INCOMPATIBILITY RELATIONSHIPS IN CERTAIN COMPLEX-HETEROZYGOTES OF OENOTHERA

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INCOMPATIBILITY RELATIONSHIPS IN CERTAIN COMPLEX-HETEROZYGOTES OF OENOTHERAI

MARY E. SCHULTZ

Department of Botany, University of Michigan, Ann Arbor, Michigan Received March 6 , 1962

H E North American oenotheras of the subgenus Euoenothera have been Tclassified by CLELAND (1950) and his associates into nine phylogenetic groups on the basis of their cytogenetic behavior. Each group is composed of a large number of relatively isolated races each of which breeds true and represents generally a single genotype. The present study involves mainly the phylogenetic groups designated by CLELAND as biennis group 2 and biennis group 3, and to a some- what lesser extent biennis group 1.

The oenotheras of these groups are self -pollinating permanent translocation heterozygotes. Multiple reciprocal translocation results in all of the 14 chromosomes forming at meiosis a single ring in which the chromosomes of maternal and paternal origin alternate. Adjacent members of the ring go to opposite poles, which means that all maternal chromosomes are separated from all paternal chromosomes. Consequently, only two kinds of gametes are formed by an individual and these are genetically identical with the gametes from which it originated. Two chromosome complements, therefore, are transmitted intact from generation to generation, each of these in effect composing a single linkage group which RENNER calls a “complex.”

Outcrossing data show that one complex, designated as “alpha,” is regularly transmitted through the egg and the other complex, or “beta,” is transmitted predominantly through the pollen. These data also show that in some races the beta complex, as well as the alpha, may be transmitted through the egg and both complexes may come through the pollen in varying frequencies. I n nature, however, these self-pollinating forms generally produce only heterozygous combinations of these complexes, or “alpha.betas.” Individuals which combine two identical complexes, that is, “alpha.alphas” and “beta.betas,” are rarely or never formed from selfing. Their absence is attributed to lethals which prevent the occurrence of the homozygous forms. In the alpha.betas these lethals are balanced; the structural heterozygosity of the alpha.betas prevents the lethals from segregating. Although the naturally occurring forms are heterozygous for the alpha and beta complexes, STEINER ( 1956, 1957) produced structurally homozygous alpha.alpha and beta.beta combinations by intercrossing biennis 1 races. The alphaalpha hybrids are self-incompatible. The beta.betas, on the other hand, are self-fertile.

This paper is from a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the University of Michigan. Cost of extra pages paid for by the Department of Botany.

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820 MARY E. SCHULTZ

STEINER showed that the self-incompatibility of the alphaalphas results from a n inhibition of pollen tube growth and can be explained on the basis of the gametophytic oppositional system first proposed by PRELL (1921 ) but better known from the work of EAST and MANGELSDORF (1925) on Nicotiana sanderae. This system involves a single locus, S, which exists in a very large number of allelic forms. The data supported the hypothesis that the alpha complex of each biennis 1 race carries a different allele. The control of the pollen reaction is gametophytic and the two alleles in the style act individually with no interaction. Pollen tube growth is inhibited in a style carrying the same S allele as the pollen tube; or stated more generally and in reference to these oenotheras, pollen tubes fail to grow down a style in which tube and style contain the same biennis 1 alpha complex.

I n self-pollinations the S alleles effectively eliminate the alpha pollen and only the beta pollen with its Sf allele is functional. Both kinds of pollen are functional in cross-pollinations because the alpha pollen carries a different S allele and the beta has no incompatibility allele.

If the alpha complexes of other groups of complex-heterozygotes in Euoeno- thera carry S alleles, an incompatibility mechanism will explain the pollen lethal in these forms. The purpose of the present investigation is to determine whether incompatibility alleles are present in the alpha complexes of biennis 2 and biennis 3 and if present to study their behavior in outcrosses.

MATERIALS AND METHODS

The transmission of the alpha complex through the pollen was studied in 14 biennis 2 and biennis 3 races seed of which was obtained from D R . RALPH E.

CLELAND and DR. HARRY T. STINSON. In the summer of 1956 these races were intercrossed in nearly all of the possible combinations and each was also used as the female parent in crosses with biennis 1 races and alpha.alpha combinations. The hybrids were identified phenotypically and confirmed cytologically on the basis of the chromosome configurations in the pollen mother cells at metaphase. In all instances the compatibility tests include both the observation of pollen tube growth in stained style preparations and seed set. The results obtained from style preparations, which proved to be a critical test of compatibility, represent crosses made on emasculated flowers removed from the plant and placed in a coistant temperature oven set at ) "C. All compatibility tests were performed at least four times with the same plants as well as with different plants having the same genetic constitution. Although these forms are self-pollinating, selfs were hand-pollinated to assure an adequate supply of pollen. Flowers used to test cross-compatibilities were emasculated 24-30 hours before pollination. Ten to 24 hours after pollination the style was fixed in acetic alcohol, stained with lactic acid-IKI ( STEINER 1957), and observed for pollen tube growth.

