Quantitative trait loci and candidate genes associated with starch pasting viscosity characteristics in cassava (Manihot esculenta Crantz).

Starch pasting viscosity is an important quality trait in cassava (Manihot esculenta Crantz) cultivars. The aim here was to identify loci and candidate genes associated with the starch pasting viscosity. Quantitative trait loci (QTL) mapping for seven pasting viscosity parameters was carried out using 100 lines of an F1 mapping population from a cross between two cassava cultivars Huay Bong 60 and Hanatee. Starch samples were obtained from roots of cassava grown in 2008 and 2009 at Rayong, and in 2009 at Lop Buri province, Thailand. The traits showed continuous distribution among the F1 progeny with transgressive variation. Fifteen QTL were identified from mean trait data, with Logarithm of Odds (LOD) values from 2.77-13.01 and phenotype variations explained (PVE) from10.0-48.4%. In addition, 48 QTL were identified in separate environments. The LOD values ranged from 2.55-8.68 and explained 6.6-43.7% of phenotype variation. The loci were located on 19 linkage groups. The most important QTL for pasting temperature (PT) (qPT.1LG1) from mean trait values showed largest effect with highest LOD value (13.01) and PVE (48.4%). The QTL co-localised with PT and pasting time (PTi) loci that were identified in separate environments. Candidate genes were identified within the QTL peak regions. However, the major genes of interest, encoding the family of glycosyl or glucosyl transferases and hydrolases, were located at the periphery of QTL peaks. The loci identified could be effectively applied in breeding programmes to improve cassava starch quality. Alleles of candidate genes should be further studied in order to better understand their effects on starch quality traits.

Characterization of microsatellite markers in cassava based on microsatellite-AFLP technique.

We developed molecular markers for cassava based on the microsatellite-amplified fragment length polymorphism (M-AFLP) technique. Twenty primer pairs were developed and used for the analysis of 48 samples of Manihot species, consisting of M. esculenta (33), M. esculenta ssp flabellifolia (3), M. chlorosticta (3), M. carthaginensis (3), M. filamentosa (3), and M. tristis (3). Nine microsatellite loci that were polymorphic among these Manihot species were identified, giving 32 polymorphic alleles and from two to seven alleles per locus. Unbiased and direct count heterozygosity varied from 0.0233 to 0.7924 and 0.0000 to 0.7083, respectively. There was significant deviation (P < 0.05) from Hardy-Weinberg equilibrium at five loci. Genotypic data from the Manihot species were subjected to genetic diversity analysis. We found that M. chlorosticta and M. esculenta ssp flabellifolia were the closest populations, while M. filamentosa and M. esculenta ssp flabellifolia were the most divergent. Considering within M. esculenta, the samples from Nigeria and Fiji were the most closely related, while those from Venezuela and of unknown origin were the most divergent. We conclude that the M-AFLP technique is an effective method for generating microsatellite markers that are useful for genetic diversity analysis in Manihot species.

Microsatellite and mitochondrial haplotype diversity reveals population differentiation in the tiger shrimp (Penaeus monodon) in the Indo-Pacific region.

The black tiger shrimp (Penaeus monodon) is an ecologically and economically important penaeid species and is widely distributed in the Indo-Pacific region. Here we investigated the genetic diversity of P. monodon (n = 355) from eight geographical regions by genotyping at 10 microsatellite loci. The average observed heterozygosity at various loci ranged from 0.638 to 0.743, indicating a high level of genetic variability in this region. Significant departures from Hardy-Weinberg equilibrium caused by heterozygote deficiency were recorded for most loci and populations. Pairwise F(ST) and R(ST) values revealed genetic differentiation among the populations. Evidence from the assignment test showed that the populations in the West Indian Ocean were unique, whereas other populations examined were partially admixed. In addition, the non-metric multidimensional scaling analysis indicated the presence of three geographic groups in the Indo-Pacific region, i.e. the African populations, a population from western Thailand and the remaining populations as a whole. We also sequenced and analysed the mitochondrial control region (mtCR) in these shrimp stocks to determine whether the nuclear and mitochondrial genomes show a similar pattern of genetic differentiation. A total of 262 haplotypes were identified, and nucleotide divergence among haplotypes ranged from 0.2% to 16.3%. Haplotype diversity was high in all populations, with a range from 0.969 to 1. Phylogenetic analysis using the mtCR data revealed that the West Indian Ocean populations were genetically differentiated from the West Pacific populations, consistent with the microsatellite data. These results should have implications for aquaculture management and conservation of aquatic diversity.

