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Half tetrad analysis in angiosperms
J. theor. Biol. (1980) 86, 315-322
Half Tetrad Analysis in Angiosperms DANIEL University
J. SCHOEN
Department of Botany, of California, Berkeley, CA 94720, U.S.A.
(Received 23 November
1979, and in revised form 31 March
1980)
A biochemical technique involving analysis of endosperm is proposed for detecting meiotic crossovers and for gene mapping in angiosperms with bisporic embryo sacs. In bisporic embryo sac development two spores resulting from meiosis-II division of the same meiosis-I daughter cell contribute two thirds of the total genetic information to the triploid endosperm nucleus, the other third coming from a sperm in the fertilizing pollen grain. In controlled crosses where the marker gene codes for allozymes with phenotypes sensitive to gene dosage, the maternal meiotic contribution to the endosperm nucleus may be determined, thereby allowing crossovers between the marker locus and the centromere to be detected.
1. Introduction
The powerful genetic tool of tetrad analysis which allows genetic study of crossovers and provides a means of genetic mapping has hitherto been restricted to fungi and single-celled algae. This is unfortunate since in higher plants there is a lack of information on recombination due to meiotic crossing over. For example, almost all of our knowledge about genetic and environmental factors controlling crossover rates in plants comes from work on fungi such as Neurospora and Schizophylium (Catchside, 1968, 1975; Jha, 1969; Landner, 1970,197l; Nakamura, 1966; Simchen & Stamberg, 1969a,b; and others). Little is known about the genetics of crossover rates in higher plants (see Allard, 1963, for one example). Such knowledge would at present be especially welcome as it may contribute to an understanding of the evolution of recombination rates. Maynard Smith (1978) points to a lack of information on variance and response to selection of crossover rates. Models that he (Maynard Smith, 1979) and others (Charlesworth, 1976; Strobeck, Maynard Smith & Charlesworth, 1976) have forwarded to explain the evolution of recombination rates have assumed the existence of some genetic variance for the phenomenon, yet most of our knowledge in this area is derived from a small number of species. Others (Stebbins, 1958; Grant, meiotic
315
0022~5193/80/180315+08
$02.00/O
@ 1980 Academic Press Inc. (London) Ltd.
316
D.
J.
SCHOEN
1958) have suggested that long-lived organisms should have lower recombination rates (presumably with less variance) than short-lived organisms, but to date this hypothesis remains untested. The detection of crossovers between marker loci and centromeres would of course in itself be of value in mapping genes. A simpler mapping procedure might serve as a taxonomic or phylogenetic tool by allowing comparisons of genome organization in closely related taxa. In this paper I outline the basis for a new, but at this stage, untested, technique for detecting meiotic crossovers and for gene mapping in higher plants. The procedure involves the electrophoretic analysis of the genetic equivalents of half tetrads, and thus requires only a single gene marker for detection of a crossover between the marker locus and the centromere. It is applicable to plants with either long or short generation times. 2. Principles
of Crossover
Detection
in Bisporic
Angiosperms
Four requirements must be met by a plant in order for it to be employed in the proposed technique: (1) the plant must have bisporic embryo sac development (see below); (2) free endosperm must be present in the seed; (3) a polymorphic locus expressed in the endosperm and producing allozymes is the marker phenotype; and (4) the marker phenotype must be sensitive to allele dosage. Bisporic embryo sac development (Foster & Gifford, 1974; Johri, 1963; Maheshwari, 1963) begins with meiosis-I of a megaspore mother cell in the ovary of a developing flower (Fig. 1). Depending upon the plant species either the micropylar or chalazal daughter cell aborts following meiosis-I. The surviving daughter cell completes meiosis-II, leading to a cell with two haploid nuclei. Cell wall formation does not follow the second meiotic division, and each of the two haploid nuclei undergo two successive mitotic divisions to produce two quartets of nuclei, one at the micropylar end, and the other at the chalazal end of the embryo sac. Three of the micropylar nuclei differentiate into the egg apparatus, and three of the chalazal nuclei differentiate into the antipodals. The remaining two nuclei, one from each quartet (together containing half the genetic information of the original tetrad), become the pair of polar nuclei, and upon fertilization by a sperm from the pollen parent, the triploid endosperm nucleus. The endosperm, or nutritive tissue of the seed, is derived by successive mitotic divisions from the endosperm nucleus. Some possible consequences of bisporic embryo sac development for the alleles of two loci located on the same pair of homologous chromosomes are illustrated in Fig. 1. At locus A, first division segregation of the alleles
HALF
TETRAD
ANALYSIS
IN
ANGIOSPERMS
317
Meiosis II
Meiosis I
micropylar daughter cell aborts
megaspore mother cell
daughter
cell
FIG. 1. Bisporic embryo sac development and its consequences for genotypes of polar nuclei. (a)-(c), Megasporogenesis. (d)-(f), megametogenesis.
