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GENETICS |
The Hebrew University, Faculty of Agricultural, Food and Environmental Quality Sciences, PO Box 12, Rehovot 76100, Israel
1 Corresponding author: cahaner{at}agri.huji.ac.il
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: ascites syndrome • divergently selected line • major gene • test cross • complementary gene interaction
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In several studies, the tendency of broilers to develop AS was found to be under genetic control. Published estimates of heritability range from 0.1 to 0.7 for the incidence of AS, with higher estimates obtained in trials in which higher %AS were observed (Lubritz et al., 1995; De Greef et al., 2001a,b; Moghadam et al., 2001; Druyan et al., 2007a,b).
Successful breeding against AS susceptibility was conducted by Wideman and French (1999, 2000) in a full-pedigreed elite commercial broiler breeder line. Only those males and females that did not develop AS after AS-inducing surgery (unilateral pulmonary artery occlusion) were used for reproduction. After 2 cycles of such selection, %AS among males that were exposed to cool temperatures (14°C) from 17 to 49 d of age was reduced to 4%, compared with 15% after 1 cycle of this selection, and 31% in the base population. That study demonstrated the feasibility of selection based on mortality of AS-susceptible (AS-S) individuals under a protocol of high-challenge ascites-inducing conditions (AIC).
Divergent selection for AS mortality was conducted by Anthony and coworkers (Anthony and Balog, 2003; Balog et al., 2003). Ascites syndrome was induced in a hypobaric chamber with partial vacuum to reduce the oxygen content to the partial pressure found at 2,900 m above sea level. Under these AIC, incidence of AS was 66% in the base population, an elite commercial broiler breeder line. After 10 generations of divergent sire-family selection, %AS increased to approximately 90% in the AS-S line and decreased to approximately 20% in the AS-resistant (AS-R) line, thus reaching a divergence of approximately 70% (Anthony and Balog, 2003).
Similarly successful divergent selection was applied by Druyan et al. (2007a). Selection was initiated in the year 2000 in a base population that was derived from a commercial broiler dam line. The first cycle of selection was based on progeny testing for AS mortality under on-farm, low-challenge AIC. Two additional cycles of full-pedigree progeny testing were conducted under an experimental high-challenge AIC protocol (Druyan et al., 2007a,b). Two divergent lines were established, AS-S and AS-R, with, respectively, 95 and 5% of AS (a divergence of 90%) when reared together under the same high-challenge AIC (Druyan et al., 2007a).
Wideman and French (2000) concluded that a gene or genes were involved in the response to a 2-cycle selection against AS susceptibility. Single-gene inheritance was also suggested by Navarro et al. (2006). The latter authors performed a complex segregation analysis of data on oxygen saturation of the hemoglobin in arterial blood (SaO2), a trait known to be closely related to AS (Julian and Mirsalimi, 1992; Druyan et al., 2007b). Data on SaO2 from 12,000 males in fully pedigreed populations of a male line that has been closed for 30 to 40 generations were available for that study. The results suggested that a single diallelic dominant locus was responsible for 90% of the genetic variation in SaO2, with high levels of SaO2 indicating AS resistance, whereas low levels indicated AS susceptibility.
Druyan et al. (2007a) noted that the extremely rapid divergence between their selected AS-S and AS-R lines may suggest the involvement of one or several major genes. Moreover, analysis of AS segregation within families in the selected lines suggested that the dominant allele was responsible for AS resistance. It was therefore hypothesized that the difference between the AS-S and AS-R lines is controlled by a major gene (A) with dominance of the allele for resistance. According to this hypothesis, individuals in the AS-R line are either homozygous or heterozygous for the major gene (i.e., AA or Aa), whereas the individuals in the AS-S line are homozygous recessive for the suggested gene (aa). The current study was designed to test this hypothesis by analyzing the segregation among test-cross progeny of heterozygous (Aa) sires from the AS-R line and from the AS-R x AS-S cross, mated to recessive homozygous (aa) dams from the AS-S line.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The birds for the current study were derived from the AS-S and AS-R selected lines (Druyan et al., 2007a). These lines were divergently selected from a commercial broiler dam line based on progeny testing. In each generation, full-pedigree progeny were produced in each line in several consecutive hatches and tested under a high-challenge AIC protocol (Druyan et al. 2007b) to determine susceptibility or resistance to AS.
