Genetics of fat tissue accumulation in pigs: a comparative approach

Fatness traits are important in pig production since they influence meat quality and fattening efficiency. On the other hand, excessive fat accumulation in humans has become a serious health problem due to worldwide spread of obesity. Since the pig is also considered as an animal model for numerous human diseases, including obesity and metabolic syndrome, comparative genomic studies may bring new insights into genetics of fatness/obesity. Input of genetic factors into phenotypic variability of these traits is rather high and the heritability coefficient (h ²) of these traits oscillates around 0.5. Genome scanning revealed the presence of more than 500 QTLs for fatness in the pig genome. In addition to QTL studies, many candidate gene polymorphisms have been analyzed in terms of their associations with pig fatness, including genes encoding leptin (LEP) and its receptor (LEPR), insulin-like growth factor 2 (IGF-2), fatty acid-binding proteins (FABP3 andFABP4), melanocortin receptor type 4 (MC4R), and theFTO (fat mass and obesity-associated) gene. Among them, a confirmed effect on pig fatness was found for a well-known polymorphism of theIGF-2 gene. In humans the strongest association with predisposition to obesity was shown for polymorphism of theFTO gene, while in pigs such an association seems to be doubtful. The development of functional genomics has revealed a large number of genes whose expression is associated with fat accumulation and lipid metabolism, so far not studied extensively in terms of the association of their polymorphism with pig fatness. Recently, epigenomic mechanisms, mainly RNA interference, have been considered as a potential source of information on genetic input into the fat accumulation process. The rather limited progress in studies focused on the identification of gene polymorphism related with fatness traits shows that their genetic background is highly complex.     

Genetic variation at bx1 controls DIMBOA content in maize

The main hydroxamic acid in maize (Zea mays L.) is 2-4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA). DIMBOA confers resistance to leaf-feeding by several corn borers. Most genes involved in the DIMBOA metabolic pathway are located on the short arm of chromosome 4, and quantitative trait loci (QTLs) involved in maize resistance to leaf-feeding by corn borers have been localized to that region. However, the low resolution of QTL linkage mapping does not allow convincing proof that genetic variation at bx loci was responsible for the variability for resistance. This study addressed the following objectives: to determine the QTLs involved in DIMBOA synthesis across genetically divergent maize inbreds using eight RIL families from the nested association mapping population, to check the stability of QTLs for DIMBOA content across years by evaluating two of those RIL families in 2 years, and to test the involvement of bx1 by performing association mapping with a panel of 281 diverse inbred lines. QTLs were stable across different environments. A genetic model including eight markers explained approximately 34% of phenotypic variability across eight RIL families and the position of the largest QTL co-localizes with the majority of structural genes of the DIMBOA pathway. Candidate association analysis determined that sequence polymorphisms at bx1 greatly affects variation of DIMBOA content in a diverse panel of maize inbreds, but the specific causal polymorphism or polymorphisms responsible for the QTL detected in the region 4.01 were not identified. This result may be because the causal polymorphism(s) were not sequenced, identity is masked by linkage disequilibrium, adjustments for population structure reduce significance of causal polymorphisms or multiple causal polymorphisms affecting bx1 segregate among inbred lines.     

Genome-wide interval mapping using SNPs identifies new QTL for growth,body composition and several physiological variables in an F2 intercross between fat and lean chicken lines

Background

For decades, genetic improvement based on measuring growth and body composition traits has been successfully applied in the production of meat-type chickens. However, this conventional approach is hindered by antagonistic genetic correlations between some traits and the high cost of measuring body composition traits. Marker-assisted selection should overcome these problems by selecting loci that have effects on either one trait only or on more than one trait but with a favorable genetic correlation. In the present study, identification of such loci was done by genotyping an F₂ intercross between fat and lean lines divergently selected for abdominal fatness genotyped with a medium-density genetic map (120 microsatellites and 1302 single nucleotide polymorphisms). Genome scan linkage analyses were performed for growth (body weight at 1, 3, 5, and 7 weeks, and shank length and diameter at 9 weeks), body composition at 9 weeks (abdominal fat weight and percentage, breast muscle weight and percentage, and thigh weight and percentage), and for several physiological measurements at 7 weeks in the fasting state, i.e. body temperature and plasma levels of IGF-I, NEFA and glucose. Interval mapping analyses were performed with the QTLMap software, including single-trait analyses with single and multiple QTL on the same chromosome.

