Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Polymorphism (biology) wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Population genetics wikipedia , lookup
Dominance (genetics) wikipedia , lookup
Designer baby wikipedia , lookup
Microsatellite wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Genome (book) wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
X-inactivation wikipedia , lookup
Neocentromere wikipedia , lookup
History of genetic engineering wikipedia , lookup
vested from the plant and planted in the greenhouse. Seven plants were obtained and selfed. Selfed seeds from the greenhouse-grown plants were planted in the field in 1993 at the rate of one seed per 30 cm. Stands were not thinned after planting. Approximately 1 month after panicle emergence, plants were rated for panicle characteristics. Pollen was collected from one vigorous plant with a mutant phenotype during the 1993 evaluation. A sample of the pollen was used to pollinate the cytoplasmic male-sterile pearl millet, Tift 23DA*. The rest of the pollen was dried and stored (Hanna et al. 1983), and used to pollinate the Tift 23DA, X mutant selection hybrids to produce testcross progeny. Selfed seed of the mutant selection from 1993 were grown in the greenhouse to determine if the trait was expressed in greenhousegrown plants. Testcross progeny and remnant F, seed of Tift 23DA, X the 1993 mutant selection were grown in the field in 1994 as described above. Panicle characteristics of plants were evaluated approximately 1 month after panicle emergence. Observed ratios of normal/mutant plants in the F2 and testcross progenies were tested to Mendelian ratios with the X2 test. F2 progenies were evaluated with a heterogeneity x2 test to determine if segregation was consistent among families. Results and Discussion Progeny derived from open-pollinated seed from the original mutant had normal panicle characteristics. This indicated that the trait was either recessive, environmentally sensitive to expression, or not heritable. Because pearl millet is cross-pollinated, the plants derived from selfing the putative open-pollinated progenies in the greenhouse were assumed to be F2 progeny derived from random crosses between the mutant phenotype and the surrounding normal plants. In the field in 1993, segregation of panicle phenotype fit a 3:1 normal/mutant ratio in six of the seven F2 families (Table 1). The heterogeneity x2 value was significant, indicating that not all families segregated similarly. Family 4 affected the heterogeneity x2 value, but not the pooled x2 value. When data from family 4 was eliminated, the heterogeneity x2 was not significant (.25 < P < .10). Modifying genes that may have been derived from the unidentified random pollen source Table 1. Segregation of a bomeotic panicle mutation in progeny of pearl millet crosses Number of plants with panicle phenotype Generation" F2 Family 1 Family 2 Family 3 Family 4 Family 5 Family 6 Family 7 Pooled Heterogeneity Testcross Mutant Ex ted ratio 39 582 12 17 72 22 61 17 14 215 83 89 Normal 61 30 209 35 152 56 X2 P 3:1 3:1 3:1 3:1 3:1 3:1 3:1 3:1 2.854 3.128 0.058 5.620 1.504 0.114 0.057 1.639 13.335 .10 -.05 .10 -.05 .90 -.75 .025-.01* .25 -.10 .75 -.50 .90 -.75 .10 -.25 .05 -.025* 1:1 0.209 .50 -.75 " F2 families obtained by selfing plants grown from open-pollinated seed taken from original mutant observed in the field. Testcross progeny obtained from the cross (Tift 23DA, X mutant selection) X mutant selection. ' Significant. may have affected expression of the trait in this family. Selfed progeny of the vigorous mutant selected in 1993 expressed the mutant phenotype in the greenhouse in the winter greenhouse cycle of 1993-1994, indicating that the expression of the trait was not sensitive to environmental conditions. Panicle characteristics segregated in a 1:1 normal/mutant phenotypic ratio in the testcross progeny grown in the field in 1994 (Table 1). All F, plants of Tift 23DA4 X the 1993 mutant selection had normal panicles. It can be concluded from the segregation ratios that the mutant phenotype is controlled by a recessive allele at a single locus. The designation of phm, for phylloid homeotic mutant, is proposed for this allele. The mutant phenotype is more strongly expressed on the apical portion