Development of polymorphic markers from expressed sequence tags of Manihot esculenta Crantz.

In this study, 49 primers were designed from sequences containing di-, tri-, tetra-, penta- and hexanucleotide motifs with a minimum of four repeats and presence of motif size polymorphisms (insertion/deletion) from cassava (Manihot esculenta Crantz) expressed sequence tags deposited in public sequence database. Each locus was subsequently screened on 29 M. esculenta Crantz obtained from 15 different countries. Cross-amplification was tested with M. esculenta Crantz (ssp. flabellifolia) and four different Manihot species, M. chlorosticta, M. carthaginensis, M. filamentosa and M. tristis. Of these, nine loci showed polymorphic profiles within M. esculenta Crantz, which revealed two to four alleles per locus. The average unbiased and direct count heterozygosities were 0.4901 and 0.5674, respectively.

Genomic analysis of the rhg1 locus: candidate genes that underlie soybean resistance to the cyst nematode.

The rhg1 gene or genes lie at a recessive or co-dominant locus, necessary for resistance to all Hg types of the soybean (Glycine max (L.) Merr.) cyst nematode (Heterodera glycines I.). The aim here was to identify nucleotide changes within a candidate gene found at the rhg1 locus that were capable of altering resistance to Hg types 0 (race 3). A 1.5 +/- 0.25 cM region of chromosome 18 (linkage group G) was shown to encompass rhg1 using recombination events from four near isogenic line populations and nine DNA markers. The DNA markers anchored two bacterial artificial chromosome (BAC) clones 21d9 and 73p6. A single receptor like kinase (RLK; leucine rich repeat-transmembrane-protein kinase) candidate resistance gene was amplified from both BACs using redundant primers. The DNA sequence showed nine alleles of the RLK at Rhg1 in the soybean germplasm. Markers designed to detect alleles showed perfect association between allele 1 and resistance to soybean cyst nematode Hg types 0 in three segregating populations, fifteen additional selected recombination events and twenty-two Plant Introductions. A quantitative trait nucleotide (QTN) [corrected] in the RLK at rhg1 was inferred that alters A87 to V87 in the context of H274 rather than N274. [corrected] Contiguous DNA sequence of 315 kbp of chromosome 18 (about 2 cM) contained additional gene candidates that may modulate resistance to other Hg-types including a variant laccase, a hydrogen-sodium ion antiport and two proteins of unknown function. A molecular basis for recessive and co-dominant resistance that involves interactions among paralagous disease-resistance genes was inferred that would improve methods for developing new nematode-resistant soybean cultivars.

Genomic analysis of a region encompassing QRfs1 and QRfs2: genes that underlie soybean resistance to sudden death syndrome.

Candidate genes were identified for two loci, QRfs2 providing resistance to the leaf scorch called soybean (Glycine max (L.) Merr.) sudden death syndrome (SDS) and QRfs1 providing resistance to root infection by the causal pathogen Fusarium solani f.sp. glycines. The 7.5 +/- 0.5 cM region of chromosome 18 (linkage group G) was shown to encompass a cluster of resistance loci using recombination events from 4 near-isogenic line populations and 9 DNA markers. The DNA markers anchored 9 physical map contigs (7 are shown on the soybean Gbrowse, 2 are unpublished), 45 BAC end sequences (41 in Gbrowse), and contiguous DNA sequences of 315, 127, and 110 kbp. Gene density was high at 1 gene per 7 kbp only around the already sequenced regions. Three to 4 gene-rich islands were inferred to be distributed across the entire 7.5 cM or 3.5 Mbp showing that genes are clustered in the soybean genome. Candidate resistance genes were identified and a molecular basis for interactions among the disease resistance genes in the cluster inferred.

Quantitative trait loci in Two Soybean Recombinant Inbred Line Populations Segregating for Yield and Disease Resistance.