occurs, so that only one of the two alleles present in the heterozygous megaspore mother cell is represented in the surviving daughter cell of meiosis-I and pair of polar nuclei. However, at locus B, a crossover occurs between the locus and the centromere, resulting in second division segregation of the alleles at that locus, so that both the parental B locus alleles are represented in the surviving daughter cell of meiosis-I and pair of polar nuclei. Thus, the occurrence of different alleles at the same locus in the two
318
D.
J.
SCHOEN
polar nuclei of a single embryo sac in a heterozygous parent indicates that a crossover has taken place between that locus and the centromere during megasporogenesis. The problem of isolating and genotyping the polar nuclei can be resolved by assaying endosperm instead of the polar nuclei. The paternal genetic contribution to the endosperm genotype can be distinguished and subtracted from the polar nuclei contribution if a pollen parent homozygous for a known pair of alleles at the marker locus is used in the cross. 3. Selection of Parents
Pollen parents homozygous for a known pair of alleles at the marker locus could be obtained through inbred lines, however, with long-lived or selfincompatible plants it may be more desirable to select the appropriate parents from natural populations. Endosperm genotypes of potential pollen parents (in hermaphrodites) and potential seed parents in natural populations can be inferred by determining endosperm genotypes of seeds. For example, for a parent homozygous at the marker locus, the genetic contributions of polar nuclei to the endosperm genotypes of its seeds will consist of those resulting from both first and second division segregation of the marker alleles (Table 1, rows I-III). Among the contributions due to first division segregation of the marker alleles, approximately one-half will be homozygous for one of the alleles, and the other half will be homozygous for the alternative allele. This is due to random attachment of the spindle apparatus TABLE
Polar nuclei contributions
1
to endosperm genotypes Genotypes meiosis-I
Seed parent
(1) 01) (III) (IV) W)
genotype
AlAz AlAz Al.42 Al.41 AzAz
of surviving daughter cell and of polar nuclei contribution endosperm genotype
of to
A,A,t AA,+ A1.42: Al-41 AA2
Seed parents are either heterozygous or homozygous at diallelic marker locus A. Heterozygous parents produce three types of polar nuclei contributions (rows I-III). The two types of homozygous parents each produce one type of polar nuclei contribution to endosperm genotypes (rows IV and V). t First division segregation of marker alleles in megaspore mother cell. $ Second division segregation of marker alleles in megaspore mother cell.
HALF
TETRAD
ANALYSIS
IN
ANGIOSPERMS
319
to the homologous chromosomes at meiosis-I (and thus, random movement of the homologs into either of the daughter cells of meiosis-I), followed by survival of only one of the daughter cells during bisporic embryo sac development. Therefore, after pollination, a parent heterozygous at the marker locus is expected to produce two classes of seed resulting from first division segregation: (1) those with endosperm genotypes having double or triple doses of one marker allele; and (2) those with endosperm genotypes having double or triple doses of the alternative marker allele. Recovery of these two classes of seed from a single parent identifies the parent as a heterozygote. A homozygous parent can produce only seeds having double or triple doses of one marker allele (Table 1, rows IV and V). To distinguish between the four combinations of two alleles possible at a diallelic locus in triploid tissue, it is necessary to employ a marker with a phenotype sensitive to allele dosage (e.g. alcohol dehydrogenase, catalase, leucine amino peptidase, esterase, and phosphoglucoisomerase) (Scandalios, 1969; Nielsen & Frydenberg, 1971; Nielsen & Scandalios, 1974; Ott & Scandalios, 1978; and Schoen, unpubl. data). Allele dosage can be determined by quantifying the relative staining intensities of the reaction product-stain complex bands formed following electrophoresis and staining. For instance, at a locus with two codominant alleles, two types of heterozygotes are expected-AlAlA* and AIAzAz. If gene A codes for a monomeric enzyme with allele Al coding for the fast migrating form and allele A2 coding for the slow migrating form, the genotype A 1A 1AZwill be expressed as two bands on a gel, the fast migrating band having twice the staining intensity of the slow migrating band (Scandalios, 1969). The genotype AIAzAz will produce a similar two-banded phenotype, except that the staining intensities of the bands will be the reverse of that seen in the former genotype.