RESULTS

Synthesis of a l p h a l p h a combinations

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COMPLEX HETEROZYGOTES 821

races of biennis 2 and biennis 3 produced seven different alphaalpha combinations. Two races belonging to biennis 3 transmitted the alpha complex through the pollen: Linville in four crosses and Newfound Gap in two crosses. Victorini, a biennis 2 race, transmitted alpha through the pollen in one cross.

The alphaalpha combinations could be distinguished from the alpha.betas phenotypically, and in all but one culture they reached maturity earlier than the alpha.betas. The one exception was for a culture composed entirely of alpha.alphas in which such a comparison could not be made. Identification of these combinations were confirmed cytologically. Beta.beta homozygotes were not found among these progenies.

All classes of progeny were selfed. The synthesized alphabetas were self- fertile as they are in nature. The alpha.alphas, on the other hand, failed to produce seed upon selfing. Style preparations were made at various intervals during a 48-hour period following self-pollination, and in all cases no pollen tubes were observed in the stylar tissue. In several instances the pollen grains had not germinated, while in others germination occurred but the pollen tubes failed to penetrate the stigma. It is upon the self-incompatibility of these alphaalpha combinations that this study is focused.

Crosses using biennis 1 alpha.alphas as pollen parents: Crosses in which biennis 2 and biennis 3 races were used as the seed parent and biennis 1 alpha.alphas as the pollen parent gave progenies composed almost entirely of alpha.alpha combinations. A single beta.alpha was found in one culture due to the transmission of the beta complex through the egg. The results of these crosses are listed in Table 1. These alpha.alpha combinations were also found to be self-incompatible. Pollen tubes carrying a biennis 1 alpha complex were not expected to grow in styles carrying the same complex since they are known to carry SI alleles (STEINER 1956, 1957). This is evidence that biennis 2 and biennis 3 alpha complexes also possess a self-incompatibility system.

Each culture listed in Table 1 was composed of two compatibility classes according to the SI alleles contributed by the pollen parent. Each complex possesses a different SI allele so that in a cross such as Coudersport I11 x aHot

TABLE 1

Occurrence of alphadphas in crosses with biennis 2 and biennis 3 races as seed parents and biennis I alphaalphas as pollen parents

Culture no. cross lla /3rr

738 Coudersport I11 x aHot SpringsaBirch Tree 1 38 1 739 Coudersport I11 x &amp Peary EaBirch Tree 2 7 0

741 Smethport x aHot SpringsaBirch Tree I 40 0

740 Smethport x aPaducah.aBirch Tree 1 4!9 0

733 Waterbury x aHot Springs.aBirch Tree 1 4.3 0

734 Waterbury x &amp Peary E.aBirch Tree 2 49 0

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a22 MARY E. SCHULTZ

SpringmBirch Tree 1 approximately equal numbers of the following combinations occurred among the progeny:

acoudersport III.aHot Springs (S,) acoudersport 111-aBirch Tree 1 ( S5)

Inasmuch as the pollen parents are structural homozygotes the combinations produced by this type of cross could not be cytologically confirmed. That is, both biennis 1 alpha complexes combined in the pollen parent have identical chromosome end arrangements so that all progeny of a cross have the same metaphase configurations. Also, pairing and random segregation of chromosomes occurs in the structural homozygotes giving considerable phenotypic variation within a single culture; thus phenotype could not be used as a criterion for identification. The progenies were analyzed on the basis of biennis S , alleles. Since intact biennis 1 alpha complexes are no longer involved, the identification is made only of the S, allele that originated with each complex. However, in order to avoid what would appear to be an unnecessary introduction of symbols the SI alleles will be referred to in this analysis by their complex name.

The identification of the biennis 1 S allele of each hybrid was made in the following way. Each culture was divided into two compatibility classes by pollinating all members with a test plant from the same progeny. Plants which were incompatible with the pollen from the test plant composed the first class and those which were compatible the second class. The specific biennis 1 S allele represented in the hybrids of each class VI as then identified through analysis of

their compatibility reactions with members of different cultures.

Crosses using biennis 2 and biennis 3 alphaalphas as pollen parents: In the following summer progeny were grown from crosses in which biennis 2 and biennis 3 races were used as the seed parent and alpha.alpha combinations belonging to the same groups as the polleii parent. The races are: Coudersport I, Coudersport 11, Tonawanda I, Tonawanda 11, and Williamsville. Pollen was used from the following combinations:

acoudersport 1II.aLinville aSmethportaLinville aVictorini.aLinville @Waterbury alinville.

The method used in analyzing the progeny of the seven-paired, or structurally homozygous, biennis 1 alpha.alphas was unnecessary here. All alphadphas in the preceding list show a circle of 14 chrcjmosomes at metaphase, thus the complexes combined in these forms were transmitted intact. The progenies were phenotypically distinct and could be confirmed cytologically.

