Patent Application
20060288433
Pursuant to 37 C.F.R. §1.52(e)(1)(iii), a compact disc containing an electronic version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. A second compact disc is submitted and is an identical copy of the first compact disc. The discs are labeled “copy 1” and “copy 2,” respectively, and each disc contains one file entitled “2183-7695US sequence listing.ST25.txt” which is 170 KB and created on Jul. 17, 2006.
The invention relates generally to biotechnology and, more particularly, to methods for selecting breeding animals or animals destined for slaughter having desired genotypic or potential phenotypic properties, in particular, related to muscle mass and/or fat deposition.
Breeding schemes for domestic animals have so far focused on farm performance traits and carcass quality. This has resulted in substantial improvements in traits like reproductive success, milk production, lean/fat ratio, prolificacy, growth rate and feed efficiency. Relatively simple performance test data have been the basis for these improvements, and selected traits were assumed to be influenced by a large number of genes, each of small effect (the infinitesimal gene model). There are now some important changes occurring in this area. First, the breeding goal of some breeding organizations has begun to include meat quality attributes in addition to the “traditional” production traits. Secondly, evidence is accumulating that current and new breeding goal traits may involve relatively large effects (known as major genes), as opposed to the infinitesimal model that has been relied on so far.
Modern DNA-technologies provide the opportunity to exploit these major genes, and this approach is a very promising route for the improvement of meat quality, especially since direct meat quality assessment is not viable for potential breeding animals. Also for other traits such as lean/fat ratio, growth rate and feed efficiency, modern DNA technology can be very effective. Also these traits are not always easy to measure in the living animal.
The evidence for several of the major genes was originally obtained using segregation analysis, i.e., without any DNA marker information. Afterwards, molecular studies were performed to detect the location of these genes on the genetic map. In practice, and except for alleles of very large effect, DNA studies are required to dissect the genetic nature of most traits of economic importance. DNA markers can be used to localize genes or alleles responsible for qualitative traits like coat color, and they can also be used to detect genes or alleles with substantial effects on quantitative traits like growth rate, IMF etc. In this case, the approach is referred to as QTL (quantitative trait locus) mapping, wherein a QTL comprises at least a part of the nucleic acid genome of an animal where genetic information capable of influencing the quantitative trait (in the animal or in its offspring) is located. Information at DNA level can not only help to fix a specific major gene in a population, but also assist in the selection of a quantitative trait which is already selected for. Molecular information in addition to phenotypic data can increase the accuracy of selection and therefore the selection response.
Improving meat quality or carcass quality is not just about changing levels of traits like tenderness or marbling, but it is also about increasing uniformity. The existence of major genes provides excellent opportunities for improving meat quality because it allows large steps to be made in the desired direction. Secondly, it will help to reduce variation, since we can fix relevant genes in our products. Another aspect is that selecting for major genes allows differentiation for specific markets. Studies are underway in several species, particularly, pigs, sheep, deer and beef cattle.
In particular, intense selection for meat production has resulted in animals with extreme muscularity and leanness in several livestock species. In recent years, it has become feasible to map and clone several of the genes causing these phenotypes, paving the way towards more efficient marker-assisted selection, targeted drug development (performance enhancing products) and transgenesis. Mutations in the ryanodine receptor (Fuji et al., 1991; MacLennan and Phillips, 1993) and myostatin (Grobet et al., 1997; Kambadur et al., 1997; McPherron and Lee, 1997) have been shown to cause muscular hypertrophies in pigs and cattle respectively, while genes with major effects on muscularity and/or fat deposition have, for instance, been mapped to pig chromosome 4 (Andersson et al., 1994) and sheep chromosome 18 (Cocket et al., 1996).
However, although there have been successes in identifying QTLs, the information is currently of limited use within commercial breeding programs. Many workers in this field conclude that it is necessary to identify the particular genes underlying the QTL. This is a substantial task, as the QTL region is usually relatively large and may contain many genes. Identification of the relevant genes from the many that may be involved thus remains a significant hurdle in farm animals.
In one aspect, the invention provides a method for selecting a domestic animal for having desired genotypic or potential phenotypic properties comprising testing the animal for the presence of a parentally imprinted qualitative or quantitative trait locus (QTL). Herein, a domestic animal is defined as an animal being selected or having been derived from an animal having been selected for having desired genotypic or potential phenotypic properties.
Domestic animals provide a rich resource of genetic and phenotypic variation; traditionally, domestication involves selecting an animal or its offspring for having desired genotypic or potential phenotypic properties. This selection process has in the past century been facilitated by a growing understanding and utilization of the laws of Mendelian inheritance. One of the major problems in breeding programs of domestic animals is the negative genetic correlation between reproductive capacity and production traits. This is, for example, the case in cattle (a high milk production generally results in slim cows and bulls) poultry, broiler lines have a low level of egg production and layers have generally very low muscle growth), pigs (very prolific sows are, in general, fat and have comparatively less meat) or sheep (high prolific breeds have low carcass quality and vice versa). The invention now provides that knowledge of the parental imprinting character of various traits allows selection of, for example, sire lines, homozygous for a paternally imprinted QTL, for example, linked with muscle production or growth; the selection for such traits can thus be less stringent in dam lines in favor of the reproductive quality. The phenomenon of genetic or parental imprinting has never been utilized in selecting domestic animals, it was never considered feasible to employ this elusive genetic characteristic in practical breeding programs. The invention provides a breeding program, wherein knowledge of the parental imprinting character of a desired trait, as demonstrated herein, results in a breeding program, for example in a BLUP program, with a modified animal model. This increases the accuracy of the breeding value estimation and speeds up selection compared to conventional breeding programs. Until now, the effect of a parentally imprinted trait in the estimation of a conventional BLUP program was neglected; using and understanding the parental character of the desired trait, as provided by the invention, allows selecting on parental imprinting, even without DNA testing. For example, selecting genes characterized by paternal imprinting is provided to help increase uniformity; a (terminal) parent homozygous for the “good or wanted” alleles will pass them to all offspring, regardless of the other parent's alleles, and the offspring will all express the desired parent's alleles. This results in more uniform offspring. Alleles that are interesting or favorable from the maternal side are often the ones that have opposite effects to alleles from the paternal side. For example, in meat animals, such as pigs, alleles linked with meat quality traits, such as intra-muscular fat or muscle mass, could be fixed in the dam lines while alleles linked with reduced back fat could be fixed in the sire lines. Other desirable combinations are, for example, fertility and/or milk yield in the female line with growth rates and/or muscle mass in the male lines.
In a preferred embodiment, the invention provides a method for selecting a domestic animal for having desired genotypic or potential phenotypic properties comprising testing a nucleic acid sample from the animal for the presence of a parentally imprinted quantitative trait locus (QTL). A nucleic acid sample can, in general, be obtained from various parts of the animal's body by methods known in the art. Traditional samples for the purpose of nucleic acid testing are blood samples or skin or mucosal surface samples, but samples from other tissues can be used as well, particularly, sperm samples, oocyte or embryo samples can be used. In such a sample, the presence and/or sequence of a specific nucleic acid, be it DNA or RNA, can be determined with methods known in the art, such as hybridization or nucleic acid amplification or sequencing techniques known in the art. The invention provides testing such a sample for the presence of nucleic acid wherein a QTL or allele associated therewith is associated with the phenomenon of parental imprinting, for example where it is determined whether a paternal or maternal allele of the QTL is capable of being predominantly expressed in the animal.
The purpose of breeding programs in livestock is to enhance the performances of animals by improving their genetic composition. In essence, this improvement accrues by increasing the frequency of the most favorable alleles for the genes influencing the performance characteristics of interest. These genes are referred to as QTL. Until the beginning of the nineties, genetic improvement was achieved via the use of biometrical methods, but without molecular knowledge of the underlying QTL.
Since the beginning of the Nineties, and due to recent developments in genomics, it is conceivable to identify the QTL underlying a trait of interest. The invention now provides identifying and using parentally imprinted QTLs which are useful for selecting animals by mapping quantitative trait loci. Again, the phenomenon of genetic or paternal imprinting has never been utilized in selecting domestic animals, it was never considered feasible to employ this elusive genetic characteristic in practical breeding programs. For example, Kovacs and Kloting (Biochem. Mol. Biol. Int. 44:399-405, 1998), where parental imprinting is not mentioned, and not suggested, found linkage of a trait in female rats, but not in males, suggesting a possible sex specificity associated with a chromosomal region, which of course excludes parental imprinting, a phenomenon wherein the imprinted trait of one parent is preferably but gender-aspecifically expressed in his or her offspring.