Test-Cross of Heterozygous Sires from the AS-R Line
Progeny of 8 sires in the S2 generation of the AS-R line were tested under the high-challenge AIC protocol (Druyan et al., 2007b) to check whether the sires were homozygous (AA) or heterozygous (Aa) for the presumed major dominant gene for AS resistance. Four families segregated to progeny with AS and healthy progeny, suggesting that these sires were heterozygous (numbers 43, 45, 62, and 63; Table 1
). Nine male progeny from these families (S3 generation) were used as sires of the first set of test-cross families, because half of them were expected to be heterozygous. Each of these 9 males was test-crossed to 4 AS-S dams, assumed to be homozygous (aa), to produce sire families. Male progeny of these 9 families, from 3 consecutive hatches, were reared under the high-challenge AIC protocol to determine susceptibility or resistance to AS for each of them.
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The sires of the second set of test-cross families were produced by crossing selected males from the AS-R line with selected females from the AS-S line. The selected males and females were previously confirmed, by within-line matings, to produce nonsegregating progeny when they were tested under AIC. The AS-R males, numbers 33, 38, and 44, were selected because they produced only healthy (i.e., non-AS) progeny (Table 1
). Similarly, all the progeny of the selected AS-S females developed AS (data not shown). Thus, it could be assumed that the crossed AS-R and AS-S parents were homozygous (AA and aa, respectively); hence, their F1 progeny were expected to be heterozygous (Aa). Eight F1 males were used as sires; each of them was test-crossed to 4 AS-S females (dams), assumed to be homozygous (aa), to produce sire families. Male progeny of these 8 families, from 3 consecutive hatches, were reared under the high-challenge AIC protocol to determine susceptibility or resistance to AS for each progeny.
Experimental High-Challenge AIC Protocol
All the male chicks from the 2 sets of test-cross sire families were brooded under standard brooding conditions on a concrete floor covered with wood shavings. At 19 d of age (d 19), the chicks were placed in individual cages with cool air (18 to 20°C) blown on them by fans at about 3 m/s. The growth rate of these chicks was enhanced by using an accelerated feeding program, comprising prestarter feed from d 0 to 4 (instead of d 0 to 10), starter feed from d 5 to 14 (instead of d 11 to 21), grower feed from d 15 to 24 (instead of d 22 to 33), and a high-energy pelleted finisher feed from d 25 to 44. The contents of CP (%) and energy (cal/kg of ME) in these 4 diets were 22 and 3,100, 20.5 and 3,125, 19.5 and 3,150, and 18.3 and 3,225, respectively. Feed and water were provided ad libitum. The growth rate was further enhanced by exposure to 23 h/d of light from hatch to the end of each trial, on d 44.
Diagnosis of AS
Throughout the AIC phase (d 19 to 44), all dead chicks were necropsied and examined to determine the cause of death. Chicks with ascitic fluid or hydropericardium were diagnosed as having died because of AS and their phenotype was recorded as being AS. The few birds that died from other causes were excluded from the data. On d 44, all surviving birds were killed by cervical dislocation, necropsied, and examined. Birds showing ascitic fluid or hydropericardium were also diagnosed as AS; the "healthy" phenotype was recorded for all other birds not showing any AS manifestations.
Statistical Analysis
The data for each family consisted of the number of progeny in each of the 2 phenotypes, AS or healthy, following exposure to the high-challenge AIC. The agreement between the proportion of AS and healthy individuals within each family, and an expected ratio of 1:1 or 3:1, was tested by 
2 tests that were conducted by using JMP software (SAS Institute, 2005).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In 5 of the 9 test-cross sire families, approximately 50% of the progeny developed AS and 50% remained healthy, and this 1:1 ratio was confirmed by a 2 test (P > 0.4; Table 2). These results were in agreement with the assumption that the sires, numbers 1515, 8626, 8634, 8645, and 8667, were indeed heterozygous (Aa) in the suggested major gene A. Furthermore, these results supported the hypothesis that the difference in %AS between the AS-R and AS-S lines was due to a single major gene with dominance of the allele for AS resistance.