of the panicle. Generally, the imperfect male floret is normally formed, whereas the primary perfect floret is transformed into vegetative leaves with a meristematic region. If environmental conditions are favorable for further development, small poorly developed plants are formed. This atypical development can be classified as a phylloid homeotic mutant as defined by Acquaah et al. (1992). No other comparable homeotic mutant of pearl millet has been described previously. The present, apparently unique homeotic mutant will be useful as an easily identifiable marker in linkage studies, and may be of value in studies of floral morphogenesis. Pollen can be collected easily from the homozygous recessive phenotype, and thus it can be readily used as the pollen parent in crosses. Because of its erratic floral development within the panicle, it is difficult to use as a female parent in crosses. Seed of the homozygous recessive mutant will be maintained by the author, and limited quantities are available upon request. From the U.S. Department of Agriculture, Agricultural Research Service, University of Georgia Coastal Plain Experiment Station, Tifton, GA 31793. This article is the result of a cooperative investigation of USDA-ARS and the University of Georgia, College of Agricultural and Environmental Sciences, Agricultural Experiment Stations. I thank A. Hornbuckle for his technical assistance. The Journal of Heredity 1996:87(1) References Acquaah G, Saunders JW, and Ewart LC, 1992. Homeotic floral mutations. Plant Breed Rev 9:63-99. Hanna WW, Wells HD, Burton GW, and Monson WG, 1983. Long-term pollen storage of pearl millet. Crop Sci 23:174-175. Koduru PRK and Rao MK, 1983. Genetics of qualitative traits and linkage studies in pearl millet. Z Pflanzenzuchtg 90:1-22. Kumar KA and Andrews DJ, 1993. Genetics of qualitative traits in pearl millet: a review. Crop Sci 33:1-20. Received December 12, 1994 Accepted July 7, 1995 Corresponding Editor: Prem Jauhar Linkage Analysis of Endogenous Viral Element 1, Blue Eggshell, and Pea Comb Loci in Chickens J. R. Bartlett, C. P. Jones, and E. J. Smith Linkage analyses were conducted among endogenous viral element 1 (ev1), pea comb (P), and blue eggshell (O) loci, mapped to the short arm of chromosome 1 in chickens. The F, males were pro- Brief Communications 6 7 Figure 1. Analysis of evi amplification products for birds produced from a test cross of F, to Rhode Island Red hens. Lane Mb_ is a 100-bp ladder. Homozygous evi* or evl~ have either a 300- or 510-bp DNA fragment. Heterozygous evi birds have both fragments. duced from a cross between pea comb Araucana males negative for ev1 and known to be homozygous at the blue eggshell locus, and single comb White Leghorn females positive for ev1 and laying white shelled eggs. Recombination data were based on birds from a test cross of three F, birds, each mated to 10 single combed, brown eggshell laying Rhode Island Red (RIR) hens, negative for ev1 integration. Birds were screened for ev1 by DNA amplification using the polymerase chain reaction (PCR). Analyses of 207 progeny segregating for linkage between ev1 and P showed an estimated average map distance of 4.0 cM. Linkage analyses based on 44 progeny resulted in estimates of map distances of 1.8 and 4.1 cM between ev1 and O and P and O loci, respectively. Based on these estimates, the order of these three loci on the short arm of chromosome 1 can be inferred to be centromere, P, ev1, and O. As researchers move to develop a consensus chicken genetic map, the integration of molecular and classical maps provides a unique and difficult challenge. This difficulty is primarily due to the use of different reference populations on which the classical and molecular maps are based. An integrated linkage map provides obvious advantages. These advantages could include the use of morphological, rather 6 8 Trie Journal of Heredity 1996:87(1) than molecular markers, in marker-assisted selection programs if both are linked to genes with significant effect on a trait of economic importance in poultry. Endogenous viral elements, with sequences homologous to avian leukosis virus, have been reported to be stably integrated in many chicken populations, with a Mendelian mode of inheritance from one generation to the next (Smith 1986; Tereba