Results

Sixty-seven QTL were detected, most of which had never been described before. Of these 67 QTL, 47 were detected by single-QTL analyses and 20 by multiple-QTL analyses, which underlines the importance of using different statistical models. Close analysis of the genes located in the defined intervals identified several relevant functional candidates, such as ACACA for abdominal fatness, GHSR and GAS1 for breast muscle weight, DCRX and ASPSCR1 for plasma glucose content, and ChEBP for shank diameter.

Conclusions

The medium-density genetic map enabled us to genotype new regions of the chicken genome (including micro-chromosomes) that influenced the traits investigated. With this marker density, confidence intervals were sufficiently small (14 cM on average) to search for candidate genes. Altogether, this new information provides a valuable starting point for the identification of causative genes responsible for important QTL controlling growth, body composition and metabolic traits in the broiler chicken.     

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> complex

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.     

Genetic linkage maps of Populus nigra L. including AFLPs, SSRs, SNPs, and sex trait

Black poplar (Populus nigra L.) is a tree of ecological and economic interest. A better knowledge of P. nigra genome is needed for an effective protection and use of its genetic resources. The main objective of this study is the construction of a highly informative genetic map of P. nigra species including genes of adaptive and economic interest. Two genotypes originated from contrasted natural Italian populations were crossed to generate a F₁ mapping pedigree of 165 individuals. Amplification fragment length polymorphism (AFLP), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP) markers were used to genotype 92 F₁ individuals, and the pseudo-test-cross strategy was applied for linkage analysis. The female parent map included 368 markers (274 AFLPs, 91 SSRs, and 3 SNPs) and spanned 2,104 cM with 20 linkage groups, and the male parent map, including 317 markers (205 AFLPs, 106 SSRs, 5 SNPs, and sex trait), spanned 2,453 cM with 23 main linkage groups. The sex, as morphological trait, was mapped on the linkage group XIX of the male parent map. The generated maps are among the most informative in SSRs when compared to the Populus maps published so far and allow a complete alignment with the 19 haploid chromosomes of Populus sequence genome. These genetic maps provide informative tools for a better understanding of P. nigra genome structure and genetic improvement of this ecologically and economically important European tree species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Epistatic effect between ACACA and FABP2 gene on abdominal fat traits in broilers

Epistasis is generally defined as the interaction between two or more genes or their mRNA or protein products to influence a single trait. Experimental evidence suggested that epistasis could be important in the determination of the genetic architecture of complex traits in domestic animals. Acetyl-coenzyme A carboxylase alpha (ACACA) and fatty acid binding protein 2 (FABP2) are both key factors of lipogenesis and transport. They may play a crucial role in the weight variability of abdominal adipose tissue in the growing chicken. In this study, the polymorphisms of c.2292GA in ACACA and c.-561AC in FABP2 were detected among individuals from two broiler lines which were divergently selected for abdominal fat content. Epistasis between the two SNPs on abdominal fat weight (AFW) and abdominal fat percentage (AFP) was analyzed. The additive × additive epistatic components between these two SNPs were found significant or suggestively significant on both AFW and AFP in lean lines of the 9th and 10th generation; whereas, it was not significantly associated with either AFW or AFP in fat lines. At the same time, there were not any other significant epistatic components found in both generations or in both lines. Significant epistatic effects between these two SNPs found only in the lean lines could partly be due to the fact that the abdominal fat traits in these two experimental lines have been greatly modified by strong artificial selection. The results suggested that the epistasis mode may be different between the lean and fat chicken lines. Our results could be helpful in further understanding the genetic interaction between candidate genes contributing to phenotypic variation of abdominal fat content in broilers.     