The invention provides the initial localization of a parentally imprinted QTL on the genome by linkage analysis with genetic markers, and the actual identification of the parentally imprinted gene(s) and causal mutations therein. Molecular knowledge of such a parentally imprinted QTL allows for more efficient breeding designs herewith provided. Applications of molecular knowledge of parentally imprinted QTLs in breeding programs include: marker-assisted segregation analysis to identify the segregation of functionally distinct parentally imprinted QTL alleles in the populations of interest, marker-assisted selection (MAS) performed within lines to enhance genetic response by increasing selection accuracy, selection intensity or by reducing the generation interval using the understanding of the phenomenon of parental imprinting, marker-assisted introgression (MAI) to efficiently transfer favorable parentally imprinted QTL alleles from a donor to a recipient population, genetic engineering of the identified parentally QTL and genetic modification of the breeding stock using transgenic technology, development of performance enhancing products using targeted drug development exploiting molecular knowledge of the QTL.
The inventors undertook two independent experiments to determine the practical use of parental imprinting of a QTL.
In a first experiment, performed in a previously described Piétrain×Large White intercross, the likelihood of the data was computed under a model of paternal (paternal allele only expressed) and maternal imprinting (maternal allele only expressed) and compared with the likelihood of the data under a model of a conventional “Mendelian” QTL. The results strikingly demonstrated that the QTL was indeed paternally expressed, the QTL allele (Piétrain or Large White) inherited from the F1 sow having no affect whatsoever on the carcass quality and quantity of the F2 offspring. It was seen that very significant lodscores were obtained when testing for the presence of a paternally expressed QTL, while there was no evidence at all for the segregation of a QTL when studying the chromosomes transmitted by the sows. The same tendency was observed for all traits showing that the same imprinted gene is responsible for the effects observed on the different traits. Table 1 reports the maximum likelihood (ML) phenotypic means for the F2 offspring sorted by inherited paternal QTL allele.
In a second experiment performed in the Wild Boar X Large White intercross, QTL analyses of body composition, fatness, meat quality, and growth traits was carried out with the chromosome 2 map using a statistical model testing for the presence of an imprinting effect. Clear evidence for a paternally expressed QTL located at the very distal tip of 2p was obtained (
Understanding the parent-of-origin effect characterizing a QTL allows for its optimal use in breeding programs. Indeed, marker-assisted segregation analysis under a model of parental imprinting will yield better estimates of QTL allele effects. Moreover, it allows for the application of specific breeding schemes to optimally exploit a QTL. In one embodiment of the invention, the most favorable QTL alleles would be fixed in breeding animal lines and, for example, used to generate commercial, crossbred males by marker-assisted selection (MAS, within lines) and marker-assisted introgression (MAI, between lines). In another embodiment, the worst QTL alleles would be fixed in the animal lines used to generate commercial crossbred females by MAS (within lines) and MAI (between lines).
In a preferred embodiment of the invention, the animal is a pig. Note, for example, that the invention provides the insight that today half of the offspring from commercially popular PiétrainX Large White crossbred boars inherit an unfavorable Large White muscle mass QTL as provided by the invention causing considerable loss, and the invention now, for example, provides the possibility to select the better half of the population in that respect. However, it is also possible to select commercial sow lines enriched with the boar's unfavorable alleles, allowing to equip the sows with other alleles that are more desirable, for example, for reproductive purposes.
In a preferred embodiment of a method provided by the invention, the QTL is located at a position corresponding to a QTL located at chromosome 2 in the pig. For example, it is known from comparative mapping data between pig and human, including bidirectional chromosome painting, that SSC2p is homologous to HSA11pter-q13.11,12 HSA11pter-q13 is known to harbor a cluster of imprinted genes: IGF2, INS2, H19, MAH2, p57KIP2, KvLQTL1, Tapa1,/CD81, Orctl2, Impt1 and Ip1. The cluster of imprinted genes located in HSA11pter-q13 is characterized by eight maternally expressed genes H19, MASH2, p57KIP2, KVLQTL1, TAPA1/CD81, ORCTL2, IMPT1 and IP1, and two paternally expressed genes: IGF2 and INS. However, Johanson et al. (Genomics 25:682-690, 1995) and Reik et al. (Trends in Genetics 13:330-334, 1997) show that the whereabouts of these loci in various animals are not clear. For example, the HSA11 and MMU7 loci do not correspond among each other, the MMU7 and the SSC2 loci do not correspond, whereas the HSA11 and SSC2 loci seem to correspond, and no guidance is given where one or more of, for example, the above-identified parentally expressed individual genes are localized on the three species' chromosomes.
Other domestic animals, such as cattle, sheep, poultry and fish, having similar regions in their genome harboring such a cluster of imprinted genes or QTLs, the invention herewith provides use of these orthologous regions of other domestic animals in applying the phenomenon of parental imprinting in breeding programs. In pigs, the cluster is mapped at around position 2p1.7 of chromosome 2, however, a method as provided by the invention employing (fragments of) the maternally or paternally expressed orthologous or homologous genes or QTLs are advantageously used in other animals as well for breeding and selecting purposes. For example, a method is provided wherein the QTL is related to the potential muscle mass and/or fat deposition, preferably with limited effects on other traits, such as meat quality and daily gain of the animal, or wherein the QTL comprises at least a part of an insulin-like growth factor-2 (IGF2) allele. Reik et al. (Trends in Genetics 13:330-334, 1997) explain that this gene in humans is related to Beckwith-Wiedemann syndrome, an apparently parentally imprinted disease syndrome most commonly seen with human fetuses, where the gene has an important role in prenatal development. No relationship is shown or suggested with postnatal development relating to muscle development or fatness in (domestic) animals.
In a preferred embodiment, the invention provides a method for selecting a pig for having desired genotypic or potential phenotypic properties comprising testing a sample from the pig for the presence of a quantitative trait locus (QTL) located at a Sus scrofa chromosome 2 mapping at position 2p1.7. In particular, the invention relates to the use of genetic markers for the telomeric end of pig chromosome 2p in marker selection (MAS) of a parentally imprinted Quantitative Trait Locus (QTL) affecting carcass yield and quality in pigs. Furthermore, the invention relates to the use of genetic markers associated with the IGF2 locus in MAS in pigs, such as polymorphisms and microsatellites and other characterizing nucleic acid sequences shown herein, such as shown in FIGS. 4 to 10. In a preferred embodiment, the invention provides a QTL located at the distal tip of Sus scrofa chromosomes 2 with effects on varies measurements of carcass quality and quantity, particularly muscle mass and fat deposition.
In a first experiment, a QTL mapping analysis was performed in a Wild Boar X Large White intercross counting 200 F2 individuals. The F2 animals were sacrificed at a live eight of at least 80 kg or at a maximum age of 190 days. Phenotypic data on birth weight, growth, fat deposition, body composition, weight of internal organs, and meat quality were collected; a detailed description of the phenotypic traits are provided by Andersson et al.1 and Andersson-Eklund et al.4
A QTL (without any significant effect on back-fat thickness) at an unspecified locus on the proximal end of chromosome 2 with moderate effect on muscle mass, and located about 30 cm away from the parentally imprinted QTL reported here, was previously reported by the inventors; whereas the QTL as now provided has a very large effect, explaining at least 20-30% of variance, making the QTL of the present invention commercially very attractive, which is even more so because the present QTL is parentally imprinted. The marker map of chromosome 2p was improved as part of this invention by adding microsatellite markers in order to cover the entire chromosome arm. The following microsatellite markers were used: Swc9, Sw2443, Sw2623, and Swr2516, all from the distal end of 2p.7 QTL analyses of body composition, fatness, meat quality, and growth traits were carried out with the new chromosome 2 map. Clear evidence for a QTL located at the very distal tip of 2p was obtained (
In a second experiment, QTL mapping was performed in a Piétrain X Large White intercross comprising 1125 F2 offspring. The Large White and Piétrain parental breeds differ for a number of economically important phenotypes. Piétrains are famous for their exceptional muscularity and leanness10 (
These experiments therefore clearly indicated the existence of a QTL with major effect on carcass quality and quantity on the telomeric end of pig chromosome arm 2p; the likely existence of an allelic series at this QTL with at least three alleles: Wild-Boar<Large White<Piétrain, and possibly more given the observed segregation within the Piétrain breed.