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). Therefore, it was expected that approximately half of the 9 sires would have the Aa genotype, a good agreement with the ratio of 5 out of 9 sires with the presumed Aa genotype. The remaining 4 sires were expected to have the AA genotype, but only in 2 families were all the progeny healthy, indicating that sire numbers 1566 and 8646 were homozygous for the dominant allele (AA). However, in 2 families (sire numbers 8617 and 8641) there was segregation, with a higher proportion of AS progeny (77.1 and 82.9%, respectively) significantly (P < 0.001) different from the expected ratio of 1:1. Thus, the segregation among the test-cross progeny of these 2 sires did not reveal their genotype, because it confirmed neither AA nor Aa; hence, they are marked as "?" in Table 2. Test-Cross of Heterozygous Sires from the AS-R x AS-S Line Cross
The progeny segregated to AS and healthy in all the families that were produced by test-crossing F1 sires (Table 3) with AS-S dams. These results are in agreement with the expected heterozygosity (Aa) of all F1 birds, which were produced by crossing homozygous sires from the selected AS-R line (presumed genotype AA) and homozygous dams from the AS-S line (presumed genotype aa). With the presumed genotype Aa in all F1 sires, their test-cross progeny were expected to segregate to AS vs. healthy according to a 1:1 ratio, based on a single dominant major gene. However, with substantially more susceptible progeny than resistant ones, the hypothesized 1:1 ratio was rejected. The deviation from the 1:1 ratio was highly significant in 6 of the 8 families, marginally significant (P = 0.083) in an additional family, and not significant (P = 0.198) in 1 family (Table 3).
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). A segregation of 3:1 among test-cross progeny is the expected ratio when the tested parent is heterozygous in 2 unlinked genes with complementary interaction between their dominant alleles, namely, that dominant alleles are needed in both genes to confer resistance to AS (Griffiths et al., 2000). Assuming the involvement of 2 major genes, with the letter A denoting 1 gene and the letter B denoting the second gene, the AS-R sires (in the line-cross) were presumably homozygous for the dominant allele in both genes (AABB), the AS-S dams were presumably homozygous for the recessive allele in both genes (aabb), and their F1 progeny were double heterozygous (AaBb). In the second round of test crosses, F1 sires with the genotype AaBb were mated to AS-S dams with the genotype aabb. Such test-cross results in 4 expected genotypes: AaBb, Aabb, aaBb, and aabb. If the 2 genes are unlinked, the 4 genotypes are expected to segregate in equal proportions, namely 1:1:1:1. However, according to a model of complementary interaction between the dominant alleles of these 2 genes, a dominant allele is needed in both genes to have the dominant phenotype (AS resistance). Accordingly, only progeny with the AaBb genotype were expected to be resistant to AS, whereas the 3 other genotypes (Aabb, aaBb, and aabb) were expected to be susceptible to AS. Thus, the average observed segregation of 25% AS-R (healthy) progeny and 75% AS-S progeny in the second set of test-cross families was in perfect agreement with the model of a complementary interaction between dominant alleles in 2 unlinked major genes.
The model of complementary interaction between 2 major genes was identified in several studies in which the genetic control of the difference between 2 phenotypes was elucidated from the segregation among test-cross progeny. In Arabidopsis, F1 plants from the cross between a line bearing the aerial-rosette (ar) phenotype and a no-aerial-rosette (nar) line were backcrossed to nar plants, and the ar:nar ratio among the back-cross progeny was 1:3. The suggestion that the aerial-rosette phenotype is controlled by complementary interaction between the dominant alleles in 2 unlinked genes was also supported by results from diallel analysis of several crosses whose progeny segregated for the ar and nar phenotypes (Grbi
and Bleecker, 1996). A similar model was found to control the resistance to beet necrotic yellow vein virus in Beta vulgaris; in 4 test-cross families there were 28% resistant progeny vs. 72% susceptible ones, as expected from 2 unlinked complementary dominant genes (Scholten et al., 1996). A model of 2 complementary dominant genes was also suggested for resistance vs. susceptibility of pea seedlings to Mycosphaerella pinodes, based on a 9:7 ratio among F2 progeny of a cross between resistant and susceptible lines (Clulow et al., 1991).