The use of comparative genome analysis and syntenic relationships allows extrapolating the position of Zn tolerance QTL regions from Arabidopsis halleri into Arabidopsis thaliana

Arabidopsis halleri is a species that has undergone natural selection for zinc (Zn) tolerance. Isolation of the quantitative trait loci (QTL) associated with this trait holds great promise for the identification of the main genes responsible for this adaptation. Using a segregating progeny produced by an interspecific cross, we previously constructed a genetic linkage map of A. halleri × A. lyrata petraea and mapped the three main QTL that confer Zn tolerance in A. halleri (Willems et al.). The goal of the present study is to compare the genetic linkage map of A. halleri × A. l. petraea to the annotated A. thaliana genome sequence to generate a tool for A. halleri genomic approaches. To achieve this aim, we constructed a genetic linkage map with 81 markers anchored on A. thaliana, including 23 genes known to be involved in metal homeostasis. First, this provided an extensive overview of the chromosomal rearrangements that have occurred since the divergence between A. thaliana and its closest relative A. halleri. Second, on the basis of the syntenic relationships assessed experimentally through this work, we transferred the QTL confidence intervals for Zn tolerance to the A. thaliana physical map, allowing access to all the genes localized in the corresponding regions. Third, we validated from the 23 genes involved in metal homeostasis the three ones localized in the QTL regions that can be considered the best candidates for conferring Zn tolerance. Nancy H. C. J. Roosens and Glenda Willems contributed equally to this paper.     

Allele-specific CAPS markers based on point mutations in resistance alleles at the pvr1 locus encoding eIF4E in Capsicum

Marker-assisted selection has been widely implemented in crop breeding and can be especially useful in cases where the traits of interest show recessive or polygenic inheritance and/or are difficult or impossible to select directly. Most indirect selection is based on DNA polymorphism linked to the target trait, resulting in error when the polymorphism recombines away from the mutation responsible for the trait and/or when the linkage between the mutation and the polymorphism is not conserved in all relevant genetic backgrounds. In this paper, we report the generation and use of molecular markers that define loci for selection using cleaved amplified polymorphic sequences (CAPS). These CAPS markers are based on nucleotide polymorphisms in the resistance gene that are perfectly correlated with disease resistance, the trait of interest. As a consequence, the possibility that the marker will not be linked to the trait in all backgrounds or that the marker will recombine away from the trait is eliminated. We have generated CAPS markers for three recessive viral resistance alleles used widely in pepper breeding, pvr1, pvr1 ¹, and pvr1 ². These markers are based on single nucleotide polymorphisms (SNPs) within the coding region of the pvr1 locus encoding an eIF4E homolog on chromosome 3. These three markers define a system of indirect selection for potyvirus resistance in Capsicum based on genomic sequence. We demonstrate the utility of this marker system using commercially significant germplasm representing two Capsicum species. Application of these markers to Capsicum improvement is discussed.     

Combining Two-Stage Testing and Interval Mapping Strategies to Detect QTL for Resistance to Bonamiosis in the European Flat Oyster Ostrea edulis

We have identified quantitative trait loci (QTL) in the flat oyster (Ostrea edulis) for resistance to Bonamia ostreae, a parasite responsible for the dramatic reduction in the aquaculture of this species. An F₂ family from a cross between a wild oyster and an individual from a family selected for resistance to bonamiosis was cultured with wild oysters injected with the parasite, leading to 20% cumulative mortality. Selective genotyping of 92 out of a total of 550 F₂ progeny (i.e., 46 heavily infected oysters that died and 46 parasite-free oysters that survived) was performed using 20 microsatellites and 34 amplification fragment length polymorphism primer pairs. Both a two-stage testing strategy and QTL interval mapping methods were used. The two-stage detection strategy had a high power with a low rate of false positives and identified nine and six probable markers linked to genes of resistance and susceptibility, respectively. Parent-specific genetic linkage maps were built for the family, spanning ten linkage groups (n = 10) with an observed genome coverage of 69–84%. Three QTL were identified by interval mapping in the first parental map and two in the second. Good concordance was observed between the results obtained after the two-stage testing strategy and QTL mapping.     

