The potential of microarrays to assist shrimp breeding and production: a review

The shrimp aquaculture industry is a relatively new livestock industry, having developed over the past 30 years. Thus, it is poised to take advantage of new technologies from the outset of selective breeding programs. This contrasts with long established livestock industries, where there are already highly specialised breeds. This review focuses specifically on the potential application of microarrays to shrimp breeding. Potential applications of microarrays in selective breeding programs are summarised. Microarrays can be used as a rapid means to generate molecular markers for genetic linkage mapping, and genetic maps have been constructed for yeast, Arabidopsis and barley using microarray technology. Microarrays can also be used in the hunt for candidate genes affecting particular traits, leading to development of perfect markers for these traits (i.e. causative mutations). However, this requires that microarray analysis be combined with genetic linkage mapping, and that substantial genomic information is available for the species in question. A novel application of microarrays is to treat gene expression as a quantitative trait in itself and to combine this with linkage mapping to identify quantitative trait loci controlling the levels of gene expression; this approach may identify higher level regulatory genes in specific pathways. Finally, patterns of gene expression observed using microarrays may themselves be treated as phenotypic traits in selection programs (e.g. a particular pattern of gene expression might be indicative of a disease tolerant individual). Microarrays are now being developed for a number of shrimp species in laboratories around the world, primarily with a focus on identifying genes involved in the immune response. However, at present, there is no central repository of shrimp genomic information, which limits the rate at which shrimp genomic research can be progressed. The application of microarrays to shrimp breeding will be extremely limited until there is a shared repository of genomic information for shrimp, and the collective will and resources to develop comprehensive genomic tools for shrimp.

Genomewide significant linkage to migrainous Hheadache on chromosome 5q21

Familial typical migraine is a common, complex disorder that shows strong familial aggregation. Using latent-class analysis (LCA), we identified subgroups of people with migraine/severe headache in a community sample of 12,245 Australian twins (60% female), drawn from two cohorts of individuals aged 23-90 years who completed an interview based on International Headache Society criteria. We report results from genomewide linkage analyses involving 756 twin families containing a total of 790 independent sib pairs ( 130 affected concordant, 324 discordant, and 336 unaffected concordant for LCA-derived migraine). Quantitative-trait linkage analysis produced evidence of significant linkage on chromosome 5q21 and suggestive linkage on chromosomes 8, 10, and 13. In addition, we replicated previously reported typical-migraine susceptibility loci on chromosomes 6p12.2-p21.1 and 1q21-q23, the latter being within 3 cM of the rare autosomal dominant familial hemiplegic migraine gene (ATP1A2), a finding which potentially implicates ATP1A2 in familial typical migraine for the first time. Linkage analyses of individual migraine symptoms for our six most interesting chromosomes provide tantalizing hints of the phenotypic and genetic complexity of migraine. Specifically, the chromosome 1 locus is most associated with phonophobia; the chromosome 5 peak is predominantly associated with pulsating headache; the chromosome 6 locus is associated with activity-prohibiting headache and photophobia; the chromosome 8 locus is associated with nausea/vomiting and moderate/severe headache; the chromosome 10 peak is most associated with phonophobia and photophobia; and the chromosome 13 peak is completely due to association with photophobia. These results will prove to be invaluable in the design and analysis of future linkage and linkage disequilibrium studies of migraine.

Butyrylcholinesterase: Association with the metabolic syndrome and identification of 2 gene loci affecting activity