The effects of the identified QTL on muscle mass and fat deposition are truly major, being of the same magnitude of those reported for the CRC locus though apparently without the associated deleterious effects on meat quality. We estimate that both loci jointly explain close to 50% of the Piétrain versus Large White breed difference for muscularity and leanness. The QTL had very large effects on lean meat content in ham and explained an astonishing 30% of the residual phenotypic variance in the F2 population. Large effects on the area of the longissumus dorsi muscle, on the weight of the heart, and on back-fat thickness (subcutaneous fat) were also noted. A moderate effect on one meat quality trait, reflectance value, was indicated. The QTL had no significant effect on abdominal fat, birth weight, growth, weight of liver, kidney, or spleen (data not shown). The Large White allele at this QTL, when compared to the Wild Boar allele, was associated with larger muscle mass and reduced back-fat thickness consistent with the difference between this breed and the Wild Boar population. The strong imprinting effect observed for all affected traits shows that a single causative locus is involved. The pleiotropic effects on skeletal muscle mass and the size of the heart appear adaptive from a physiological point of view as a larger muscle mass requires a larger cardiac output.
In a further embodiment, the invention provides a method for selecting a pig for having desired genotypic or potential phenotypic properties comprising testing a sample from the pig for the presence of a quantitative trait locus (QTL) located at a Sus scrofa chromosome 2 mapping at position 2p1.7., wherein the QTL comprises at least a part of a Sus scrofa insulin-like growth factor-2 (IGF2) allele or a genomic area closely related thereto, such as polymorphisms and microsatellites and other characterizing nucleic acid sequences shown herein, such as shown in FIGS. 4 to 10. The important role of IGF2 for prenatal development is well-documented from knock-out mice as well as from its causative role in the human Beckwith-Wiedemann syndrome. This invention demonstrates an important role for the IGF2-region also for postnatal development.
To show the role of Igf2, the inventors performed the following three experiments:
A genomic IGF2 clone was isolated by screening a porcine BAC library. FISH analysis with this BAC clone gave a strong consistent signal on the terminal part of chromosome 2p.
A polymorphic microsatellite is located in the 3′UTR of IGF2 in mice (GenBank U71085), humans (GenBank S62623), and horse (GenBank AF020598). The possible presence of a corresponding porcine microsatellite was investigated by direct sequencing of the IFG2 3′UTR using the BAC clone. A complex microsatellite was identified about 800bp downstream of the stop codon; a sequence comparison revealed that this microsatellite was identical to a previously described anonymous microsatellite, Swc9.6 This marker was used in the initial QTL mapping experiments and its location on the genetic map correspond with the most likely position of the QTL both in the Piétrain X Large White and in the Large White x Wild Boar pedigree.
Analysis of skeletal muscle and liver cDNA from ten-week-old fetuses heterozygous for a nt241 (G-A) transversion in the second exon of the porcine IGFII gene and SWC9, shows that the IGFII gene is imprinted in these tissues in the pig as well and only expressed from the paternal allele.
Based on a published porcine adult liver cDNA sequence,16 the inventors designed primer pairs allowing amplification of the entire IgfII coding sequence with 222 bp of leader and 280 bp of trailer sequence from adult skeletal muscle cDNA. Piétrain and Large White RT-PCR products were sequenced indication that the coding sequences are identical in both breeds and with the published sequence. However, a GA transition was found in the leader sequence corresponding to exon 2 in man. Following conventional nomenclature, this polymorphism will be referred to as nt241(G-A). We developed a screening test for this single nucleotide polymorphism 9(SNP) based on the ligation amplification reaction (LAR), allowing us to genotype our pedigree material. Based on these data, IgfII was shown to colocalize with the SWC9 microsatellite marker (θ=0%), therefore virtually coinciding with the most likely position of the QTL, and well within the 95% support interval for the QTL. Subsequent sequence analysis demonstrated that the microsatellite marker SWC9 is actually located within the 3′UTR of the IgfII gene.
As previously mentioned, the knowledge of this QTL provides a method for the selection of animals such as pigs with improved carcass merit. Different embodiments of the invention are envisaged, including: marker-assisted segregation analysis to identify the segregation of functionally distinct QTL alleles in the populations of interest; marker-assisted selection (MAS) performed within lines to enhance genetic response by increasing selection accuracy, selection intensity or by reducing the generation interval; marker-assisted introgression (MAI) to efficiently transfer favorable QTL alleles from a donor to a recipient population, thereby enhancing genetic response in the recipient population. Implementation of embodiments marker-assisted segregation analysis, selection (MAS) and introgression (MAI), can be performed using genetic markers that are linked to the QTL; genetic markers that are in linkage disequilibrium with the QTL, the actual causal mutations within the QTL.
As indicated above, the insulin-like growth factor 2 (IGF2) gene was mapped to the distal tip of the short arm on chromosome 2 in swine. Gene mapping studies indicated that this paternally expressed QTL at the IGF2 gene region has a large effect on back fat thickness and carcass leanness (e.g., Jeon et al. (1999), Nature Genetics 21, 157-158; Nezer et al. (1999), Nature Genetics 21, 155-156). Recently, a mutation in the regulatory region of the IGF2 gene has been identified to be the cause underlying the QTL effect on muscle growth and fat deposition (Van Laere et al. (2003) Nature 425:832-836). This single nucleotide substitution (G-A), located at position 3072 in the intron 3 of IGF2 gene, increases gene expression of IGF2 in muscle threefold, stimulates muscle growth at the expense of back fat and results in leaner swine carcass and lower back fat.
The large effect of the QTL on lean meat and back fat without influence on growth or meat quality, makes this an attractive QTL to use in the breeding program. Terminal sires have been selected to be homozygous for the lean allele (AA) in order to pass the full effect to their offspring. Field results have been reported by several authors (Scheller et al. (2002) Proceedings of the 27th Annual National Swine Improvement Federation, Des Moines, Iowa, USA; Buys, N. (2003) Proceedings of the 28th Annual National Swine Improvement Federation, Des Moines, Iowa, USA, pp 146-149).
It is generally believed that prolificacy and sow longevity is reducing as a result of the genetic selection for increased leanness and lowering fat deposition (See, e.g., P. Mathur and Y. Liu (2003), Proceedings of the 28th Annual National Swine Improvement Federation, Des Moines, Iowa, USA, pp 155-163). Body fat deposition is necessary to sustain sow reproduction performance, for example, to support adequate milk production and to limit body weight loss. The selection for leaner carcasses, demanded by the packing industry and consumers, may conflict with the prolificacy and longevity of the sow and lead to increased replacement costs of sows in pig production. The QTN (quantitative trait nucleotide) in the IGF2 gene might provide a possibility to overcome this conflict. The imprinting character of the gene might be used to produce lean slaughter pigs from fatter dams, that inherited the wild type allele from their father (genotype of Grand parent boar=GG), crossed with terminal sires being homozygous for the lean allele (AA). The objective of this experiment was to investigate a possible effect of the QTN at the IGF2 gene on prolificacy and longevity.
The details of the experiment are described in Example 5. It was found that sows that inherited the wild type allele from their father had significantly more piglets born alive, total born and weaned, while there was no effect on stillborn piglets (See, Example 5, Table 4). No effect on any of these prolificacy data could be observed when data were analyzed according to the allele inherited from the mother (maternal allele). Also, if sows from heterozygous dams were taken into account and grouped according to the maternal allele, again no significant effect on prolificacy could be observed, which was expected since the maternal allele is not expressed.
The parity or average number of cycles per sow was also found to be higher in sows that inherited G from their father as compared to those that received the A allele, which points to a beneficial effect on longevity. This is related to higher litter size, since that is a major criterion for elimination in the selection program.
Thus, the IGF2-intron3 G3072A mutation (herein also referred to as IGF2+ or A-allele; the wild type being the igf2− or G-allele) has an influence on prolificacy and longevity in sows, which allows for the possibility to use the same imprinted QTN for different selection in sire and dam lines. Terminal sires should be homozygous for the lean allele to give uniform and lean slaughter pigs, while dam lines can benefit from a selection for the wild type allele since this has a beneficial effect on prolificacy and longevity. Because of the imprinted character of the gene, selection for the fatter allele in sow lines will not influence the carcass quality of the offspring.