If the genetic divergence between the AS-R and AS-S lines was due to 2 unlinked complementary major genes, the results of the first set of test-cross families should confirm this. The sires in these test-crosses were taken from the AS-R line; therefore, it is most probable that the majority of those that produced segregating families were heterozygous in only 1 of these 2 genes, either AaBB or AABb. When such sires are crossed with aabb dams, 50% of the progeny are expected to be AaBb, hence AS-R (healthy), and the remaining 50% chicks should be AS-S because they carry the dominant allele in 1 gene only, either aaBb (from AaBB sire) or Aabb (from AABb sire). Accordingly, the genotypes of the 5 sires in the first set of test-cross families that were assumed to be Aa (Table 2) were apparently either AaBB or AABb. The 2 sires that produced 100% healthy progeny (numbers 1566 and 8646) were probably homozygous for the dominant allele in both genes (i.e., AABB). According to the model of a single major gene, no genotype could be assigned to 2 sires in the first set (numbers 8617 and 8641; Table 2). However, progeny segregation in these 2 families did not differ significantly from the 3:1 ratio of AS progeny vs. healthy progeny, indicating that these 2 sires were heterozygous in both genes (AaBb).
The segregation among the test-cross progeny in the current study suggested a model of complementary interaction between the dominant alleles of 2 unlinked major genes. This finding is in agreement with that of Wideman and French (2000), who suggested that "gene or genes involved in ascites susceptibility appear to be dominant.". Involvement of a few major genes may also explain the rapid response to the divergent selection on AS mortality that was conducted by Anthony and coworkers (Anthony and Balog, 2003; Balog et al., 2003) and by Druyan et al. (2007a). Navarro et al. (2006) suggested that a single dominant gene was responsible for the variation in SaO2, an AS-related trait. The latter study was conducted in primary breeding lines that had been selected against AS susceptibility; hence, these lines could already have been homozygous for the resistance allele in another AS-related major gene.
However, most studies considered AS susceptibility as a polygenic trait (De Greef et al., 2001a,b; Moghadam et al., 2001; Pakdel et al., 2005a,b; Rabie et al., 2005; Zerehdaran et al., 2006). Those studies were conducted under low-challenge AIC, in which individuals with a relatively low growth rate, and hence low oxygen demand, do not develop AS even if they are genetically susceptible. Therefore, under such conditions, the genes that affect growth rate are indirectly affecting the development of AS in the genetically susceptible birds. Other factors that reduce growth and oxygen demand (e.g., female gender, lower meat yield, and certain environmental conditions) also lead to partial penetrance and expressivity of susceptibility to AS. The current study, however, as in the studies by Wideman and French (2000) and Anthony and Balog (2003), was conducted under a high-challenge AIC protocol that induced AS in all susceptible individuals (Druyan et al., 2007a,b). Under such conditions, all the AS-S birds develop AS, even those with a relatively lower growth rate, thus facilitating detection of the few major genes that are directly responsible for AS susceptibility.
Wade (2001) noted that fitness traits are often controlled by 2 or 3 major genes that interact with each other (i.e., epistasis) and with the genetic background and environment. This description fits the susceptibility of broilers to AS, as well as the susceptibility of the beet to beet necrotic yellow vein virus (Scholten et al., 1996) and of pea seedlings to M. pinodes (Clulow et al., 1991).
Under no-challenge or low-challenge conditions, it is difficult to identify the genes that are directly responsible for susceptibility, because other genes and favorable environments (e.g., no pathogen exposure) may prevent the expression of susceptibility in genetically susceptible individuals. In the case of AS, being a female or bearing genes for lower growth rate or benefiting from a favorable environment (e.g., warmer niche in the chicken house) may keep the oxygen demand of genetically susceptible individuals below the threshold that initiates development of AS, thus resulting in the resistant phenotype. This situation, which apparently was common in many studies on the genetics of AS, complicated the efforts to select against AS susceptibility and to identify the genes with a direct effect on AS.
In contrast, the current study was conducted under a high-challenge AIC protocol, and by using test-crosses between the fully-divergent AS-S and AS-R lines, genetic control of this divergence was successfully elucidated. The finding that only 2 major genes were involved suggests a higher chance of detecting the AS genes in resource populations derived from crosses between the AS-S and AS-R lines, of identifying causative markers in these genes, and of using them efficiently in commercial breeding programs. Once such markers are available, high-challenge AIC will not be needed to effectively select against susceptibility to AS, because broiler breeders will easily be able to detect individual birds that carry the alleles for AS susceptibility.
Received for publication January 16, 2007. Accepted for publication July 12, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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