Background: Plasma cholinesterase activity is known to be correlated with plasma triglycerides, HDL- and LDL-cholesterol, and other features of the metabolic syndrome. A role in triglyceride metabolism has been proposed. Genetic variants that decrease activity have been studied extensively, but the factors contributing to overall variation in the population are poorly understood. We studied plasma cholinesterase activity in a sample of 2200 adult twins to assess covariation with cardiovascular risk factors and components of the metabolic syndrome, to determine the degree of genetic effects on enzyme activity, and to search for quantitative trait loci affecting activity. Methods and Results: Cholinesterase activity was lower in women than in men before the age of 50, but increased to activity values similar to those in males after that age. There were highly significant correlations with variables associated with the metabolic syndrome: plasma triglyceride, HDL- and LDL-cholesterol, apolipoprotein B and E, urate, and insulin concentrations; gamma-glutamyltransferase and aspartate and alanine aminotransferase activities; body mass index; and blood pressure. The heritability of plasma cholinesterase activity was 65%. Linkage analysis with data from the dizygotic twin pairs showed suggestive linkage on chromosome 3 at the location of the cholinesterase WHO gene and also on chromosome 5. Conclusions: Our results confirm and extend the connection between cholinesterase, cardiovascular risk factors, and metabolic syndrome. They establish a substantial heritability for plasma cholinesterase activity that might be attributable to variation near the structural gene and at an independent locus. (c) 2006 American Association for Clinical Chemistry.

Analysing diversity in sugarcane resistance gene analogues

As resistance genes have been shown to contain conserved motifs and cluster in many plant genomes, the identification of resistance gene analogues can be used as a strategy for both the discovery of DNA markers linked to disease resistance loci and the map-based cloning of disease resistance genes. Sugarcane suffers from many important diseases and an analysis of resistance gene analogues offers a means to identify DNA markers linked to resistance loci. However, sugarcane has the most complex genome of any crop plant and initially it is important to understand the extent of resistance gene analogue diversity in the sugarcane genome before genetic analysis. We review herein how more than 100 expressed sequence tags with homology to different resistance genes have been identified in sugarcane with many mapped as single-dose restriction fragment length polymorphism markers. Importantly, some of these resistance gene analogues have been shown to be linked to disease resistance genes or disease quantitative trait loci. In an attempt to more efficiently analyse additional resistance gene analogues in sugarcane, we report on experiments aimed at investigating the molecular diversity of several resistance gene analogue families using a modified form of a technique termed Ecotilling. Using Ecotilling, we were able to rapidly detect single nucleotide polymorphisms in fragments amplified by PCR from four different resistance gene analogue families, SoRP1D, SoPTO, SoXa21 and SoHs1pro-1. An analysis of a diverse set of sugarcane varieties, including modern sugarcane cultivars and several S. officinarum and S. spontaneum clones, indicated that all amplicons, apart from SoHs1pro-1, contained significant polymorphism within the gene region studied. However, a comparison among these sugarcane clones, including between the parents of two sugarcane mapping populations, indicated that most polymorphisms were multi-dose, not single-dose, preventing their genetic map location or association with disease susceptibility or resistance from being determined.

Congruence in QTL for adventitious rooting in Pinus elliottii X Pinus caribaea hybrids resolves between and within-species effects

Targeting between-species effects for improvement in synthetic hybrid populations derived from outcrossing parental tree species may be one way to increase the efficacy and predictability of hybrid breeding. We present a comparative analysis of the quantitative trait loci (QTL) which resolved between from within-species effects for adventitious rooting in two populations of hybrids between Pinus elliottii and P. caribaea, an outbred F-1 (n=287) and an inbred-like F-2 family (n=357). Most small to moderate effect QTL (each explaining 2-5% of phenotypic variation, PV) were congruent (3 out of 4 QTL in each family) and therefore considered within-species effects as they segregated in both families. A single large effect QTL (40% PV) was detected uniquely in the F-2 family and assumed to be due to a between-species effect, resulting from a genetic locus with contrasting alleles in each parental species. Oligogenic as opposed to polygenic architecture was supported in both families (60% and 20% PV explained by 4 QTL in the F-2 and F-1 respectively). The importance of adventitious rooting for adaptation to survive water-logged environments was thought in part to explain oligogenic architecture of what is believed to be a complex trait controlled by many hundreds of genes.