Thus, the invention provides a method for selecting a domestic animal for having desired genotypic properties comprising testing the animal for the presence of a parentally imprinted quantitative trait locus (QTL) or a mutation therein and to the use of an isolated and/or recombinant nucleic acid comprising a parentally imprinted quantitative trait locus (QTL) or a mutation therein or functional fragment derived thereof to select a breeding animal or animal destined for slaughter having desired genotypic or potential phenotypic properties. The test may, for instance, comprise testing a sample from the pig for the presence of a quantitative trait locus (QTL) located at a Sus scrofa chromosome 2 mapping at position 2p1.7., wherein the QTL is paternally expressed, i.e., is expressed from the paternal allele.
In particular, the genotypic or potential phenotypic properties are selected from the group consisting of muscle mass, fat deposition, lean meat, lean back fat, sow prolificacy and sow longevity. In particular, improved sow prolificacy may include such phenotypic expressions as higher teat number, more piglets born alive, higher litter size, higher number of total born and weaned piglets with no effect on stillborn piglets. Improved sow longevity may in particular include such phenotypic expressions as parity or average number of cycles per sow.
Thus, the above-described IGF2 mutation influencing lean meat also influences a number of other positive traits and allows for marker-assisted selection in opposite directions in sire and dam lines due to the parentally imprinting character of the mutation. The mutation increases muscle mass at the expense of back fat with on average 2-4% more lean meat. This effect on leanness is of the same magnitude as reported for the Halothane gene but without any of the well-known deleterious effects on meat quality and animal health. Homozygous positive terminal sires (IGF2+/IGF2+) will pass the full effect to the slaughter pigs, regardless of the genotype of the mother. Furthermore, such selection principle allows for the possibility to push a far higher proportion of lower grading pigs into the higher payment categories. The experiment described in Example 5 shows that parent sows benefit from inheriting the negative gene (igf2−) from their father: they are more prolific and have an increased longevity. Parent sows are fatter but this will have no effect on the carcass quality of the slaughter pig (see illustration in Example 5).
In a further embodiment, the invention provides a method for selecting a pig for having desired genotypic or potential phenotypic properties comprising testing a sample from the pig for the presence of a quantitative trait locus (QTL) located at a Sus scrofa chromosome 2 mapping at position 2p1.7., wherein the QTL is paternally expressed, i.e., is expressed from the paternal allele. In man and mouse, Igf2 is known to be imprinted and to be expressed exclusively from the paternal allele in several tissues. Analysis of skeletal muscle cDNA from pigs heterozygous for the SNP and/or SWC9, shows that the same imprinting holds in the pig as well. Understanding the parent-of-origin effect characterizing the QTL as provided by the invention now allows for its optimal use in breeding programs. Indeed, today half of the offspring from commercially popular Piétrain x Large White crossbred boars inherit the unfavorable Large White allele causing considerable loss. Using a method as provided by the invention avoids this problem.
The invention furthermore provides an isolated and/or recombinant nucleic acid or functional fragment derived thereof comprising a parentally imprinted quantitative trait locus (QTL) or fragment thereof capable of being predominantly expressed by one parental allele. Having such a nucleic acid as provided by the invention available allows constructing transgenic animals wherein favorable genes are capable of being exclusively or predominantly expressed by one parental allele, thereby equipping the offspring of the animal homozygous for a desired trait with desired properties related to that parental allele that is expressed.
In a preferred embodiment, the invention provides an isolated and/or recombinant nucleic acid or fragment derived thereof comprising a synthetic parentally imprinted quantitative trait locus (QTL) or functional fragment thereof derived from at least one chromosome. “Synthetic” herein describes a parentally expressed QTL wherein various elements are combined that originate from distinct locations from the genome of one or more animals. The invention provides recombinant nucleic acid wherein sequences related to parental imprinting of one QTL are combined with sequences relating to genes or favorable alleles of a second QTL. Such a gene construct is favorably used to obtain transgenic animals wherein the second QTL has been equipped with paternal imprinting, as opposed to the inheritance pattern in the native animal from which the second QTL is derived. Such a second QTL can, for example, be derived from the same chromosome where the parental imprinting region is located, but can also be derived from a different chromosome from the same or even a different species. In the pig, such a second QTL can, for example, be related to an estrogen receptor (ESR)-gene (Rothschild et al., PNAS 93, 201-201, 1996) or a FAT-QTL (Andersson, Science 263, 1771-1774, 1994) for example derived from another pig chromosome, such as chromosome 4. A second or further QTL can also be derived from another (domestic) animal or a human.
The invention furthermore provides an isolated and/or recombinant nucleic acid or functional fragment derived thereof at least partly corresponding to a QTL of a pig located at a Sus scrofa chromosome 2 mapping at position 2p1.7 wherein the QTL is related to the potential muscle mass and/or fat deposition of the pig and/or wherein the QTL comprises at least a part of a Sus scrofa insulin-like growth factor-2 (IGF2) allele, preferably at least spanning a region between INS and H19, or preferably derived from a domestic pig, such as a Piétrain, Meishan, Duroc, Landrace or Large White, or from a Wild Boar. For example, a genomic IGF2 clone was isolated by screening a porcine BAC library. FISH analysis with this BAC clone gave a strong consistent signal on the terminal part of chromosome 2p. A polymorphic microsatellite is located in the 3′UTR of IGF2 in mice (GenBank U71085), humans (GenBank S62623), and horse (GenBank AF020598). The possible presence of a corresponding porcine microsatellite was investigated by direct sequencing of the IGF2 3′UTR using the BAC clone. A complex microsatellite was identified about 800 bp downstream of the stop codon; a sequence comparison revealed that this microsatellite is identical to a previously described anonymous microsatellite, Swc9. PCR primers were designed and the microsatellite (IGF2ms) was found to be highly polymorphic with three different alleles among the two Wild Boar founders and another two among the eight Large White founders. IGF2ms was fully informative in the intercross as the breed of origin as well as the parent of origin could be determined with confidence for each allele in each F2 animal.
A linkage analysis using the intercross pedigree was carried out with IGF2ms and the microsatellites Sw2443, Sw2623, and Swr2516, all from the distal end of 2p.7 IGF2 was firmly assigned to 2p by highly significant lod scores (e.g., Z=89.0, θ=0.003 against Swr2516). Multipoint analyses, including previously typed chromosome 2 markers, revealed the following order of loci (sex-average map distances in Kosambi cM): Sw2443/Swr2516-0.3-IGF2-14.9-Sw2623-10.3-Sw256. No recombinant was observed between Sw2443 and Swr2516, and the suggested proximal location of IGF2 in relation to these loci is based on a single recombinant giving a lod score support of 0.8 for the reported order. The most distal marker in our previous QTL study, Sw256, is located about 25 cM from the distal end of the linkage group.
The invention furthermore provides use of a nucleic acid or functional fragment derived thereof according to the invention in a method according to the invention. In a preferred embodiment, use of a method according to the invention is provided to select a breeding animal or animal destined for slaughter, or embryos or semen derived from these animals for having desired genotypic or potential phenotypic properties. In particular, the invention provides such use wherein the properties are related to muscle mass and/or fat deposition. The QTL as provided by the invention may be exploited or used to improve, for example, lean meat content or back-fat thickness by marker-assisted selection within populations or by marker-assisted introgression of favorable alleles from one population to another. Examples of marker-assisted selection using the QTL as provided by the invention are use of marker-assisted segregation analysis with linked markers or with markers in disequilibrium to identify functionally distinct QTL alleles. Furthermore, identification of a causative mutation in the QTL is now possible, again leading to identifying functionally distinct QTL alleles. Such functionally distinct QTL alleles located at the distal tip of chromosome 2p with large effects on skeletal muscle mass, the size of the heart, and on back-fat thickness are also provided by the invention. The observation of a similar QTL effect in a Large White x Wild Boar as well as in a Piétrain x Large White intercross provides proof of the existence of a series of at least three distinct functional alleles. Moreover, preliminary evidence based on marker-assisted segregation analysis points towards residual segregation at this locus within the Piétrain population (data not shown). The occurrence of an allelic series as provided by the invention allows identifying causal polymorphisms which—based on the quantitative nature of the observed effect—are unlikely to be gross gene alterations but rather subtle regulatory mutations. The effects on muscle mass of the three alleles rank in the same order as the breeds in which they are found, e.g., Piétrain pigs are more muscular than Large White pigs that in turn have higher lean meat content than Wild Boars. The invention furthermore provides use of the alleles as provided by the invention for within line selection or for marker-assisted introgression using linked markers, markers in disequilibrium or alleles comprising causative mutations.