4. Crossover
Detection
in Practice
Once the endosperm genotypes of potential pollen parents and seed parents have been inferred via the method outlined above, appropriate parents can be chosen and crosses can be made for studying crossovers and for gene mapping. For example, in the cross of a seed parent heterozygous at the marker locus (e.g. A1A2) with a pollen parent homozygous at the locus (e.g. A 1A 1), each seed produced will have one of three possible endosperm genotypes. Endosperm genotypes A 1A 1Af (paternal contribution starred here and below) and AzAzAT are the result of fertilizations of polar nuclei in which no single crossover between the marker locus and centromere has occurred. On the other hand, an endosperm genotype of A1A2Af,
320
D.
J.
SCHOEN
produced as a result of the above-described cross, must be due to a crossover between the marker locus and the centromere during megasporogenesis. 5. Genetic Mapping
Frequencies of recombination between marker loci and centromeres have been used in the mapping of genes in fungi via tetrad analysis (Fincham & Day, 1971). The conceptually similar method proposed here could be used in centromere mapping in higher plants. Members of the genetically wellstudied genus Nicotiana are likely candidates for such a project as they fulfil the morphological requirements of the method. With more than one locus available for study it is possible to determine linkage relationships and to measure centromere to locus distance for each locus. Table 2 gives the most frequent classes of tetrads, together with their corresponding half tetrads, obtained following meiosis in a double heterozygote (Fincham & Day, 1971). Tetrad (i.e. ascus) classes 5,6, and 7, in fungi all correspond to one half tetrad class (i.e. one polar nuclei pair class) obtained in bisporic angiosperms. This is because half tetrad classes in this technique are distinguishable only by differences in allele dosage, and half tetrad classes 5,6, and 7 are identical in this feature. Thus, there are five classes (not seven as in tetrad analysis) obtained via the proposed technique. TABLE
Classes
of tetrads
2
and half tetrads obtained following A1A2B1B2 double heterozygote
in an
(6)
(7) A,Bl Ad% AI& Ad31
Tetrad
(1) AIB, AlBl Ad% A& (1) AIBI AlBl
(3) A,Bl AI& &BI Ad&
(2) AI& AI&
classes (4) AI& A& AIBI Ad?, Half tetrad classes
meiosis
(3) AlBl AI&
(2,%2,0)
GO, 0,2)
c&0,1,1)
or
or
0, AzBt Ad&
A& A&
(0,2,0,2)
Ad1 AzBl
(0,2,2,0)
K4z
191)
(4) Al& AZ& (1,1,0,2) or AlBl AzB, (1, 1,2,0)
(5) AIBI A& AlBl Ai%
AI& ASI AI& A81 (5)
A,B, Ad%
A,& A91 (l,l,
A;, Ad%
1, 1) or AI& A#I (1, 1, 131)
\ ALE, AZ& or AI& Ad31
Half tetrads are distinguishable only by allele dosage differences between them, as outlined in the text. Numbers in parentheses, beneath the half tetrads indicate allele dosage in the order Al, Al, B1, B2 for each half tetrad.