Spatial pattern of nucleotide polymorphism indicates molecular adaptation in the bryophyte Sphagnum fimbriatum

In organisms with haploid-dominant life cycles, natural selection is expected to be especially effective because genetic variation is exposed directly to selection. However, in spore-producing plants with high dispersal abilities, among-population migration may counteract local adaptation by continuously redistributing genetic variability. In this study, we tested for adaptation at the molecular level by comparing nucleotide polymorphism in two genes (GapC and Rpb2) in 10 European populations of the peatmoss species, Sphagnum fimbriatum with variability at nine microsatellite loci assumed to be selectively neutral. In line with previous results, the GapC and Rpb2 genes showed strikingly different patterns of nucleotide polymorphism. Neutrality tests and comparison of population differentiation based on the GapC and Rpb2 genes with neutrally evolving microsatellites using coalescent simulations supported non-neutral evolution in GapC, but neutral evolution in the Rpb2 gene. These observations and the positions of the replacement mutations in the GAPDH enzyme (coded by GapC) indicate a significant impact of replacement mutations on enzyme function. Furthermore, the geographic distribution of alternate GapC alleles and/or linked genomic regions suggests that they have had differential success in the recolonization of Europe following the Last Glacial Maximum.     

Identifying QTLs for fire-blight resistance via a European pear (<Emphasis Type="Italic">Pyrus communis</Emphasis> L.) genetic linkage map

The existence of different levels of susceptibility to fire blight (Erwinia amylovora) in European pear (Pyrus communis L.) cultivars suggests that it is possible to identify QTLs related to resistance in pear germplasm. Given the polygenic nature of this trait, we designed two genetic maps of the parental lines 'Passe Crassane' (susceptible) and 'Harrow Sweet' (resistant) using SSRs, MFLPs, AFLPs, RGAs and AFLP-RGAs markers. RGA-related markers should theoretically map in chromosome regions coding for resistance genes. The 'Passe Crassane' map includes 155 loci, for a total length of 912 cM organised in 18 linkage groups, and the 'Harrow Sweet' map 156 loci, for a total length of 930 cM divided in 19 linkage groups; both maps have a good genome coverage when compared to the more detailed apple maps. Four putative QTLs related to fire blight resistance were identified in the map. A suite of molecular markers, including two AFLP-RGAs, capable of defining resistant and susceptible haplotypes in the analysed population was developed.     

Fatness QTL on chicken chromosome 5 and interaction with sex

Quantitative trait loci (QTL) affecting fatness in male chickens were previously identified on chromosome 5 (GGA5) in a three-generation design derived from two experimental chicken lines divergently selected for abdominal fat weight. A new design, established from the same pure lines, produced 407 F2 progenies (males and females) from 4 F1-sire families. Body weight and abdominal fat were measured on the F₂ at 9 wk of age. In each sire family, selective genotyping was carried out for 48 extreme individuals for abdominal fat using seven microsatellite markers from GGA5. QTL analyses confirmed the presence of QTL for fatness on GGA5 and identified a QTL by sex interaction. By crossing one F1 sire heterozygous at the QTL with lean line dams, three recombinant backcross 1 (BC1) males were produced and their QTL genotypes were assessed in backcross 2 (BC2) progenies. These results confirmed the QTL by sex interaction identified in the F2 generation and they allow mapping of the female QTL to less than 8 Mb at the distal part of the GGA5. They also indicate that fat QTL alleles were segregating in both fat and lean lines.     

Mapping and validation of chromosome regions conferring a new boron-efficient locus in <Emphasis Type="Italic">Brassica napus</Emphasis>

Considerable genotypic variation exists in the response of different cultivars of rapeseed (Brassica napus) to B deficiency. This raises the possibility of genetic improvement of a B nutrition trait that will make the plant more tolerant to low B stress. The results of our study showed that B-efficient backcross plants had lower B concentration and more dry matter when grown at low levels of B when compared with the recurrent parent. Accordingly, we proposed that the improved B efficiency was attributed to either a high B utilization efficiency or less demand for B. The results of the genetic analysis showed that B efficiency is a dominant trait that is controlled by a single locus, namely BnBE2. By using bulked segregant analysis (BSA) in combination with amplified fragment length polymorphism (AFLP) and sequence related amplified polymorphism (SRAP) techniques, five SRAP markers and one converted single strand conformation polymorphism (SSCP) marker were identified to be linked to BnBE2 after screening 1,800 primer combinations. The six markers together with BnBE2 were mapped in a region that covered a genetic distance of 6.9 cM on a linkage group using a BC₆ population. This region was located on linkage group N14 after mapping these markers in two doubled haploid (DH) populations (TNDH and BQDH). The SRAP and AFLP markers were sequenced and found to be homologous to a BAC sequence from Brassica oleracea (CC). This finding suggested that the segment containing BnBE2 locus originated from the C genome of Brassica oleracea. Three SSR markers were identified to be linked to BnBE2 through comparative mapping. All these markers might have potential value for facilitating the pyramiding of the BnBE2 gene with other B efficient genes in order to improve the B efficiency trait and for further fine mapping of the BnBE2 gene in Brassica napus.     