The invention furthermore provides an animal selected by using a method according to the invention. For example, a pig characterized in being homozygous for an allele in a QTL located at a Sus scrofa chromosome 2 mapping at position 2p1.7 can now be selected and is thus provided by the invention. Since the QTL is related to the potential muscle mass and/or fat deposition of the pig and/or the QTL comprises at least a part of a Sus scrofa insulin-like growth factor-2 (IGF2) allele, it is possible to select promising pigs to be used for breeding or to be slaughtered. In particular, an animal according to the invention which is a male is provided. Such a male, or its sperm or an embryo derived thereof, can advantageously be used in breeding animals for creating breeding lines or for finally breeding animals destined for slaughter. In a preferred embodiment of such use as provided by the invention, a male, or its sperm, deliberately selected for being homozygous for an allele causing the extreme muscular hypertrophy and leanness, is used to produce offspring heterozygous for such an allele. Due to the allele's paternal expression, the offspring will also show the favorable traits for example related to muscle mass, even if the parent female has a different genetic background. Moreover, it is now possible to positively select the female(s) for having different traits, for example related to fertility, without having a negative effect on the muscle mass trait that is inherited from the allele from the selected male. For example, earlier such males could occasionally be seen with Piétrain pigs but, genetically, it was not understood how to most profitably use these traits in breeding programs.
Furthermore, the invention provides a transgenic animal, sperm and an embryo derived thereof, comprising a synthetic parentally imprinted QTL or functional fragment thereof as provided by the invention, i.e., it is provided by the invention to introduce a favorable recombinant allele; for example, to introduce the estrogen receptor locus related to increased litter size of an animal homozygously in a parentally imprinted region of a grandparent animal (for example, the father of a hybrid sow if the region was paternally imprinted and the grandparent was a boar); to introduce a favorable fat-related allele or muscle mass-related recombinant allele in a paternally imprinted region, and so on. Recombinant alleles that are interesting or favorable from the maternal side are often the ones that have opposite effects to alleles from the paternal side. For example, in meat animals, such as pigs, recombinant alleles linked with meat quality traits such as intramuscular fat or muscle mass could be fixed in the dam lines while recombinant alleles linked with reduced back fat could be fixed in the sire lines. Other desirable combinations are, for example, fertility and/or milk yield in the female line with growth rates and/or muscle mass in the male lines.
The invention is further explained in the detailed description without limiting the invention.
Methods
Isolation of an IGF2 BAC clone and fluorescent in situ hybridization (FISH). IGF2 primers (F:5′-GGCAAGTTCTTCCGCTAATGA-3′ (SEQ ID NO:1) and R:5′-GCACCGCAGAATTACGACAA-3′ (SEQ ID NO:2)) for PCR amplification of a part of the last exon and 3 UTR were designed on the basis of a porcine IGF2 cDNA sequence (GenBank X56094). The primers were used to screen a porcine BAC library and the clone 253G10 was isolated. Crude BAC DNA was prepared as described.24 The BAC DNA was linearized with EcoRV and purified with QIAEXII (QIAGEN GmbH, Germany). The clone was labeled with biotin-14-dATP using the GIBCO-BRL Bionick labeling system (BRL18246-015). Porcine metaphase chromosomes were obtained from pokeweed (Seromed) stimulated lymphocytes using standard techniques. The slides were aged for two days at room temperature and then kept at −20° C. until use. FISH analysis was carried out as previously described.25 The final concentration of the probe in the hybridization mix was 10 ng/μl. Repetitive sequences were suppressed with standard concentrations of porcine genomic DNA. After post-hybridization washing, the biotinylated probe was detected with two layers of avidin-FITC (Vector A-2011). The chromosomes were counterstained with 0.3 mg/ml DAPI (4,6-Diamino-2-phenylindole; Sigma D9542), which produced a G-banding like pattern. No post hybridization banding was needed, since chromosome 2 is easily recognized without banding. A total of 20 metaphase spreads were examined under an Olympus BX-60 fluorescence microscope connected to an IMAC-CCD S30 video camera and equipped with an ISIS 1.65 (Metasystems) software.
Sequence, microsatellite, and linkage analysis.
About two μg of linearized and purified BAC DNA was used for direct sequencing with 20 pmoles of primers and BigDye Terminator chemistry (Perkin Elner, USA). DNA sequencing was done from the 3′ end of the last exon towards the 3′ end of the UTR until a microsatellite was detected. A primer set (F:5′-GTTTCTCCTGTACCCACACGCATCCC-3′ (SEQ ID NO:3) and R:5′-Fluorescein-CTACAAGCTGGGCTCAGGG-3′ (SEQ ID NO:4)) was designed for the amplification of the IGF2 microsatellite, which is about 250 bp long and located approximately 800 bp downstream from the stop codon. The microsatellite was PCR amplified using fluorescently labeled primers and the genotyping was carried out using an ABI377 sequencer and the GeneScan/Genotyper software (Perkin Elmer, USA). Two-point and multipoint linkage analysis were done with the Cri-Map software.26
Animals and Phenotypic Data.
The intercross pedigree comprised two European Wild Boar males and eight Large White females, 4 F, males and 22 F, females, and 200 F2 progeny.1 The F2 animals were sacrificed at a live weight of at least 80 kg or at a maximum age of 190 days. Phenotypic data on birth weight, growth, fat deposition, body composition, weight of internal organs, and meat quality were collected; a detailed description of the phenotypic traits are provided by Andersson et al.1 and Andersson-Eklund et al.4
Statistical Analysis.
Interval mapping for the presence of QTL were carried out with a least squares method developed for the analysis of crosses between outbred lines.27 The method is based on the assumption that the two divergent lines are fixed for alternative QTL alleles. There are four possible genotypes in the F2 generation as regards the grandparental origin of the alleles at each locus. This makes it possible to fit three effects: additive, dominance, and imprinting.2 The latter is estimated as the difference between the two types of heterozygotes, the one receiving the Wild Boar allele through an F1 sire and the one receiving it from an F1 dam. An F-ratio was calculated using this model (with 3 d.f.) versus a reduced model without a QTL effect for each cM of chromosome 2. The most likely position of a QTL was obtained as the location giving the highest F-ratio. Genome-wise significance thresholds were obtained empirically by a permutation test28 as described.2 The QTL model including an imprinting effect was compared with a model without imprinting (with 1 d.f.) to test whether the imprinting effect was significant.
The statistical models also included the fixed effects and covariates that were relevant for the respective traits; see Andersson-Eklund et al.4 for a more detailed description of the statistical models used. Family was included to account for background genetic effects and maternal effects. Carcass weight was included as a covariate to discern QTL effects on correlated traits, which means that all results concerning body composition were compared at equal weights. Least-squares means for each genotype class at the IGF2 locus were estimated with a single point analysis using Procedure GLM of SAS;29 the model included the same fixed effects and covariates as used in the interval mapping analyses.
The QTL shows a clear parent of origin-specific expression and the map position coincides with that of the insulin-like growth factor II gene (IGF2), indicating IGF2 as the causative gene. A highly significant segregation distortion (excess of Wild Boar-derived alleles) was also observed at this locus. The results demonstrate an important effect of the IGF2 region on postnatal development and it is possible that the presence of a paternally expressed IGF2-linked QTL in humans and in rodent model organisms has so far been overlooked due to experimental design or statistical treatment of data. The study also has important implications for quantitative genetics theory and practical pig breeding.
IGF2 was identified as a positional candidate gene for this QTL due to the observed similarity between pig chromosome 2p and human chromosome 11p. A genomic IGF2 clone was isolated by screening a porcine BAC library. FISH analysis with this BAC clone gave a strong consistent signal on the terminal part of chromosome 2p (
A linkage analysis using the intercross pedigree was carried out with IGF2ms and the microsatellites Sw2443, Sw2623, and Swr2516, all from the distal end of 2p.7 IGF2 was firmly assigned to 2p by highly significant lod scores (e.g., Z=89.0, θ=0.003 against Swr2516). Multipoint analyses, including previously typed chromosome 2 markers,8 revealed the following order of loci (sex-average map distances in Kosambi cM): Sw2443/Swr2516-0.3-IGF2-14.9-Sw2623-10.3-Sw256. No recombinant was observed between Sw2443 and Swr2516, and the suggested proximal location of IGF2 in relation to these loci is based on a single recombinant giving a lod score support of 0.8 for the reported order. The most distal marker in our previous QTL study, Sw256, is located about 25 cM from the distal end of the linkage group.