HALF
TETRAD
ANALYSIS
IN
ANGIOSPERMS
321
Nevertheless, it is still possible to use data on the frequency of these classes together with the principles of tetrad analysis (Fincham & Day, 1971) to determine whether the loci are linked or segregating independently, and if they are linked, what their relative positions are with respect to each other and the centromere. 6. Some Limitations
Bisporic embryo sac development occurs in at least 41 angiosperm families (Maheshwari, 1955; Davis, 1966), however, only those plants having seeds with free endosperm are useable in the technique outlined. The amount of endosperm required for the technique is not large. In assaying endosperm for other types of studies I have used seeds weighing O-5 mg (dry weight) (Schoen, 1979,198O). This is below the median seed weight class for the California flora (Baker, 1972). Among the economically important families with bisporic embryo sac development are the Solanaceae and the Amaryllidaceae. In addition, many members of tropical families have bisporic embryo sacs. This would perhaps allow comparisons to be made between members of tropical and temperate floras for genetic and environmental factors controlling recombination. Another limitation is the number of known enzyme assays, and thus the number of loci employable in the technique. The ability to detect allele dosage effects with these loci may limit the application of half tetrad analysis in higher plants. Presumably, as more sophisticated electrophoretic methods are developed some of these limitations will become less severe. I thank H. G. Baker, D. Fruchter, R. Ornduff, P. Schultz, P. T. Spieth, and an anonymous reviewer for helpful advice and criticism. This work was supported by National Science Foundation Grant DEB 78-10613 and by a grant from the University of California Chancellor’s Patent Fund. REFERENCES ALLARD, R. W. (1962). Genetics 48, 1389. BAKER. H. G. (1972). ECO~OW53.997. CATCHSIDE, b. G. i1968).%1 R&cation and Recombination of Genetic Material (W. J. Peacock & R. D. Brock, eds). Canberra: Australian Academy of Science. CATCHSIDE, W. J. (1975). Aust. J. biol. Sci. 28, 213. CHARLESWORTH, B. (1976). Genetics 83, 181. DAVIS, G. L. (1966). Systematic Embryology of Angiosperms. New York: Wiley. FINCHAM. J. R. S. & DAY. P. R. (19711. Funnal Genetics. 3rd edn. London: Blackwell. FOSTER, A. S. & GIFFOR;, E. M: (19?4). Cimparatioe Ikorphology of Vascular Plants, 2nd edn. San Francisco: Freeman. GRANT, V. (1958). Cold Spring Harbor Symp. Quant. Biol. 23,337.
322
D.
J.
SCHOEN
JHA, K. K. (1969). Molec. gen. Genet. 105, 30. LANDNER, L. (1970). Molec. gen. Genet. 109,219. LANDNER, L. (1971). Heredity 27, 385. MAHESHWARI, P. (1950). An Introduction to the Embr\lology of Angiosperms. New York McGraw-Hill. MAHESHWARI, S. C. (1955). Phytomorphology 5, 67. MAYNARD SMITH, J. (1978). The Evolution of Se+. London: Cambridge University Press NAKAMURA, K. (1966). Genetica 37,235. NIELSEN, G. & FRYDENBERG, 0. (1971). Hereditas 67, 152. NIELSEN, G. & SCANDALIOS, J. G. (1974). Genetics 77.697. OTT, L. & SCANDALIOS, J. G. (1978). Genetics 89, 137. SCANDALIOS, J. G. (1969). Biochem. Genet. 3,365. SCHOEN, D. J. (1979), J. theor. Biol. 79, 543. SCHOEN, D. J. (1980). In prep. SIMCHEN,G.& STAMBERG,J. (1969a). Nature 222,329. SIMCHEN,G. & STAMBERG,J. (1969b). Heredity 24, 369. STEBBINS, G. L. (1958). Cold Spring Harbor Symp. Quant. Eiol. 23,365. STROBECK,C.,MAYNARD SMITH,J.& CHARLESWORTH.~~.(~~~~). Genetics 82,547.
Half Tetrad Analysis in Angiosperms DANIEL University
J. SCHOEN
Department of Botany, of California, Berkeley, CA 94720, U.S.A.
(Received 23 November
1979, and in revised form 31 March
1980)
A biochemical technique involving analysis of endosperm is proposed for detecting meiotic crossovers and for gene mapping in angiosperms with bisporic embryo sacs. In bisporic embryo sac development two spores resulting from meiosis-II division of the same meiosis-I daughter cell contribute two thirds of the total genetic information to the triploid endosperm nucleus, the other third coming from a sperm in the fertilizing pollen grain. In controlled crosses where the marker gene codes for allozymes with phenotypes sensitive to gene dosage, the maternal meiotic contribution to the endosperm nucleus may be determined, thereby allowing crossovers between the marker locus and the centromere to be detected.