et al. 1979). Earlier reports of work done in White Leghorns, for which most studies of evi genes have been conducted, suggested a random distribution of endogenous viral elements in the chicken genome (Crittenden 1991). More recent work, however, indicates a clustering of ev genes on chromosome 1 (Burke et al. 1994). There are conflicting reports of the chromosomal position of the endogenous viral element characterized by Astrin (1978) as evi. Based on in situ hybridization, Tereba and Astrin (1980) localized evi to the long arm of chromosome 1. More recently, observations by Bitgood and Crittenden reported in Bitgood (1993) suggested that evi is located on the short arm of chromosome 1 and is linked to the pea comb gene (P) locus at a map distance of <10 cM. Additionally, a recent report presented by Burke et al. (1994) supports the observation that evi is mapped to the short arm of chromosome 1. The objective of the present work was to evaluate linkage between evi and two other loci: O, the gene for blue eggshell, and P, previously reported to be linked and mapped to the short arm of chromosome 1 (Bitgood et al. 1980). This work provides an opportunity to further integrate the classical and molecular linkage maps of the chicken. Materials and Methods Animals and Mating Scheme The Araucona (AR), Rhode Island Red (RIR), and White Leghorn (WL) populations from which birds in the present studies were produced had been maintained for several generations as true breeding stock. Two WL males of genotype, based on evi, O, and P loci, evl+-o+-p+/evl+-o+- p+, were each mated to AR hens that were pea comb, laid blue shell eggs and were negative for evi integration and of genotype evl~-O-P/evl'-O-P. Three pea comb F, males from this mating, heterozygous at the evi (Figure 1), O, and P loci, were each mated to 10 RIR females that were single comb, laid brown shelled eggs, negative for evi, with a genotype of evl~-o+-p*l evl'o+-p*. Linkage analysis among the loci was based on data from both male and female progeny for P and evi, and for females only for P and 0, and 0 and evi. Figure 2. evi amplified PCR products analyzed on a 1.2% agarose gel and stained in ethidium bromide. Lane (Ln) B is control reaction without template. Lanes with AR, RIR, and WL prefix are amplified products from Araucona, Rhode Island Red and White Leghorn birds, respectively. Lane F,<3 is amplification product from a bird produced by mating Ln WL, birds to birds in Ln AR2. Homozygous evi* or evl~ have either a 300- or 510-bp DNA fragment. Heterozygous evi birds have both fragments. Ln Mbp is a 100-bp DNA ladder. Molecular Analysis DNA isolation. Blood collected in 0.5 M EDTA by brachial venipuncture was stored in 50-JJLI aliquots at -20°C until used. High molecular weight DNA was isolated from each 50-n-l aliquot by a modification of standard protocols (Sambrook et al. 1989). Briefly, 600 |il of lysis buffer (10 mM TrisCl, pH 8.0, 100 mM NaCl, 0.5% SDS, and 50 (jig/ml proteinase K) was added to each sample. Samples were then incubated for 6-12 h with gentle shaking at 55°C. Phenolchloroform extraction and ethanol precipitation were by conventional methods (Sambrook et al. 1989). Genotyping of birds for EVI. Birds were genotyped for evi by the polymerase chain reaction (PCR) using a modification (Smith et al. 1993) of the approach of Benkel et al. (1992). Briefly, the PCR amplification reaction was in a final volume of 50 \xi, containing 200 ng of genomic DNA as template, 200 \JM of each deoxyribonucleotide triphosphate, 2 mM MgCl2, and the primers PR-A, PR-B, and PR-C described by Benkel et al. (1992), each to a final concentration of 370, 330, and 462 ng, respectively, and 2.5 units of Taq Polymerase (Promega, Madison, WI). The reaction mixture was then overlaid with mineral oil. Cycling parameters for PCR in a PTC100 (MJ Research, Watertown, MA) were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 1 min each, at 94°C, 3 min at 49°C, and 3 min at 72°C for denaturation, annealing, and exten- Table 1. x ! analysis of segregation at the evi, O, and P loci Phenotype F° No" Endogenous viral element 1 Blue eggshell Pea comb Comb type and evi Egg color and evi Comb type and egg color 0.5 0.5 0.5 0.25 0.25 0.25 207 44 207 207 44 44 Probability 0.40 3.28 4.06 179.06 43.81 42.90 .10 .10 .10 .001 .001 .001 • Expected frequency of progeny within each phenotype from the test cross of F, males mated to Rhode Island Red females. * Total number of progeny analyzed by the polymerase chain reaction for each phenotype. sion, respectively. A final extension for 5 min was at 72°C. Twenty-five microliters of each amplification product was analyzed on a 1.2% agarose gel and stained with ethidium bromide. DNA from birds known to be positive for evi were included in the PCR analysis as a standard (a gift from Dr. Eugene Smith, Avian Disease and Oncology Lab, East Lansing, Ml). Statistical Analysis Independence of segregation and assortment at the evi, P, and O loci was tested by x2 (Green 1963). Linkage analysis was by the direct method based on the frequency of recombinant gametes as a measure of the distance between any of two loci (Ott 1991). Standard errors were estimated according to Green (1963) for a double back-cross in coupling. Results and Discussion Results of the PCR analysis of the backcross population indicated segregation at the evi locus (Figure 1) as expected from matings involving the heterozygous F, males (Figure 2) and homozygous evi' RIR hens. Birds homozygous for either evi* or evi' produced on amplification by Brief Communications 6 9 Table 2. Analysis of recombination data for linkage between evl and P Sire 4692 4691 4202 Total 79 76 52 207 N." cM 2 3 3 8 2.5 3.9 5.7 3.8 ± ± ± ± 0.06 0.06 0.07 0.04 " Where Nv is the total number of birds analyzed lor evl by the polymerase chain reaction within comb type. » Where N, is the number of recombinants for evl and P. PCR a fragment of size 300 or 510 bp as reported by Benkel et al. (1992) for evl. The co-dominance at this locus resulted in two amplified products, 300 and 510 bp, for birds heterozygous for evl. Segregation was normal at each of the evl, 0, and P loci. However, there was no random assortment among the three genes as determined by a x2 test of independence, suggesting linkage (Table 1). The deviation from the expected assortment was highly significant (P = .001). Linkage analysis between evl and P, evl and 0, and O and P for each F, male are presented in Tables 2, 3, and 4, respectively. Estimates of map distance between P and evl varied between males from 2.5 to 5.7 cM with an average of 4.0 cM (Table 2). Map distance between evl and O ranged from 0.0 to 5.5 cM with an average of 1.8 cM (Table 3). These results indicate that evl is on the short arm of chromosome 1 and linked to the P and 0 loci. This is contrary to the report by Tereba and Astrin (1980) that evl is localized to the long arm of chromosome 1, but consistent with previous reports by Burke et al. (1994) and Levin et al. (1994). Based on the recombination data from the crosses in the current work, the average map distance between P and O loci was 4.1 cM. This is consistent with previous reports suggesting linkage between these two loci. Bitgood et al. (1983) reported a map distance of 4.28 cM between these two loci. The classic work by Bruckner and Hutt (1939) tested only 35 birds and reported a map distance of 6 cM, and Table 3. Analysis of recombination data for linkage between evl and O Sire 4692 4691 4202 Total 17 9 18 44 N," cM 0 0 1 1 0 5.5 ± 0.12 2.3 ± 0.08 0 " Where Na is the total number of birds analyzed for evl by the polymerase chain reaction within eggshell color type. * Where A', is the number of recombinants for evl and 0. 7 0 The Journal of Heredity 1996:87(1) Table 4. Analysis of recombination data for linkage between 0 and P Crittenden LB, 1991. Retrovfral elements in the genome of the chicken: implications for poultry genetics and breeding. Crit Rev Poult Biol 3:73-109. Sire Green MC, 1963. Methods for testing linkage. In: Methodology in mammalian genetics (Burdette WJ, ed). San Francisco: Holden-Day. 4692 4691 4202 Total cM 17 9 18 44 1 0 1 2 6.7 ± 0.12 0.0 5.5 ± 0.12 4.5 ± 0.08 " Where A^ is the total number of birds analyzed for evl by the polymerase chain reaction within eggshell color type. " Where N, is the number of recombinants for 0 and P. Crawford (1986) calculated this distance to be 2.4 cM. Based on these results, the order of the three loci on chromosome 1 is determined to be centromere, P, evl, and O. The order of centromere, P and 0 is consistent with that reported