QTL analyses of body composition, fatness, meat quality, and growth traits were carried out with the new chromosome 2 map using a statistical model testing for the possible presence of an imprinting effect as expected for IGF2. Clear evidence for a paternally expressed QTL located at the very distal tip of 2p was obtained (
The IGF2-linked QTL and the FAT1 QTL on chromosome 4.1,9 are by far the two loci with the largest effect on body composition and fatness segregating in this Wild Boar intercross. The IGF2 QTL controls primarily muscle mass whereas FAT1 has major effects on fat deposition including abdominal fat, a trait that was not affected by the IGF2 QTL (
A highly significant segregation distortion was observed in the IGF2 region (excess of Wild Boar-derived alleles) as shown in Table 1 (χ2=11.7, d.f.=2; P=0.003). The frequency of Wild Boar-derived IGF2 alleles was 59% in contrast to the expected 50% and there was twice as many “Wild Boar” as “Large White” homozygotes. This deviation was observed with all three loci at the distal tip and is thus not due to typing errors. The effect was also observed with other loci but the degree of distortion decreased as a function of the distance to the distal tip of the chromosome. Blood samples for DNA preparation were collected at 12 weeks of age and we are convinced that the deviation from expected Mendelian ratios was present at birth as the number of animals lost prior to blood sampling was not sufficient to cause a deviation of this magnitude. No other of the more than 250 loci analyzed in this pedigree show such a marked segregation distortion (L. Andersson, unpublished). The segregation distortion did not show an imprinting effect, as the frequencies of the two reciprocal types of heterozygotes were identical (Table 1). This does not exclude the possibility that the QTL effects and the segregation distortion are controlled by the same locus. The segregation distortion may be due to meiotic drive favoring the paternally expressed allele during gametogenesis, as the F, parents were all sired by Wild Boar males. Another possibility is that the segregation distortion may be due to codominant expression of the maternal and paternal allele in some tissues and/or during a critical period of embryo development. Biallelic IGF2 expression has been reported to occur to some extent during human developments10,11 and interestingly a strong influence of the parental species background on IGF2 expression was recently found in a cross between Mus musculus and Mus spretus.12 It is also interesting that a VNTR polymorphism at the insulin gene, which is very closely linked to IGF2, is associated with size at birth in humans.13 It is possible that the IGF2-linked QTL in pigs has a minor effect on birth weight but in our data it was far from significant (
This study is an advance in the general knowledge concerning the biological importance of the IGF2 locus. The important role of IGF2 for prenatal development is well-documented from knock-out mice,14 as well as from its causative role in the human Beckwith-Wiedemann syndrome.15 This study demonstrates an important role for the IGF2-region also for postnatal development. It should be stressed that our intercross between outbred populations is particularly powerful to detect QTL with a parent of origin-specific effect on a multifactorial trait. This is because multiple alleles (or haplotypes) are segregating and we could deduce whether a heterozygous F2 animal received the Wild Boar allele from the F1 male or female. It is quite possible that the segregation of a paternally expressed IGF2-linked QTL affecting a trait like obesity has been overlooked in human studies or in intercrosses between inbred rodent populations because of experimental design or statistical treatment of data. An imprinting effect cannot be detected in an intercross between two inbred lines as only two alleles are segregating at each locus. Our result has therefore significant bearings on the future analysis of the association between genetic polymorphism in the insulin-IGF2 region and Type I diabetes,16 obesity,17 and variation in birth weight13 in humans, as well as for the genetic dissection of complex traits using inbred rodent models. A major impetus for generating an intercross between the domestic pig and its wild ancestor was to explore the possibilities to map and identify major loci that have responded to selection. We have now showed that two single QTLs on chromosome 2 (this study) and 41,2 explain as much as one third of the phenotypic variance for lean meat content in the F2 generation. This is a gross deviation from the underlying assumption in the classical infinitesimal model in quantitative genetics theory namely that quantitative traits are controlled by an infinite number of loci each with an infinitesimal effect. If a large proportion of the genetic difference between two divergent populations (e.g., Wild Boar and Large White) is controlled by a few loci, one would assume that selection would quickly fix QTL alleles with large effects leading to a selection plateau. However, this is not the experience in animal breeding programs or selection experiments where good persistent long-term selection responses are generally obtained, provided that the effective population size is reasonably large.18 A possible explanation for this paradox is that QTL alleles controlling a large proportion of genetic differences between two populations may be due to several consecutive mutations; this may be mutations in the same gene or at several closely linked genes affecting the same trait. It has been argued that new mutations contribute substantially to long-term selection responses,19 but the genomic distribution of such mutations is unknown.
The search for a single causative mutation is the paradigm with regard to the analysis of genetic defects in mice and monogenic disorders in humans. We propose that this may not be the case for loci that have been under selection for a large number of generations in domestic animals, crops, or natural populations. This hypothesis predicts the presence of multiple alleles at major QTL. It gains some support from our recent characterization of porcine coat color variation. We have found that both the alleles for dominant white color and for black-spotting differ from the corresponding wild type alleles by at least two consecutive mutations with phenotypic effects at the KIT and MC1R loci, respectively.20, 21 In this context it is highly interesting that in the accompanying example we have identified a third allele at the IGF2-linked QTL. The effects on muscle mass of the three alleles rank in the same order as the breeds in which they are found i.e., Piétrain pigs are more muscular than Large White pigs that in turn have higher lean meat content than Wild Boars.
There are good reasons to decide that IGF2 is the causative gene for the now reported QTL. Firstly, there is a perfect agreement in map localization (
The results have several important implications for the pig breeding industry. They show that genetic imprinting is not an esoteric academic question but need to be considered in practical breeding programs. The detection of three different alleles in Wild Boar, Large White, and Piétrain populations indicates that further alleles at the IGF2-linked QTL segregate within commercial populations. The paternal expression of the QTL facilitates its detection using large paternal half-sib families as the female contribution can be ignored. The QTL is exploited to improve lean meat content by marker-assisted selection within populations or by marker-assisted introgression of favorable alleles from one population to another.
Methods
Pedigree material: The pedigree material utilized to map QTL was selected from a previously described Piétrain x Large White F2 pedigree comprising>1,800 individuals.6, 7 To assemble this F2 material, 27 Piétrain boars were mated to 20 Large White sows to generate an F1 generation comprising 456 individuals. 31 F1 boars were mated to unrelated 82 F1 sows from 1984 to 1989, yielding a total of 1862 F2 offspring. F1 boars were mated on average to 7 females, and F1 sows to an average of 2.7 males. Average offspring per boar were 60 and per sow 23.
Phenotypic information: (i) Data collection: A total of 21 distinct phenotypes were recorded in the F2 generation.6, 7 These included:
five growth traits: birth weight (g), weaning weight (Kg), grower weight (Kg), finisher weight (Kg) and average daily gain (ADG; Kg/day; grower to finisher period);
two body proportion measurements: carcass length (cm); and a conformation score (0 to 10 scale; reference 6);
ten measurements of carcass composition obtained by dissection of the chilled carcasses 24 hours after slaughter. These include measurements of muscularity: % ham (weight hams/carcass weight), % loin (weight loin/carcass weight), % shoulder (weight shoulder/carcass weight), % lean cuts (% ham+% loin+% shoulder); and measurements of fatness: average back-fat thickness (BFT; cm), % back fat (weight back fat/carcass weight), % belly (weight belly/carcass weight), % leaf fat (weight leaf fat/carcass weight), % jowl (weight jowl/carcass weight), and “% fat cuts” (% back fat+% belly+% leaf fat+% jowl).
four meat quality measurements: pH LDi (Longissimus dorsi 1 hour after slaughter), pH LD24 (Longissimus dorsi 24 hours after slaughter), pH GI (Gracilis 1 hour after slaughter) and pH G24 (Gracilis 24 hours after slaughter). (ii)Data processing: Individual phenotypes were preadjusted for fixed effects (sire, dam, CRC genotype, sex, year-season, parity) and covariates (litter size, birth weight, weaning weight, grower weight, finisher weight) that proved to significantly affect the corresponding trait. Variables included in the model were selected by stepwise regression.