1. Introduction
The powerful genetic tool of tetrad analysis which allows genetic study of crossovers and provides a means of genetic mapping has hitherto been restricted to fungi and single-celled algae. This is unfortunate since in higher plants there is a lack of information on recombination due to meiotic crossing over. For example, almost all of our knowledge about genetic and environmental factors controlling crossover rates in plants comes from work on fungi such as Neurospora and Schizophylium (Catchside, 1968, 1975; Jha, 1969; Landner, 1970,197l; Nakamura, 1966; Simchen & Stamberg, 1969a,b; and others). Little is known about the genetics of crossover rates in higher plants (see Allard, 1963, for one example). Such knowledge would at present be especially welcome as it may contribute to an understanding of the evolution of recombination rates. Maynard Smith (1978) points to a lack of information on variance and response to selection of crossover rates. Models that he (Maynard Smith, 1979) and others (Charlesworth, 1976; Strobeck, Maynard Smith & Charlesworth, 1976) have forwarded to explain the evolution of recombination rates have assumed the existence of some genetic variance for the phenomenon, yet most of our knowledge in this area is derived from a small number of species. Others (Stebbins, 1958; Grant, meiotic
315
0022~5193/80/180315+08
$02.00/O
@ 1980 Academic Press Inc. (London) Ltd.
316
D.
J.
SCHOEN
1958) have suggested that long-lived organisms should have lower recombination rates (presumably with less variance) than short-lived organisms, but to date this hypothesis remains untested. The detection of crossovers between marker loci and centromeres would of course in itself be of value in mapping genes. A simpler mapping procedure might serve as a taxonomic or phylogenetic tool by allowing comparisons of genome organization in closely related taxa. In this paper I outline the basis for a new, but at this stage, untested, technique for detecting meiotic crossovers and for gene mapping in higher plants. The procedure involves the electrophoretic analysis of the genetic equivalents of half tetrads, and thus requires only a single gene marker for detection of a crossover between the marker locus and the centromere. It is applicable to plants with either long or short generation times. 2. Principles
of Crossover
Detection
in Bisporic
Angiosperms
Four requirements must be met by a plant in order for it to be employed in the proposed technique: (1) the plant must have bisporic embryo sac development (see below); (2) free endosperm must be present in the seed; (3) a polymorphic locus expressed in the endosperm and producing allozymes is the marker phenotype; and (4) the marker phenotype must be sensitive to allele dosage. Bisporic embryo sac development (Foster & Gifford, 1974; Johri, 1963; Maheshwari, 1963) begins with meiosis-I of a megaspore mother cell in the ovary of a developing flower (Fig. 1). Depending upon the plant species either the micropylar or chalazal daughter cell aborts following meiosis-I. The surviving daughter cell completes meiosis-II, leading to a cell with two haploid nuclei. Cell wall formation does not follow the second meiotic division, and each of the two haploid nuclei undergo two successive mitotic divisions to produce two quartets of nuclei, one at the micropylar end, and the other at the chalazal end of the embryo sac. Three of the micropylar nuclei differentiate into the egg apparatus, and three of the chalazal nuclei differentiate into the antipodals. The remaining two nuclei, one from each quartet (together containing half the genetic information of the original tetrad), become the pair of polar nuclei, and upon fertilization by a sperm from the pollen parent, the triploid endosperm nucleus. The endosperm, or nutritive tissue of the seed, is derived by successive mitotic divisions from the endosperm nucleus. Some possible consequences of bisporic embryo sac development for the alleles of two loci located on the same pair of homologous chromosomes are illustrated in Fig. 1. At locus A, first division segregation of the alleles
HALF
TETRAD
ANALYSIS
IN
ANGIOSPERMS
317
Meiosis II
Meiosis I
micropylar daughter cell aborts
megaspore mother cell
daughter
cell
FIG. 1. Bisporic embryo sac development and its consequences for genotypes of polar nuclei. (a)-(c), Megasporogenesis. (d)-(f), megametogenesis.
occurs, so that only one of the two alleles present in the heterozygous megaspore mother cell is represented in the surviving daughter cell of meiosis-I and pair of polar nuclei. However, at locus B, a crossover occurs between the locus and the centromere, resulting in second division segregation of the alleles at that locus, so that both the parental B locus alleles are represented in the surviving daughter cell of meiosis-I and pair of polar nuclei. Thus, the occurrence of different alleles at the same locus in the two
318
D.
J.