by Bitgood (1985). However, due to the relatively small number of hens included in the analysis, the map distance and order of 0 and evl may need to be confirmed. From the Department of Agricultural Sciences, Tuskegee University, Tuskegee, AL 36088. We are grateful to Drs. T. F. Savage, Oregon State University, J. Bitgood, University of Wisconsin, and M. Egnin and S. Nahashon of Tuskegee University for suggestions and review of the manuscript, and to Paul Drummond for technical assistance. This work was funded in part by the U.S. Agency for International Development grant number PCE-5053-G-OO4017-00 and NSF HRD 94-50385. The Journal of Heredity 1996:87(1) References Astrin S, 1978. Endogenous viral genes of the White Leghorn chicken: common site of residence and sites associated with specific phenotypes of viral gene expression. Proc Natl Acad Sci USA 75:5941-5945. Benkel BF, Mucha J, and Gavora JS, 1992. A new diagnostic method for the detection of endogenous Rousassociated virus-type provirus in chickens. Poult Sci 71:1520-1526. Bitgood JJ, 1985. Locating pea comb and blue egg in relation to the centromere of chromosome 1 in the chicken. Poult Sci 64:1411-1414. Bitgood JJ, 1993. The genetic map of the chicken and availability of genetically diverse stocks. In: Manipulation of the chicken genome (Etches RJ and Verrinder Gibbins AM, eds). Boca Raton, Florida: CRC Press; 6179. Bitgood JJ, Otis JS, and Shoffner RN, 1983. Refined linkage value for pea comb and blue egg: lack of effect of pea comb, blue egg and naked neck on age at first egg in the domestic fowl. Poult Sci 62:235-238. Bitgood JJ, Shoffner RN, Otis JS, and Briles WE, 1980. Mapping of the genes for pea comb, blue egg, barring, silver, and blood groups A, E, H, and P in the domestic fowl. Poult Sci 59:1686-1693. Bruckner JH and Hutt FB, 1939. Linkage of pea comb and blue egg in the fowl. Science 90:88-89. Burke D, Ponce de Leon FA, and Smith EJ, 1994. Simultaneous assignment of eu/,3,6 and 12 loci in White Leghorn chickens. Poult Sci 73:4. Crawford RD, 1986. Linkage between pea comb and melanotic plumage loci in chickens. Poult Sci 65:1859— 1862. Crittenden LB, 1981. Exogenous and endogenous leukosis virus genes—a review. Avian Pathol 10:101-112. Levin I, Santangelo L, Cheng H, Crittenden LB, and Dodgson JB, 1994. An autosomal genetic linkage map of the chicken. J Hered 85:79-85. Ott J, 1991. Analysis of human genetic linkage. Baltimore: Johns Hopkins University Press. Sambrook J, Fritsch EF, and Maniatis T, 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press. Smith EJ, 1986. Endogenous avian leukemia viruses. In: Avian leukosis (De Boer GF, ed). Boston: Martinus Nijhoff; 101-120. Smith EJ, Bartlett JR, and Scott TR, 1993. Molecular analysis of chicken lines divergently selected for aflatoxin B, resistance. Poult Sci 72:79. Tereba A and Astrin SM, 1980. Chromosomal localization of evl, a frequently occurring endogenous retrovirus locus in White Leghorn chicken by in situ hybridization. J Virol 35:888-894. Tereba A, Lai MMC, and Murti KG, 1979. Chromosome 1 contains the endogenous RAV-O retrovirus sequence in chicken cells. Proc Natl Acad Sci USA 76:6486-6490. Received December 29, 1994 Accepted July 7, 1995 Corresponding Editor: Lyman Crittenden Genetic Regulation of Border Zone Formation in Female Mastomys (Praomys coucha) Adrenal Cortex S. Tanaka, M. Nozaki-Ukai, J. Kitoh, and A. Matsuzawa The unique border zone between the zona fasciculata and z. reticularis of the female adrenal cortex is formed in the wild-colored inbred mastomys (Praomys coucha) strain, MWC, but never in the chamois-colored inbred strain, MCC. This clear strainspecific trait was genetically analyzed using F,, F2, and backcross progenies produced between MWC and MCC. Reciprocal crosses gave no significant differences in the phenotypic ratio of F, or F2 progeny. Border zone formation was detected in 0% of F, females, 25.8% of F2 females, 0% of backcross females between F, and MCC, and 47.7% of backcross females between F, and MWC. From these results, it was concluded that border zone formation in the female MWC adrenal is regulated by a single autosomal recessive gene and this gene was named bzf (border zone formation). A specific border zone between the zona fasciculata and z. reticularis of the female