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Research Note: Linkage Relationship Between the Pea Comb (P) and Extended Black (E) Loci of the Chicken1
Research Note: Linkage Relationship Between the Pea Comb (P) and Extended Black (E) Loci of the Chicken1 J. ROBERT SMYTH, Jr. and F. ABEL PONCE de LEON Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, Massachusetts 01003 (Received for publication July 22, 1991) ABSTRACT The linkage relationship between the autosomal genes, extended black (E) and pea comb (P), were estimated from the cross E/eY P/p+ X eb/eY p+/p+ in the coupling phase. Reciprocal crosses yielded a total of 545 chicks and indicated that the E and P loci were genetically linked by 45.5 ± 2.14 (SE) crossover units (P<.05). The two reciprocals resulted in similar estimates (45.6 and 45.3), although significant individually only at the .10 level of probability. Intra-allelic segregation at both loci closely approximated the expected 1:1 ratio. These data provide additional evidence that the E and P loci are linked in linkage Group III on Chromosome 1. (Key words: autosomal linkage, extended black, pea comb, linkage Group III, chickens) 1992 Poultry Science 71508-210
INTRODUCTION There is increased interest in the physical mapping of the chicken to help facilitate the molecular analyses and manipulations of its genetic material. The number of mapped loci in the fowl is relatively low in light of the large number of mutations that have been reported. Not surprisingly, the Z chromosome has the most complete linkage map with 17 loci, but linkage Group III on Chromosome 1 is the only autosome with more than a few mapped loci (see Bitgood and Somes, 1990, for review). As many as 14 loci have been assigned to Chromosome 1. Several important additions have been made to linkage Group III recently by Carefoot (1987, 1988, 1990) resulting from a series of studies on the genetics of secondary plumage patterns (see Smyth, 1990, for review). Carefoot (1988) reported the map distances between the pea comb (P), dark brown (Db), melanotic (Ml), plumage pattern (Pg), and naked neck
1 This study was supported by the Massachusetts Experiment Station.
(Na) loci to be 32, 10, 10, and 26 units, respectively. More recently, Carefoot (1990) reported that the E plumage color locus also belongs in this group, because he found it to be linked to the P locus by approximately 34 crossover units. This represented the first chromosomal location for the multiple allelic E locus, whose important contributions to plumage color genetics have been much studied (see Smyth, 1990, for review). Morejohn (1955) previously reported no linkage between the E allele, eY, and the P locus from a testcross involving 62 gametes. However, as pointed out by Carefoot (1990), recessive wheaten (eY) can be difficult to identify on certain genetic backgrounds. Therefore, the data of Morejohn are not only limited, but suspect. The E locus was reported to be linked to the plumage color gene, mottling, by Brumbaugh and Hollander (1965), but further study indicated that this relationship was not repeatable 0. A. Brumbaugh, School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, personal communication). Adjunct to studies on the genetics of the E locus, an attempt was made to clarify
208
RESEARCH NOTE
209
TABLE 1. Results of a two-point linkage test between extended black (E) and pea comb (P)
Mating
n
E-P
Gamete types (n) Ep+ eP2 ep +2
Cross-over percentage3
Probability
Redprocal l 4 Reciprocal 2 5 Pooled
331 214 545
90 58 148
71 51 122
45.62 ± 2.75 45.32 ± 3.42 45.50 ± 2.14
>.05 >.05 <.05
80 46 126
90 59 149
^Recombinant parent (E/e^ P/p+) in coupling phase mated to e°/ef p+/p+ tester. Non-E class (mixed ^/ef and e?/ef). % ±SE. 4 E/«y P/p+ male x eb/ey p+/p+ female. 5 h e /& p+/p+ male x E M P/p+ female. 2
further the linkage relationships between the P and E loci. The present report presents additional evidence that the E and P loci are linked on Chromosome 1. MATERIALS AND METHODS The linkage testcross analysis reported herein was derived from the progeny of reciprocal crosses between birds heterozygous for extended black and pea comb (E/ eyp/p+) and a recessive type tester (e b / eyp+/p+). The E and P loci were located on the same chromosome, so the cross was made in coupling. Although heterozygous for the brown (efy and recessive wheaten («y) alleles at the E locus, the pertinent segregation was E versus non-E, because E is dominant to all other E alleles. The E / black or near-black chicks were readily separated from the non- E at hatching, the latter having either yellow (e v /e v ) or stripped tan (e b /e v ) down. Pea comb was also described at 1 day of age. Although pea comb can be difficult to determine at this age (Crawford, 1961), the accuracy was checked by rearing the first hatch of 112 chicks to 12 wk of age with only one error in classification. This high degree of accuracy is probably due to the relatively small comb size of non-Leghorn birds used in the current study. The large combs of the Leghorn types can make classification more difficult because of the excessive development of the middle blade in the triple-bladed pea comb (Punnett, 1923). Because this problem was not experienced in the present study, subsequent hatches were classified only at 1 day of age. Further verification of phenotypic accu-
