sources of germplasm with altered fatty acid profiles.

Genotype x Environment Interactions, Stability, and Agronomic Performance of Soybean with Altered Fatty Acid Profiles.

There has been a major effort to produce soybean [Glycine max (L.) Merr.] lines with modified fatty acid profiles in order to improve quality and develop new uses for soybean oil. Utilization of the lines depends on their agronomic traits and stability of the fatty acid profiles in diverse environments. The objectives of this study were to (i) evaluate the influence of years and locations on the fatty acid composition of soybean genotypes with unique fatty acid profiles, (ii) determine which fatty acids and fatty acid profiles are the most stable, and (iii) evaluate agronomic and seed quality traits of mutant soybean lines. Genotypes were evaluated over three years (1996, 1997, and 1998) at four locations in Southern Ontario, Canada. Year effects had the largest impact on all fatty acid levels. Location effects were significant only for oleic and linolenic acids. Genotype x year interaction effect was significant for all fatty acids whereas genotype x location and genotype x year x location interaction effects were significant only for oleic, linoleic, and linolenic acids. Mutants with reduced or elevated palmitic, elevated oleic, or reduced linolenic acid concentrations exhibited average or higher stability than lines with normal levels of these fatty acids. Therefore, these lines may be suitable for growing in a wide range of environments. Maturity, plant height, lodging, seed size, and seed quality were significantly different between mutants and cultivars. Seed yield was significantly reduced in mutants compared to cultivars.