SCHOEN
polar nuclei of a single embryo sac in a heterozygous parent indicates that a crossover has taken place between that locus and the centromere during megasporogenesis. The problem of isolating and genotyping the polar nuclei can be resolved by assaying endosperm instead of the polar nuclei. The paternal genetic contribution to the endosperm genotype can be distinguished and subtracted from the polar nuclei contribution if a pollen parent homozygous for a known pair of alleles at the marker locus is used in the cross. 3. Selection of Parents
Pollen parents homozygous for a known pair of alleles at the marker locus could be obtained through inbred lines, however, with long-lived or selfincompatible plants it may be more desirable to select the appropriate parents from natural populations. Endosperm genotypes of potential pollen parents (in hermaphrodites) and potential seed parents in natural populations can be inferred by determining endosperm genotypes of seeds. For example, for a parent homozygous at the marker locus, the genetic contributions of polar nuclei to the endosperm genotypes of its seeds will consist of those resulting from both first and second division segregation of the marker alleles (Table 1, rows I-III). Among the contributions due to first division segregation of the marker alleles, approximately one-half will be homozygous for one of the alleles, and the other half will be homozygous for the alternative allele. This is due to random attachment of the spindle apparatus TABLE
Polar nuclei contributions
1
to endosperm genotypes Genotypes meiosis-I
Seed parent
(1) 01) (III) (IV) W)
genotype
AlAz AlAz Al.42 Al.41 AzAz
of surviving daughter cell and of polar nuclei contribution endosperm genotype
of to
A,A,t AA,+ A1.42: Al-41 AA2
Seed parents are either heterozygous or homozygous at diallelic marker locus A. Heterozygous parents produce three types of polar nuclei contributions (rows I-III). The two types of homozygous parents each produce one type of polar nuclei contribution to endosperm genotypes (rows IV and V). t First division segregation of marker alleles in megaspore mother cell. $ Second division segregation of marker alleles in megaspore mother cell.
HALF
TETRAD
ANALYSIS
IN
ANGIOSPERMS
319
to the homologous chromosomes at meiosis-I (and thus, random movement of the homologs into either of the daughter cells of meiosis-I), followed by survival of only one of the daughter cells during bisporic embryo sac development. Therefore, after pollination, a parent heterozygous at the marker locus is expected to produce two classes of seed resulting from first division segregation: (1) those with endosperm genotypes having double or triple doses of one marker allele; and (2) those with endosperm genotypes having double or triple doses of the alternative marker allele. Recovery of these two classes of seed from a single parent identifies the parent as a heterozygote. A homozygous parent can produce only seeds having double or triple doses of one marker allele (Table 1, rows IV and V). To distinguish between the four combinations of two alleles possible at a diallelic locus in triploid tissue, it is necessary to employ a marker with a phenotype sensitive to allele dosage (e.g. alcohol dehydrogenase, catalase, leucine amino peptidase, esterase, and phosphoglucoisomerase) (Scandalios, 1969; Nielsen & Frydenberg, 1971; Nielsen & Scandalios, 1974; Ott & Scandalios, 1978; and Schoen, unpubl. data). Allele dosage can be determined by quantifying the relative staining intensities of the reaction product-stain complex bands formed following electrophoresis and staining. For instance, at a locus with two codominant alleles, two types of heterozygotes are expected-AlAlA* and AIAzAz. If gene A codes for a monomeric enzyme with allele Al coding for the fast migrating form and allele A2 coding for the slow migrating form, the genotype A 1A 1AZwill be expressed as two bands on a gel, the fast migrating band having twice the staining intensity of the slow migrating band (Scandalios, 1969). The genotype AIAzAz will produce a similar two-banded phenotype, except that the staining intensities of the bands will be the reverse of that seen in the former genotype.
4. Crossover
Detection
in Practice
Once the endosperm genotypes of potential pollen parents and seed parents have been inferred via the method outlined above, appropriate parents can be chosen and crosses can be made for studying crossovers and for gene mapping. For example, in the cross of a seed parent heterozygous at the marker locus (e.g. A1A2) with a pollen parent homozygous at the locus (e.g. A 1A 1), each seed produced will have one of three possible endosperm genotypes. Endosperm genotypes A 1A 1Af (paternal contribution starred here and below) and AzAzAT are the result of fertilizations of polar nuclei in which no single crossover between the marker locus and centromere has occurred. On the other hand, an endosperm genotype of A1A2Af,
320
D.
J.