Genetic improvement of lucerne for anthracnose (Colletotrichum trifolii) resistance

Anthracnose, caused by Colletotrichum trifolii, is one of the most serious diseases influencing lucerne persistence and productivity in eastern Australia. The disease is largely controlled by plant resistance; however, new pathotypes of C. trifolii have developed in Australia, seriously limiting the productive life of susceptible cultivars. This paper describes an incompletely recessive and quantitatively inherited resistance to C. trifolii identified in a clone (W116) from cv. Sequel. S-1, F-1, F-2 and backcross populations of W116 and D (highly susceptible clone) were studied for their reaction to C. trifolii race 1. Resistance was found to be quantitatively inherited, and quantitative trait loci associated with resistance and susceptibility were identified in a backcross population (D x W116) x D using random amplified polymorphic DNA and amplified fragment length polymorphic markers. A multi-locus region on linkage group 4 was found to contribute significantly to the resistance phenotype. The application of DNA markers to allow exploitation of this quantitatively inherited resistance in lucerne breeding is discussed.

Novel Genetic Determinants of Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a devastating diabetes complication, with known heritability not fully revealed by previous genetics studies. We performed the largest genome-wide association study of type 1 DKD to date, in a 13-cohort consortium of 15,590 individuals of European ancestry genotyped on the Illumina HumanCoreExome Beadchip, which allows exploration of coding variation in addition to genomic markers.

As prior work has shown that different characterizations of the DKD phenotype highlight distinct genetic associations, we investigated a spectrum of DKD definitions based on proteinuria and renal function criteria. Controls were DKD-free after a minimum of 15 years diabetes duration; cases had diabetes for at least 10 years prior to DKD diagnosis. We also performed a quantitative trait analysis of estimated glomerular filtration rate in all participants.

Our top finding was a missense mutation in COL4A3, rs55703767 (Asp326Tyr); the minor allele is common in Europeans (20%) and East Asians (13%) but not Africans (2%). This SNP had a genome-wide significant association with traditionally defined DKD (macroalbuminuria or end-stage renal disease [ESRD], (OR= 0.79, P=1.9×10-9), and a suggestive association with macroalbuminuria (OR= 0.79, P=1.6×10-6) and ESRD (OR= 0.79, P=4.5×10-5) individually. Though its PolyPhen score is 0.3 (benign), this SNP has been implicated as a splice site disruptor.

The COL4A3 gene encodes the alpha 3 subunit of Type IV collagen, the major structural component of basement membranes. Pathogenic mutations in COL4A3 have been identified in thin basement membrane nephropathy, familial focal segmental glomerulosclerosis, and Alport syndrome. A proxy (r2=0.6) for rs55703767 had no significant associations in the CKDGen consortium, suggesting its pathogenicity occurs solely in the setting of hyperglycemia.

By significantly increasing sample size we have discovered a novel locus underlying DKD risk, paving the way for better understanding of pathology, prevention, and treatment.

Nonparametric Evaluation of Quantitative Traits in Population-Based Association Studies when the Genetic Model is Unknown

Statistical association between a single nucleotide polymorphism (SNP) genotype and a quantitative trait in genome-wide association studies is usually assessed using a linear regression model, or, in the case of non-normally distributed trait values, using the Kruskal-Wallis test. While linear regression models assume an additive mode of inheritance via equi-distant genotype scores, Kruskal-Wallis test merely tests global differences in trait values associated with the three genotype groups. Both approaches thus exhibit suboptimal power when the underlying inheritance mode is dominant or recessive. Furthermore, these tests do not perform well in the common situations when only a few trait values are available in a rare genotype category (disbalance), or when the values associated with the three genotype categories exhibit unequal variance (variance heterogeneity). We propose a maximum test based on Marcus-type multiple contrast test for relative effect sizes. This test allows model-specific testing of either dominant, additive or recessive mode of inheritance, and it is robust against variance heterogeneity. We show how to obtain mode-specific simultaneous confidence intervals for the relative effect sizes to aid in interpreting the biological relevance of the results. Further, we discuss the use of a related all-pairwise comparisons contrast test with range preserving confidence intervals as an alternative to Kruskal-Wallis heterogeneity test. We applied the proposed maximum test to the Bogalusa Heart Study dataset, and gained a remarkable increase in the power to detect association, particularly for rare genotypes. Our simulation study also demonstrated that the proposed non-parametric tests control family-wise error rate in the presence of non-normality and variance heterogeneity contrary to the standard parametric approaches. We provide a publicly available R library nparcomp that can be used to estimate simultaneous confidence intervals or compatible multiplicity-adjusted p-values associated with the proposed maximum test.