Molecular makers linked to quantitative trait loci (QTL) can assist soybean [Glycine max (L.) Merr.] breeders to combine traits of low heritability, such as yield, with disease resistance. The objective of this study was to identify markers linked to yield QTL in two recombinant inbred line (RIL) populations ['Essex' x 'Forrest' (ExF; n = 100) and 'Flyer' x 'Hartwig' (FxH; n = 94)] that also segregate for soybean cyst nematode (SCN) resistance genes (rhg1 and Rhg4). Each population was yield tested in four environments between 1996 and 1999. The resistant parents produced lower yields. Heritability of yield across four environments was 47% for ExF and 57% for FxH. Yield was normally distributed in both populations. High yielding, SCN resistant transgressive segregants were not observed. In the ExF RIL population, 134 microsatellite markers were compared against yield by ANOVA and MAPMAKER QTL. Regions associated with yield were identified by SATT294 on linkage group (LG.) C1 (P = 0.006, R(2) = 10%), SATT440 on LG. I (P = 0.007, R(2) = 10%), and SATT337 on LG. K (P = 0.004, R(2) = 10%). Essex provided the beneficial allele at SATT337. Mean yields among FxH RILs were compared against 33 microsatellite markers from LG. K. In addition 136 markers from randomly selected LGs were compared with extreme phenotypes by bulk segregant analysis. Two regions on LG. K (20 cM apart) associated with yield were identified by SATT326 (P = 0.0004, R(2) = 15%) and SATT539 (P = 0.0008, R(2) = 14%). Flyer provided both beneficial alleles. Both populations revealed a yield QTL in the interval (5 cM) between SATT337 and SATT326. These populations may share a common allele for yield in this region, given that about 40% of Flyer genome derived from Essex.

A pyramid of loci for partial resistance to Fusarium solani f. sp. glycines maintains Myo-inositol-1-phosphate synthase expression in soybean roots.

Myo-inositol 1-phosphate synthase (MIPS; EC 5.5.1.4) converts glucose 6-phosphate to myo-inositol 1-phosphate in the presence of NAD(+). It catalyzes the first step in the synthesis of myo-inositol and pinitol, and is a rate limiting step in the de novo biosynthesis of inositol in eukaryotes. Therefore, MIPS is involved in biotic and abiotic stress via Ca(2+) signalling. Seedlings of four soybean genotypes were inoculated with Fusarium solani f. sp. glycines, the causative agent of sudden death syndrome (SDS), and differentially abundant mRNAs were identified by differential display. The genotypes carried either zero, two, four or six alleles of the quantitative trait loci (QTLs) that control resistance to SDS in an additive manner. The mRNA abundance of MIPS did not decrease following inoculation in a recombinant inbred line (RIL 23) containing all six resistance alleles of the QTLs conferring resistance to SDS of soybean. However, the abundance of MIPS mRNA was decreased in genotypes containing four, two or no resistance alleles. The specific activity of the MIPS enzyme in vitro followed the same pattern across genotypes. The IP(3) content in the inoculated roots of genotypes with two, four or six resistance alleles were higher compared to the non-inoculated root. The results suggests that a non-additive effect on transcription and translation of MIPS is established in RIL 23 roots by pyramiding six QTLs for resistance to SDS. A role of MIPS in the partial resistance or response of soybean roots to F. solani infection is suggested.

Conversion of AFLP bands into high-throughput DNA markers.

The conversion of AFLP bands into polymorphic sequence-tagged-site (STS) markers is necessary for high-throughput genotype scoring. Technical hurdles that must be overcome arise from genome complexity (particularly sequence duplication), from the low-molecular-weight nature of the AFLP bands and from the location of the polymorphism within the AFLP band. We generated six STS markers from ten AFLP bands (four AFLPs were from co-dominant pairs of bands) in soybean (Glycine max). The markers were all linked to one of two loci, rhg1 on linkage group G and Rhg4 on linkage group A2, that confer resistance to the soybean cyst nematode (Heterodera glycines I.). When the polymorphic AFLP band sequence contained a duplicated sequence or could not be converted to a locus-specific STS marker, direct sequencing of BAC clones anchored to a physical map generated locus-specific flanking sequences at the polymorphic locus. When the polymorphism was adjacent to the restriction site used in the AFLP analysis, single primer extension was performed to reconstruct the polymorphism. The six converted AFLP markers represented 996 bp of sequence from alleles of each of two cultivars and identified eight insertions or deletions, two microsatellites and eight single-nucleotide polymorphisms (SNPs). The polymorphic sequences were used to design a non-electrophoretic, fluorometric assay (based on the TaqMan technology) and/or develop electrophoretic STS markers for high-throughput genotype determination during marker-assisted breeding for resistance to cyst nematode. We conclude that the converted AFLP markers contained polymorphism at a 10- to 20-fold higher frequency than expected for adapted soybean cultivars and that the efficiency of AFLP band conversion to STS can be improved using BAC libraries and physical maps. The method provides an efficient tool for SNP and STS discovery suitable for marker-assisted breeding and genomics.