Marker genotyping: Primer pairs utilized for PCR amplification of microsatellite markers are as described.19 Marker genotyping was performed as previously described.20 Genotypes at the CRC and MyoD loci were determined using conventional methods as described.1, 12 The LAR test for the Igf2 SNP was developed according to Baron et al.21 using a primer pair for PCR amplification (5′-CCCCTGAACTTGAGGACGAGCAGCC-3′ (SEQ ID NO:5); 5′-ATCGCTGTGGGCTGGGTGGGCTGCC-3′ (SEQ ID NO:6)) and a set of three primers for the LAR step (5′-FAM-CGCCCCAGCTGCCCCCCAG-3′ (SEQ ID NO:7); 5′-HEX-CGCCCCA GCTGCCCCCCAA-3′ (SEQ ID NO:8); 5′-CCTGAGCTGCAGCAGGCCAG-3′ (SEQ ID NO:9)).
Map construction: Marker maps were constructed using the TWOPOINT, BUILD and CHROMPIC options of the CRIMAP package.22 To allow utilization of this package, full-sib families related via the boar or sow were disconnected and treated independently. By doing so, some potentially usable information was neglected, yielding, however, unbiased estimates of recombination rates.
QTL mapping: (i) Mapping Mendelian QTL: Conventional QTL mapping was performed using a multipoint maximum likelihood method. The applied model assumed one segregating QTL per chromosome, and fixation of alternate QTL alleles in the respective parental lines, Piétrain (P) and Large White (LW). A specific analysis program had to be developed to account for the missing genotypes of the parental generation, resulting in the fact that the parental origin of the F1 chromosomes could not be determined. Using a typical “interval mapping” strategy, a hypothetical QTL was moved along the marker map using user-defined steps. At each position, the likelihood (L) of the pedigree data was computed as:
where
is the sum over all possible marker-QTL phase combinations of the F1 generation. As there are two possible phases for each parent (left chromosome P or right chromosome P), there is a total of 2′ combinations for r F1 parents.
is the product over the n F2
is the sum, for the ith F2 offspring, over the four possible QTL genotypes: P/P, P/LW, LW/P and LW/LW
P(G|Mi,θ,φ) is the probability of the considered QTL genotype, given (i) Mi: the marker genotype of the ith F2 offspring and its F1 parents, (ii): the vector of recombination rates between adjacent markers and between the hypothetical QTL and its flanking markers, and (iii) θ the considered marker-QTL phase combination of the F1 parents.
Recombination rates and marker linkage phase of F1 parents are assumed to be known when computing this probability. Both were determined using CRIMAP in the map construction phase (see above).
P(yi|G) is the probability of the phenotypic value of (yi) of offspring i, given the QTL genotype under consideration. This probability is computed from the normal density function:
where μG is the phenotypic mean of the considered QTL genotype (PP, PL, LP or LL) and σ2 the residual variance σ2 was considered to be the same for the four QTL genotypic classes.
The values of μPP, μPP, μPL=μLP, μLL and σ2 maximizing L were determined using the GEMINI optimization routine.23
The likelihood obtained under this alternative H1 hypothesis was compared with the likelihood obtained under the null hypothesis H0 of no QTL, in which the phenotypic means of the four QTL genotypic classes were forced to be identical. The difference between the logarithms of the corresponding likelihoods yields a lodscore measuring the evidence in favor of a QTL at the corresponding map position.
(ii) Significance thresholds: Following Lander & Botstein,24 lodscore thresholds (7) associated with a chosen genome-wise significance level, were computed such that:
α=(C+9.21GT)χ22(4.6T)
where C corresponds to the number of chromosomes (=19), G corresponds to the length of the genome in Morgans (=29), and χ22(4.6T) denotes one minus the cumulative distribution function of the chi-squared distribution with 2 d.f. Single point 2In(LR) were assumed to be distributed as a chi-squared distribution with two degrees of freedom, as we were fitting both an additive and dominance component. To account for the fact that we were analyzing multiple traits, significance levels were adjusted by applying a Bonferoni correction corresponding to the effective number of independent traits that were analyzed. This effective number was estimated at 16 following the approach described by Spelman et al.25 Altogether, this allowed us to set the lodscore threshold associated with an experiment-wise significance level of 5% at 5.8. When attempting to confirm the identified QTL in an independent sample, the same approach was used, however, setting C at 1, G at 25 cM and correcting for the analysis of 4.5 independent traits (as only six traits were analyzed in this sample). This yielded a lodscore threshold associated with a Type I error of 5% of 2.
(iii). Testing for an imprinted QTL: To test for an imprinted QTL, we assumed that only the QTL alleles transmitted by the parent of a given sex would have an effect on phenotype, the QTL alleles transmitted by the other parent being “neutral.” The likelihood of the pedigree data under this hypothesis was computed using equation 1. To compute P(yi|G), however, the phenotypic means of the four QTL genotypes were set at μPP=μPL=μP and μLP=μLL=μL to test for a QTL for which the paternal allele only is expressed, and μPP=μLP=μP and μPL=μLL=μL to test for a QTL for which the maternal allele only is expressed. It is assumed in this notation that the first subscript refers to the paternal allele, the second subscript to the maternal allele. H0 was defined as the null-hypothesis of no QTL, H1 testing the presence of a Mendelian QTL; H2 testing the presence of a paternally expressed QTL, and H3 testing the presence of a maternally expressed QTL.
RT-PCR: Total RNA was extracted from skeletal muscle according to Chirgwin et al.26 RT-PCR was performed using the Gene-Amp RNA PCR Kit (Perkin-Elmer) The PCR products were purified using QiaQuick PCR Purification kit (Qiagen) and sequenced using Dye terminator Cycle Sequencing Ready Reaction (Perkin Elmer) and an ABI373 automatic sequencer.
In Example 2, we report the identification of a QTL with major effect on muscle mass and fat deposition mapping to porcine 2p1.7 The QTL shows clear evidence for parental imprinting strongly suggesting the involvement of the Igf2 locus.
A Piétrain X Large White intercross comprising 1125 F2 offspring was generated as described.6, 7 The Large White and Piétrain parental breeds differ for a number of economically important phenotypes. Piétrains are famed for their exceptional muscularity and leanness8 (
In order to map QTL underlying the genetic differences between these breeds, we undertook a whole genome scan using microsatellite markers on an initial sample of 677 F2 individuals. Analysis of pig chromosome 2 using a ML multipoint algorithm, revealed highly significant lodscores (up to 20) for six of the 12 phenotypes measuring muscularity and fat deposition at the distal end of the short arm of chromosome 2 (
Bidirectional chromosome painting establishes a correspondence between SSC2p and HSA11pter-q13.9, 10 At least two serious candidate genes map to this region in man: the myogenic basic helix-loop-helix factor, MyoD, maps to HSA11p15.4, while Igf2 maps to HSA11p15.5 MyoD is a well known key regulator of myogenesis and is one of the first myogenic markers to be switched on during development.11 A previously described amplified sequence polymorphism in the porcine MyoD gene12 proved to segregate in our F2 material, which was entirely genotyped for this marker. Linkage analysis positioned the MyoD gene in the SW240-SW776 (odds >1000) interval, therefore well outside the lod-2 drop off support interval for the QTL (
Igf2 therefore appeared as a strong positional allele having the observed QTL effect. In man and mouse, IgO is known to be imprinted and to be expressed exclusively from the paternal allele in several tissues.15 Analysis of skeletal muscle cDNA from pigs heterozygous for the SNP and/or SWC9, shows that the same imprinting holds in this tissue in the pig as well (
These data therefore confirmed our hypothesis of the involvement of an imprinted gene expressed exclusively from the paternal allele. The fact that the identified chromosomal segment coincides precisely with an imprinted domain documented in man and mice strongly implicates the orthologous region in pigs. At least seven imprinted genes mapping to this domain have been documented (Igf2, Ins2, H19, Mash2, p57KIP2, KνLQTL1 and TDAGS1) (reference 15 an Andrew Feinberg, personal communication). Amongst these, only Igf2 and Ins2 are paternally expressed. While we cannot exclude that the observed QTL effect is due to an as of yet unidentified imprinted gene in this region, its reported effects on myogenesis in vitro and in vivo13 strongly implicate Igf2. Particularly the muscular hypertrophy observed in transgenic mice overexpressing Igf2 from a muscle specific promoter is in support of this hypothesis (Nadia Rosenthal, personal communication. Note that allelic variants of the INS VNTR have recently been shown to be associated with size at birth in man,16 and that the same VNTR has been shown to affect the level of Igf2 expression.17
The observation of the same QTL effect in a Large White x Wild Boar intercross indicates the existence of a series of at least three distinct functional alleles. Moreover, preliminary evidence based on marker-assisted segregation analysis points towards residual segregation at this locus within the Piétrain population (data not shown). The occurrence of an allelic series might be invaluable in identifying the causal polymorphisms which—based on the quantitative nature of the observed effect—are unlikely to be gross gene alterations but rather subtle regulatory mutations.