SCHOEN
produced as a result of the above-described cross, must be due to a crossover between the marker locus and the centromere during megasporogenesis. 5. Genetic Mapping
Frequencies of recombination between marker loci and centromeres have been used in the mapping of genes in fungi via tetrad analysis (Fincham & Day, 1971). The conceptually similar method proposed here could be used in centromere mapping in higher plants. Members of the genetically wellstudied genus Nicotiana are likely candidates for such a project as they fulfil the morphological requirements of the method. With more than one locus available for study it is possible to determine linkage relationships and to measure centromere to locus distance for each locus. Table 2 gives the most frequent classes of tetrads, together with their corresponding half tetrads, obtained following meiosis in a double heterozygote (Fincham & Day, 1971). Tetrad (i.e. ascus) classes 5,6, and 7, in fungi all correspond to one half tetrad class (i.e. one polar nuclei pair class) obtained in bisporic angiosperms. This is because half tetrad classes in this technique are distinguishable only by differences in allele dosage, and half tetrad classes 5,6, and 7 are identical in this feature. Thus, there are five classes (not seven as in tetrad analysis) obtained via the proposed technique. TABLE
Classes
of tetrads
2
and half tetrads obtained following A1A2B1B2 double heterozygote
in an
(6)
(7) A,Bl Ad% AI& Ad31
Tetrad
(1) AIB, AlBl Ad% A& (1) AIBI AlBl
(3) A,Bl AI& &BI Ad&
(2) AI& AI&
classes (4) AI& A& AIBI Ad?, Half tetrad classes
meiosis
(3) AlBl AI&
(2,%2,0)
GO, 0,2)
c&0,1,1)
or
or
0, AzBt Ad&
A& A&
(0,2,0,2)
Ad1 AzBl
(0,2,2,0)
K4z
191)
(4) Al& AZ& (1,1,0,2) or AlBl AzB, (1, 1,2,0)
(5) AIBI A& AlBl Ai%
AI& ASI AI& A81 (5)
A,B, Ad%
A,& A91 (l,l,
A;, Ad%
1, 1) or AI& A#I (1, 1, 131)
\ ALE, AZ& or AI& Ad31
Half tetrads are distinguishable only by allele dosage differences between them, as outlined in the text. Numbers in parentheses, beneath the half tetrads indicate allele dosage in the order Al, Al, B1, B2 for each half tetrad.
HALF
TETRAD
ANALYSIS
IN
ANGIOSPERMS
321
Nevertheless, it is still possible to use data on the frequency of these classes together with the principles of tetrad analysis (Fincham & Day, 1971) to determine whether the loci are linked or segregating independently, and if they are linked, what their relative positions are with respect to each other and the centromere. 6. Some Limitations
Bisporic embryo sac development occurs in at least 41 angiosperm families (Maheshwari, 1955; Davis, 1966), however, only those plants having seeds with free endosperm are useable in the technique outlined. The amount of endosperm required for the technique is not large. In assaying endosperm for other types of studies I have used seeds weighing O-5 mg (dry weight) (Schoen, 1979,198O). This is below the median seed weight class for the California flora (Baker, 1972). Among the economically important families with bisporic embryo sac development are the Solanaceae and the Amaryllidaceae. In addition, many members of tropical families have bisporic embryo sacs. This would perhaps allow comparisons to be made between members of tropical and temperate floras for genetic and environmental factors controlling recombination. Another limitation is the number of known enzyme assays, and thus the number of loci employable in the technique. The ability to detect allele dosage effects with these loci may limit the application of half tetrad analysis in higher plants. Presumably, as more sophisticated electrophoretic methods are developed some of these limitations will become less severe. I thank H. G. Baker, D. Fruchter, R. Ornduff, P. Schultz, P. T. Spieth, and an anonymous reviewer for helpful advice and criticism. This work was supported by National Science Foundation Grant DEB 78-10613 and by a grant from the University of California Chancellor’s Patent Fund. REFERENCES ALLARD, R. W. (1962). Genetics 48, 1389. BAKER. H. G. (1972). ECO~OW53.997. CATCHSIDE, b. G. i1968).%1 R&cation and Recombination of Genetic Material (W. J. Peacock & R. D. Brock, eds). Canberra: Australian Academy of Science. CATCHSIDE, W. J. (1975). Aust. J. biol. Sci. 28, 213. CHARLESWORTH, B. (1976). Genetics 83, 181. DAVIS, G. L. (1966). Systematic Embryology of Angiosperms. New York: Wiley. FINCHAM. J. R. S. & DAY. P. R. (19711. Funnal Genetics. 3rd edn. London: Blackwell. FOSTER, A. S. & GIFFOR;, E. M: (19?4). Cimparatioe Ikorphology of Vascular Plants, 2nd edn. San Francisco: Freeman. GRANT, V. (1958). Cold Spring Harbor Symp. Quant. Biol. 23,337.
322
D.
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