Evidence for different QTL underlying the immune and hypersensitive responses of Eucalyptus globulus to the rust pathogen Puccinia psidii

The rust Puccinia psidii infects many species in the family Myrtaceae. Native to South America, the pathogen has recently entered Australia which has a rich Myrtaceous flora, including trees of the ecologically and economically important genus Eucalyptus. We studied the genetic basis of variation in rust resistance in Eucalyptus globulus, the main plantation eucalypt in Australia. Quantitative trait loci (QTL) analysis was undertaken using 218 genotypes of an outcross F2 mapping family, phenotyped by controlled inoculation of their open pollinated progeny with the strain of P. psidii found in Australia. QTL analyses were conducted using a binary classification of individuals with no symptoms (immune) versus those with disease symptoms, and in a separate analysis dividing plants with disease symptoms into those exhibiting the hypersensitive response versus those with more severe symptoms. Four QTL were identified, two influencing whether a plant exhibited symptoms (Ppr2 and Ppr3), and two influencing the presence or absence of a hypersensitive reaction (Ppr4 and Ppr5). These QTL mapped to four different linkage groups, none of which overlap with Ppr1, the major QTL previously identified for rust resistance in Eucalyptus grandis. Candidate genes within the QTL regions are presented and possible mechanisms discussed. Together with past findings, our results suggest that P. psidii resistance in eucalypts is quantitative in nature and influenced by the complex interaction of multiple loci of variable effect.

Can survival of European flat oysters following experimental infection with Bonamia ostreae be predicted using QTLs?

The present study identifies quantitative trait loci (QTLs) in response to an experimental infection with the parasite responsible for bonamiosis, Bonamia ostreae, in two segregating families of the European flat oyster, Ostrea edulis. We first constructed a genetic-linkage map for each studied family and improved the existing genetic-linkage map for the European flat oyster with a set of SNP markers. This latter map now combines the best accuracy and the best estimate of the genome coverage available for an oyster species. Secondly, by comparing the QTLs detected in this study with those previously published for O. edulis in similar experimental conditions, we identified several potential QTLs that were identical between the different families, and also new specific QTLs. We also detected, within the confidence interval of several QTL regions, some previously predicted candidate genes differentially expressed during an infection with B. ostreae, providing new candidate genome regions which should now be studied more specifically.

Stay-green Drought Adaptation Trait Enhances Sorghum Production in Sub-tropical Australia, Central-Western India and Sub-Saharan Africa

Drought during grain filling is a common challenge for sorghum production in north-eastern Australia, central-western India, and sub-Saharan Africa. We show that the stay-green drought adaptation trait enhances sorghum grain yield under post-anthesis drought in these three regions. A positive relationship between stay-green and yield was generally found in breeding trials in north-eastern Australia that sampled 1668 unique hybrid combinations and 23 environments. Physiological studies in Australia also found that introgressing four individual stay-green (Stg1–4) quantitative trait loci (QTLs) into a senescent background reduced water demand before flowering and hence increased water supply during grain filling, resulting in higher grain yield relative to the senescent control. Studies in India found that various Stg QTLs affected both transpiration and transpiration efficiency, although these effects depended on the interaction between genetic background (S35 and R16) and individual QTLs. The yield variation unexplained by harvest index was related to transpiration efficiency in S35 (R2 = 0.29) and R16 (R2 = 0.72), and was related to total water extracted in S35 (R2 = 0.41) but not in R16. Finally, sixty-eight stay-green enriched lines were evaluated in six countries in sub-Saharan Africa during the 2013/14 season. Analysis of the data from Kenya indicates that stay-green and grain size were positively correlated at two sites: Kiboko (high yielding, r2=0.25) and Masongaleni (low yielding, r2=0.37). Together, these studies suggest that stay-green is a beneficial trait for sorghum production in the semi-arid tropics and is a consequence of traits altering the plant water budget.