racy is the close approximation of segregation ratios to the expected 1:1 for the alleles at both the E and P loci. Reciprocal testcross matings w e r e made. A total of five biweekly settings were made resulting in a total of 545 chicks that were classified as resulting from one of the four gamete types: EP, Ep+, eP, ep+. Note that the e symbol was used in the present study to designate either eb or e v . Detection and estimation of linkage was carried out by the method of maximum-likelihood as described by Green (1963). RESULTS AND DISCUSSION The results of the matings designed to test for genetic linkage between the E and P loci are presented in Table 1. The two reciprocal crosses resulted in 545 test gametes and these pooled data yielded a recombination percentage of 45.50 ± 2.14 (SE), which differed significantly (P<.05) from independent assortment. Therefore, there is a 95% probability that the true value for the linkage lies between 41.22 and 49.78%. Although both reciprocals were in close agreement (45.62 ± 2.75 and 45.32 ± 3.42) neither by itself quite reached the P = .05 significant level for linkage. These data support the report of linkage between E and P by Carefoot (1990). Although the numbers of gametes involved in the tatter's study was smaller (152) than in the present study, they were derived from two separate crosses that were in relatively close agreement (35.1 and 30.9% recombination). In the present study, the number of offspring examined
210
SMYTH, JR. AND PONCE DE LEON
approximates the 600 proposed by Bitgood and Somes (1990) as needed to prove linkage of 45 map units at the 5% level of probability. There is some discrepancy between the mean map distance of 45.5 found in the present study and the approximate 34 map units reported by Carefoot (1990). This suggests that the true distance probably lies somewhere between these values. The present study contributes no information on the map location of £ in respect to P and the other mapped loci in linkage Group III (see Bitgood and Somes, 1990, for review). Carefoot (1990) has proposed that E and sleepy eye (se) are on opposite sides of P based on earlier studies (Somes, 1988), indicating that se is loosely linked to P but E and se segregate independently. This would place E on the opposite side of the centromere from P and suggests that E should be linked on the Q arm with Db (dark brown), t (tardy), and several other loci presumably located in this region. Although the E alleles have been readily available in the stocks used over the years in linkage studies of chickens, there is a paucity of data involving the E locus. Extended black (E) was an obvious choice for inclusion in these studies. It is hypothesized that this could be due to the then little understood complexities of the genetics of the E alleles (see Smyth, 1990, for review). For example, a cross of extended black stocks to produce a heterozygote followed by the testcross mating back to the recessive stock could result in considerable difficulty in identifying some of the E alleles. This would be particularly true if the original black phenotype was actually a birchen (ER) with the addition of eu-
melanizing modifiers. Inability to classify the E locus segregation in the testcross with confidence could have resulted in data involving extended black not to have been published.
REFERENCES Bitgood, J. J., and R. G. Somes, Jr., 1990. Linkage relationships and gene mapping. Pages 469-495 in: Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier, New York, NY. Brumbaugh, J. A., and W. F. Hollander, 1965. A further study of the E pattern locus in the fowl. Iowa State J. Sci. 40:51-64. Carefoot, W. C, 1987. Relative positions of the loci of the peacomb (P), eumelanin restrictor (Db), eumelanin extension (Ml), and plumage pattern (Pg) genes of the domestic fowl. Br. Poult. Sci. 28:347-350. Carefoot, W. C , 1988. Inheritance of the spangled plumage pattern and the marbled chick down phenotypes of the Silver-Spangled Hamburgh bantam. Br. Poult. Sci. 29:785-790. Carefoot, W. C, 1990. Test for linkage between the eumelanin dilution blue (B0, the extended black (E) allele at the E-locus and the linked peacomb (P) and eumelanin extension (Ml) genes in the domestic fowl. Br. Poult. Sci. 31:465-472. Crawford, R. D., 1961. Breast ridge, associated with pea and walnut comb, as an aid in identifying comb type. Poultry Sci. 40262-263. Green, M. C, 1963. Methods for testing linkage. Pages 56-82 in: Methodology in Mammalian Genetics. W. J. Burdette, ed. Holden-Day, Inc., San Francisco, CA. Morejohn, G. V., 1955. Plumage color allelism in the Red Jungle Fowl (Gallus gallus) and related domestic forms. Genetics 40:519-530. Punnett, R. C , 1923. Heredity in Poultry. Macmillan and Co., Limited, London, England. Smyth, J. R., Jr., 1990. Genetics of plumage, skin and eye pigmentation in chickens. Pages 109-167 in: Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier, New York, NY. Somes, R. G., Jr., 1988. International Registry of Poultry Genetic Stocks. Bulletin 476, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT.