Use of single-primer DNA amplifications in genetic studies of peanut (Arachis hypogaea L.)

A recent approach to detecting genetic polymorphism involves the amplification of genomic DNA using single primers of arbitrary sequence. When separated electrophoretically in agarose gels, the amplification products give banding patterns that can be scored for genetic variation. The objective of this research was to apply these techniques to cultivated peanut (Arachis hypogaea L.) and related wild species to determine whether such an approach would be feasible for the construction of a genetic linkage map in peanut or for systematic studies of the genus. Two peanut cultivars, 25 unadapted germplasm lines of A. hypogaea, the wild allotetraploid progenitor of cultivated peanut (A. monticola), A. glabrata (a tetraploid species from section Rhizomatosae), and 29 diploid wild species of Arachis were evaluated for variability using primers of arbitrary sequence to amplify segments of genomic DNA. No variation in banding pattern was observed among the cultivars and germplasm lines of A. hypogaea, whereas the wild Arachis species were uniquely identified with most primers tested. Bands were scored (+/–) in the wild species and the PAUP computer program for phylogenetic analysis and the HyperRFLP program for genetic distance analysis were used to generate dendrograms showing genetic relationships among the diploid Arachis species evaluated. The two analyses produced nearly identical dendrograms of species relationships. In addition, approximately 100 F₂ progeny from each of two interspecific crosses were evaluated for segregation of banding patterns. Although normal segregation was observed among the F₂ progeny from both crosses, banding patterns were quite complex and undesirable for use in genetic mapping. The dominant behavior of the markers prevented the differentiation of heterozygotes from homozygotes with certainty, limiting the usefulness of arbitrary primer amplification products as markers in the construction of a genetic linkage map in peanut.     

Marker-assisted introgression of blackmold resistance QTL alleles from wild Lycopersicon cheesmanii to cultivated tomato (L. esculentum) and evaluation of QTL phenotypic effects

Blackmold, caused by the fungus Alternaria alternata, is a major ripe fruit disease of processing tomatoes. Previously, we found blackmold resistance in a wild tomato (Lycopersicon cheesmanii) and quantitative trait loci (QTL) for resistance were mapped in an interspecific population. Five QTLs were selected for introgression from L. cheesmanii into cultivated tomato using marker-assisted selection (MAS). Restriction fragment length polymorphism and PCR-based markers flanking, and within, the chromosomal regions containing QTLs were used for MAS during backcross and selfing generations. BC₁ plants heterozygous at the QTLs, and subsequent BC₁S₁ and BC₁S₂ lines possessing different homozygous combinations of alleles at the target QTLs, were identified using DNA markers. Field experiments were conducted in 1998 (with 80 marker-selected BC₁S₂ lines) and 1999 (with 151 marker-selected BC₁S₂ and BC₁S₃ lines) at three California locations. Blackmold resistance was assessed during both years, and horticultural traits were evaluated in 1999. The BC₁S₂ and BC₁S₃ lines containing L. cheesmanii alleles at the QTLs were associated with a large genetic variance for resistance to blackmold and moderate heritability, suggesting that significant genetic gain may be achieved by selection in this genetic material. L. cheesmanii alleles at three of the five introgressed QTLs showed a significant, positive effect on blackmold resistance. A QTL on chromosome 2 had the largest positive effect on blackmold resistance, alone and in combination with other QTLs, and was also associated with earliness, a positive horticultural trait. The other four QTLs were associated primarily with negative horticultural traits. Fine mapping QTLs using near isogenic lines could help determine if such trait associations are due to linkage drag or pleiotropy.