Genetic diversity of stay-green sorghums and their derivatives revealed by microsatellites

The genetic variability of 28 sorghum genotypes of known senescence phenotype was investigated using 66 SSR markers well-distributed across the sorghum genome. The genotypes of a number of lines from breeding programmes for stay-green were also determined. This included lines selected phenotypically for stay-green and also RSG 03123, a marker-assisted backcross progeny of R16 (recurrent parent) and B35 (stay-green donor). A total of 419 alleles were detected with a mean of 6.2 per locus. The number of alleles ranged from one for Xtxp94 to 14 for Xtxp88. Chromosome SBI-10 had the highest mean number of alleles (8.33), while SBI-05 had the lowest (4.17). The PIC values obtained ranged from zero to 0.89 in Xtxp94 and Xtxp88, respectively, with a mean of 0.68. On a chromosome basis, mean PIC values were highest in SBI-10 (0.81) and lowest in SBI-05 (0.53). Most of the alleles from B35 in RSG 03123 were found on chromosomes SBI-01, SBI-02 and SBI-03, confirming the successful introgression of quantitative trait loci associated with stay-green from B35 into the senescent background R16. However, the alternative stay-green genetic sources were found to be distinct based on either all the SSRs employed or using only those associated with the stay-green trait in B35. Therefore, the physiological and biochemical basis of each stay-green source should be evaluated in order to enhance the understanding of the functioning of the trait in the various backgrounds. These genetic sources of stay-green could provide a valuable resource for improving this trait in sorghum breeding programmes.

Sustainable deployment of QTLs conferring quantitative resistance to crops: first lessons from a stochastic model

International audience

Identification of regulatory polymorphisms associated with breast cancer risk

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Evaluation of breeding strategies for polledness in dairy cattle using a newly developed simulation framework for quantitative and Mendelian traits

Background: Intensified selection of polled individuals has recently gained importance in predominantly horned dairy cattle breeds as an alternative to routine dehorning. The status quo of the current polled breeding pool of genetically-closely related artificial insemination sires with lower breeding values for performance traits raises questions regarding the effects of intensified selection based on this founder pool. Methods: We developed a stochastic simulation framework that combines the stochastic simulation software QMSim and a self-designed R program named QUALsim that acts as an external extension. Two traits were simulated in a dairy cattle population for 25 generations: one quantitative (QMSim) and one qualitative trait with Mendelian inheritance (i.e. polledness, QUALsim). The assignment scheme for qualitative trait genotypes initiated realistic initial breeding situations regarding allele frequencies, true breeding values for the quantitative trait and genetic relatedness. Intensified selection for polled cattle was achieved using an approach that weights estimated breeding values in the animal best linear unbiased prediction model for the quantitative trait depending on genotypes or phenotypes for the polled trait with a user-defined weighting factor. Results: Selection response for the polled trait was highest in the selection scheme based on genotypes. Selection based on phenotypes led to significantly lower allele frequencies for polled. The male selection path played a significantly greater role for a fast dissemination of polled alleles compared to female selection strategies. Fixation of the polled allele implies selection based on polled genotypes among males. In comparison to a base breeding scenario that does not take polledness into account, intensive selection for polled substantially reduced genetic gain for this quantitative trait after 25 generations. Reducing selection intensity for polled males while maintaining strong selection intensity among females, simultaneously decreased losses in genetic gain and achieved a final allele frequency of 0.93 for polled. Conclusions: A fast transition to a completely polled population through intensified selection for polled was in contradiction to the preservation of high genetic gain for the quantitative trait. Selection on male polled genotypes with moderate weighting, and selection on female polled phenotypes with high weighting, could be a suitable compromise regarding all important breeding aspects.