The effects of the identified QTL on muscle mass and fat deposition are truly major, being of the same magnitude of those reported for the CRC locus6, 7 though apparently without the associated deleterious effects on meat quality. We estimate that both loci jointly explain close to 50% of the Piétrain versus Large White breed difference for muscularity and leanness. Understanding the parent-of-origin effect characterizing this locus will allow for its optimal use in breeding programs. Indeed, today half of the offspring from commercially popular Piétrain x Large White crossbred boars inherit the unfavorable Large White allele causing considerable loss.
The QTL described in this work is the second example of a gene affecting muscle development in livestock species that exhibits a non-Mendelian inheritance pattern. Indeed, we have previously shown that the callipyge locus (related to the qualitative trait wherein muscles are doubled) is characterized by polar overdominance in which only the heterozygous individuals that inherit the CLPG mutation from their sire express the double-muscling phenotype.5 This demonstrates that parent-of-origin effects affecting genes underlying production traits in livestock might be relatively common.
The invention provides an imprinted QTL with major effect on muscle mass mapping to the IGF2 locus in the pig, and use of the QTL as tool in marker-assisted selection. To fine tune this tool for marker-assisted selection, as well as to further identify a causal mutation, we have further generated a reference sequence encompassing the entire porcine IGF2 sequence as well as that from flanking genes.
To achieve this, we screened a porcine BAC library with IGF2 probes and identified two BACs. BAC-PIGF2-1 proved to contain the INS and IGF2 genes, while BAC-PIGF2-2 proved to contain the IGF2 and H19 genes. The NotI map as well as the relative position of the two BACs is shown in
For BAC-PIGF2-2, the 24 Kb NotI fragment not present in BAC-PIGF2-1 was subcloned and sequenced using the EZ::TN transposon approach and ABI automatic sequencers. Resulting sequences were assembled using the Phred-Phrap-Consed program suit, yielding seven distinct contigs (
Based on the reference sequence obtained as described in Example 1, we resequenced part of the IGF2 and flanking loci from genomic DNA isolated from Piétrain, Large White and Wild Boar individuals, allowing identification of DNA sequence polymorphisms such as reported in
This example shows presently described unique inheritance method of paternal imprinting, wherein only the gene inherited from the father is expressed, and wherein the gene inherited from the mother is a silent gene and has no effect on the carcass quality of the offspring.
Material and Methods
Animals. The animals used in this experiment are purebred animals belonging to three different closed dam lines based on Large White and Landrace breeds. From 1999 until 2005 blood samples were collected from all nucleus sows and boars. Genotypic frequencies per line were calculated on 555 sows in total.
Measurements. Individual blood samples are linked to individual phenotypes. For all sows the following parameters were recorded: total born, live born, stillborn and weaned piglets per litter. At test weight of 110 kg carcass measures were performed on live animals using Piglog 105 (including back fat 1, back fat 2 (3rd-4th rib), loin eye depth and lean meat percentage).
Genotyping. DNA was extracted from the pig blood samples using the Wizard Genomic DNA purification kit according to procedures provided by the manufacturer (Promega, Madison Wis., US). An allelic discrimination assay was performed using the ABI Prism 7700 sequence detection system (Applied Biosystems). The final concentrations used in the 5 μl master mix were: 2.5 μl Taqman Universal PCR Master Mix, NoAmpErase Ung (Applied Biosystems, Foster City, Calif.), 1× Assay Mix, 10 ng DNA and 2,375 μl H2O (Foster City, Calif.).
Statistical analyses. The statistical analysis was performed using the statistical software SAS. The gene frequencies were calculated from PROC FREQ. IGF2 effects were analyzed using SAS PROC GLM with paternal or maternal allele as class variables and taking into account parity and sire. For the calculation of the effect of the IGF2 mutation on the traits measured, a subset of data was made in which only sows that originate from sires that are heterozygous for the IGF2 mutation were used. Sows that inherited the G allele were compared with those that inherited the A allele. Another subset of data was made in which only sows from heterozygous dams were retained. In this dataset the effect of the maternal allele was analyzed.
Results and Discussion. Allelic frequencies are presented in Table 3. All three dam lines segregate for the IGF2 mutation, although frequencies differ according to the line.
A subset of data was made in which only sows derived from heterozygous sires that segregate in the population were retained. A comparison was made between sows that inherited the A or the G allele from their father.
Sows that inherited the wild type allele from their father had significantly more piglets born alive, total born and weaned, while there was no effect on stillborn piglets (Table 4). If the same dataset was analyzed according to the allele inherited from the mother (maternal allele) no effect on any of these prolificacy data could be observed. A second subset of data was created in which only sows from heterozygous dams were taken into account and grouped according to the maternal allele. Again, no significant effect on prolificacy could be observed, which was expected since the maternal allele is not expressed.
The parity or average number of cycles per sow was also higher in sows that inherited G from their father as compared to those that received the A allele, which points to a beneficial effect on longevity. This is related to higher litter size, since that is a major criterion for elimination in the selection program.
The effect of the paternal allele for IGF2 was also analyzed on conformation measures at ca. 110 kg live weight. These data are presented in Table 5.
Although no significant effects of IGF2 paternal allele on Piglog results could be observed, there is a tendency towards higher muscularity and lower back fat in the sows that inherited the A allele form their father. The fact that this difference is not significant could be due to the low number of animals on the one hand, and the use of a threshold value on back fat in the selection program on the other.
These results show an influence of the IGF2-intron3 G3072A mutation on prolificacy and longevity in sows. This opens the possibilities to use the same imprinted QTN for different selection in sire and dam lines. Terminal sires should be homozygous for the lean allele to give uniform and lean slaughter pigs, while dam lines can benefit from a selection for the wild type allele since this has a beneficial effect on prolificacy and longevity. Because of the imprinted character of the gene, selection for the fatter allele in sow lines will not influence the carcass quality of the offspring.
A suitable marker-assisted selection program for the IGF2 mutation may now be represented as depicted in
Literature Cited in Example 1:
26. Chirgwin J. M., A. E. Przybyla, R. J. MacDonald and W. J. Rutter (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299.
*P < 0.05;
**P < 0.01;
***P < 0.001
1Only the traits for which the QTL peak was in the IGF2 region (0-10 cM) and the test statistic reached the nominal significance threshold of F = 3.9 are included.
2“QTL” is the test statistic for the presence of a QTL under a genetic model with additive, dominance, and imprinting effects (3 d.f.) while “Imprinting” is the test statistic for the presence of an imprinting effect (1 d.f.), both obtained at the position of the QTL peak. Genome-wise,
3In cM from the distal end of 2p; IGF2 is located at 0.3 cM.
4The reduction in the residual variance of the F2 population effected by inclusion of an imprinted QTL at the given position.
5Means and standard errors estimated at the IGF2 locus by classifying the genotypes according to the population and parent of origin of each allele. W and L represent alleles derived from the Wild Boar and Large White founders, respectively; superscript P and M represent a paternal and maternal
R
R
*Model taking parity and sire into account, NS = not significant P > 0.05
*Model taking sire into account. NS = not significant P > 0.05
| Number | Date | Country | Kind |
|---|---|---|---|
| 98204291.3 | Dec 1998 | EP | regional |
This application is a continuation in part of U.S. Ser. No. 09/868,732, filed on Nov. 1, 2001, which is a national stage entry under 35 U.S.C. §371 of International Application No. PCT/EP99/10209, filed on Dec. 16, 1999, and published as International Publication No. WO 00/36143, in English, on Jun. 22, 2000, which claims the benefit of European Patent Application EP 98204291.3, filed on Dec. 16, 1998, the contents of the entirety of each of which are incorporated herein by this reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09868732 | Nov 2001 | US |
| Child | 11357733 | Aug 2006 | US |