INTRODUCTION There is increased interest in the physical mapping of the chicken to help facilitate the molecular analyses and manipulations of its genetic material. The number of mapped loci in the fowl is relatively low in light of the large number of mutations that have been reported. Not surprisingly, the Z chromosome has the most complete linkage map with 17 loci, but linkage Group III on Chromosome 1 is the only autosome with more than a few mapped loci (see Bitgood and Somes, 1990, for review). As many as 14 loci have been assigned to Chromosome 1. Several important additions have been made to linkage Group III recently by Carefoot (1987, 1988, 1990) resulting from a series of studies on the genetics of secondary plumage patterns (see Smyth, 1990, for review). Carefoot (1988) reported the map distances between the pea comb (P), dark brown (Db), melanotic (Ml), plumage pattern (Pg), and naked neck
1 This study was supported by the Massachusetts Experiment Station.
(Na) loci to be 32, 10, 10, and 26 units, respectively. More recently, Carefoot (1990) reported that the E plumage color locus also belongs in this group, because he found it to be linked to the P locus by approximately 34 crossover units. This represented the first chromosomal location for the multiple allelic E locus, whose important contributions to plumage color genetics have been much studied (see Smyth, 1990, for review). Morejohn (1955) previously reported no linkage between the E allele, eY, and the P locus from a testcross involving 62 gametes. However, as pointed out by Carefoot (1990), recessive wheaten (eY) can be difficult to identify on certain genetic backgrounds. Therefore, the data of Morejohn are not only limited, but suspect. The E locus was reported to be linked to the plumage color gene, mottling, by Brumbaugh and Hollander (1965), but further study indicated that this relationship was not repeatable 0. A. Brumbaugh, School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, personal communication). Adjunct to studies on the genetics of the E locus, an attempt was made to clarify
208
RESEARCH NOTE
209
TABLE 1. Results of a two-point linkage test between extended black (E) and pea comb (P)
Mating
n
E-P
Gamete types (n) Ep+ eP2 ep +2
Cross-over percentage3
Probability
Redprocal l 4 Reciprocal 2 5 Pooled
331 214 545
90 58 148
71 51 122
45.62 ± 2.75 45.32 ± 3.42 45.50 ± 2.14
>.05 >.05 <.05
80 46 126
90 59 149
^Recombinant parent (E/e^ P/p+) in coupling phase mated to e°/ef p+/p+ tester. Non-E class (mixed ^/ef and e?/ef). % ±SE. 4 E/«y P/p+ male x eb/ey p+/p+ female. 5 h e /& p+/p+ male x E M P/p+ female. 2
further the linkage relationships between the P and E loci. The present report presents additional evidence that the E and P loci are linked on Chromosome 1. MATERIALS AND METHODS The linkage testcross analysis reported herein was derived from the progeny of reciprocal crosses between birds heterozygous for extended black and pea comb (E/ eyp/p+) and a recessive type tester (e b / eyp+/p+). The E and P loci were located on the same chromosome, so the cross was made in coupling. Although heterozygous for the brown (efy and recessive wheaten («y) alleles at the E locus, the pertinent segregation was E versus non-E, because E is dominant to all other E alleles. The E / black or near-black chicks were readily separated from the non- E at hatching, the latter having either yellow (e v /e v ) or stripped tan (e b /e v ) down. Pea comb was also described at 1 day of age. Although pea comb can be difficult to determine at this age (Crawford, 1961), the accuracy was checked by rearing the first hatch of 112 chicks to 12 wk of age with only one error in classification. This high degree of accuracy is probably due to the relatively small comb size of non-Leghorn birds used in the current study. The large combs of the Leghorn types can make classification more difficult because of the excessive development of the middle blade in the triple-bladed pea comb (Punnett, 1923). Because this problem was not experienced in the present study, subsequent hatches were classified only at 1 day of age. Further verification of phenotypic accu-
racy is the close approximation of segregation ratios to the expected 1:1 for the alleles at both the E and P loci. Reciprocal testcross matings w e r e made. A total of five biweekly settings were made resulting in a total of 545 chicks that were classified as resulting from one of the four gamete types: EP, Ep+, eP, ep+. Note that the e symbol was used in the present study to designate either eb or e v . Detection and estimation of linkage was carried out by the method of maximum-likelihood as described by Green (1963). RESULTS AND DISCUSSION The results of the matings designed to test for genetic linkage between the E and P loci are presented in Table 1. The two reciprocal crosses resulted in 545 test gametes and these pooled data yielded a recombination percentage of 45.50 ± 2.14 (SE), which differed significantly (P<.05) from independent assortment. Therefore, there is a 95% probability that the true value for the linkage lies between 41.22 and 49.78%. Although both reciprocals were in close agreement (45.62 ± 2.75 and 45.32 ± 3.42) neither by itself quite reached the P = .05 significant level for linkage. These data support the report of linkage between E and P by Carefoot (1990). Although the numbers of gametes involved in the tatter's study was smaller (152) than in the present study, they were derived from two separate crosses that were in relatively close agreement (35.1 and 30.9% recombination). In the present study, the number of offspring examined
210
SMYTH, JR. AND PONCE DE LEON
approximates the 600 proposed by Bitgood and Somes (1990) as needed to prove linkage of 45 map units at the 5% level of probability. There is some discrepancy between the mean map distance of 45.5 found in the present study and the approximate 34 map units reported by Carefoot (1990). This suggests that the true distance probably lies somewhere between these values. The present study contributes no information on the map location of £ in respect to P and the other mapped loci in linkage Group III (see Bitgood and Somes, 1990, for review). Carefoot (1990) has proposed that E and sleepy eye (se) are on opposite sides of P based on earlier studies (Somes, 1988), indicating that se is loosely linked to P but E and se segregate independently. This would place E on the opposite side of the centromere from P and suggests that E should be linked on the Q arm with Db (dark brown), t (tardy), and several other loci presumably located in this region. Although the E alleles have been readily available in the stocks used over the years in linkage studies of chickens, there is a paucity of data involving the E locus. Extended black (E) was an obvious choice for inclusion in these studies. It is hypothesized that this could be due to the then little understood complexities of the genetics of the E alleles (see Smyth, 1990, for review). For example, a cross of extended black stocks to produce a heterozygote followed by the testcross mating back to the recessive stock could result in considerable difficulty in identifying some of the E alleles. This would be particularly true if the original black phenotype was actually a birchen (ER) with the addition of eu-
melanizing modifiers. Inability to classify the E locus segregation in the testcross with confidence could have resulted in data involving extended black not to have been published.
REFERENCES Bitgood, J. J., and R. G. Somes, Jr., 1990. Linkage relationships and gene mapping. Pages 469-495 in: Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier, New York, NY. Brumbaugh, J. A., and W. F. Hollander, 1965. A further study of the E pattern locus in the fowl. Iowa State J. Sci. 40:51-64. Carefoot, W. C, 1987. Relative positions of the loci of the peacomb (P), eumelanin restrictor (Db), eumelanin extension (Ml), and plumage pattern (Pg) genes of the domestic fowl. Br. Poult. Sci. 28:347-350. Carefoot, W. C , 1988. Inheritance of the spangled plumage pattern and the marbled chick down phenotypes of the Silver-Spangled Hamburgh bantam. Br. Poult. Sci. 29:785-790. Carefoot, W. C, 1990. Test for linkage between the eumelanin dilution blue (B0, the extended black (E) allele at the E-locus and the linked peacomb (P) and eumelanin extension (Ml) genes in the domestic fowl. Br. Poult. Sci. 31:465-472. Crawford, R. D., 1961. Breast ridge, associated with pea and walnut comb, as an aid in identifying comb type. Poultry Sci. 40262-263. Green, M. C, 1963. Methods for testing linkage. Pages 56-82 in: Methodology in Mammalian Genetics. W. J. Burdette, ed. Holden-Day, Inc., San Francisco, CA. Morejohn, G. V., 1955. Plumage color allelism in the Red Jungle Fowl (Gallus gallus) and related domestic forms. Genetics 40:519-530. Punnett, R. C , 1923. Heredity in Poultry. Macmillan and Co., Limited, London, England. Smyth, J. R., Jr., 1990. Genetics of plumage, skin and eye pigmentation in chickens. Pages 109-167 in: Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier, New York, NY. Somes, R. G., Jr., 1988. International Registry of Poultry Genetic Stocks. Bulletin 476, Storrs Agricultural Experiment Station, University of Connecticut, Storrs, CT.