Alpha Linville was transmitted through the pollen exclusively in these crosses. Alpha Coudersport 111, asmethport, LuVictorini, and &Waterbury failed as pollen complexes. All alpha.alpha combinations produced by these crosses were self- incompatible and each culture represented a single compatibility class. An analy-

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C O M P L E X HETEROZYGOTES 823

TABLE 2

Analysis of progenies from crosses of biennis group 2 and biennis group 3 races with alpha.alpha combinations of the same phylogenetic groups as pollen parents

Progeny

Crosses Expected combinations Observed

Tonawanda I x acoudersport IIIaLinville

Tonawanda I x aSmethportaLinville

Tonawanda I x cuVictorini.aLinville

Tonawanda I1 x cuWaterburyaLinville

Tonawanda I1 x acoudersport IIIaLinville

Tonawanda I1 x cuSmethportaLinville

Tonawanda I1 x avictorinialinville

Coudersport I x aSmethportaLinville

Coudersport I x aVictorini.aLinville

Coudersport I1 x acoudersport IIIaLinville

Coudersport I1 x aSmethportdinville

Coudersport I1 x aVictorinidinville

Williamsville x aWaterburyaLinville

Williamsville x acoudersport IIIaLinville

Williamsville x aSmethport.cuLinville

Williamsville x aVictoriniaLinville

aTonawanda IaCoudersport I11

aTonawanda IaLinville

aTonawanda I d m e t h p o r t aTonawanda IaLinville

aTonawanda IaVictodni aTonawanda 1.aLinville

aTonawanda IIaWaterbury aTonawanda IIaLinville

aTonawanda IIaCoudersport I11

aTonawanda IIaLinville

aTonawanda 1I.aSmethpox-t aTonawanda IIaLinville

aTonawanda IIaVictorini aTonawanda 1I.aLinville

acoudersport I d m e t h p o r t acoudersport IaLinville

acoudersport IaVictorini acoudersport IaLinville

acoudersport IIaCoudersport I11

aCoudersport IIaLinville

acoudersport IIaSmethport acoudersport IIaLinville

aCoudersport IIaVictorini acoudersport IIaLinville

aWilliamsvilleaWaterbury aWilliamsvilleaLinville

aWilliamsville&oudersport I11

aWilliamsvilleaLinville aWilliamsvilleaSmethport aWilliamsvilleaLinville cuWilliamsvilleaVictorini aWilliamsvilleaLinville 0 1 0 19 0 16 0 3 0 21 0 18 0 13 0 35 0 25 0 49 0 9 0 25 0 8 0 8 0 21 0 14.

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824 MARY E. SCHULTZ

The alpha complex was transmitted through the pollen by biennis 1 races in a number of crosses to races belonging to biennis 2 and biennis 3 . The alpha.alpha combinations produced were also self-incompatible. The results of these crosses are listed in Table 3.

Having established the presence of self-incompatibility alleles in the alpha complexes of biennis 2 and biennis 3 , an attempt was then made to determine whether the mechanism operating here is the same as previously reported in the alpha complexes of biennis 1 (STEINER 1956, 1957). The self-incompatibility of certain biennis 1 alpha complexes is due to a single S locus which is occupied by a different allele in each complex. The pollen reaction is gametophytic with independent action of the alleles in the stylar tissue. On this basis all different alpha-alpha hybrids should be cross-compatible whether they are unlike in one or both complexes, unless they carry identical alleles. That is, if pollen from asmethport-aLinville is placed on the stigma of awaterburyalinville, only one kind of pollen, asmethport, should grow to produce two classes of progeny, providing, of course, both kinds of eggs are formed. These expected classes are aWaterburyaSmethport and aLinville.aSmethport. Alpha Linville pollen should not grow since that complex is present in the style. If pollen from aSmethporta Linville is placed upon stigmas of aIndian River-@Newfound Gap both kinds of pollen should grow, since they carry complexes not present in the style. Four compatibility classes would be expect,ed among the progeny, namely, aIndian RiveraSmethport, aIndian RiveraLinville, aNewfound Gap&methport, and aNewfound Gap.aLinville.

Compatibility reactions of t he alpha complexes in outcrosses

Crosses between alphaalpha combinations having one complex in common: Several series of crosses were made, each involving hybrids having one complex

TABLE 3

Progenies from crosses to biennis 2 and biennis 3 races using biennis 2 races as pollen parents

Total

Culluie no. (.ross NIY .B progeny

760 761 762 763 765 769 7 70 771 772 773 776 777 750 753

Waterbury x Camp Peary E

Coudersport I11 x Camp Peary E

Linville x Camp Peary E Smethport x Camp Peary E Victorini x Camp Peary E

Coudersport I11 x Birch Tree 2 Waterbury x Paducah Elma V x Paducah Smethport x Paducah Coudersport I11 x Paducah Elma V x Birch Tree 1 Newfound Gap x Birch Tree 1 Victorini x Birch Tree 1 Smethport x Birch Tree 1

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COMPLEX HETEROZYGOTES 825 in common. In series 1 the common complex was aLinville. I n the summer of 1957 the following genotypes were crossed in all possible combinations:

acoudersport 1II.aLinville aSmethportwLinville aVictoriniwLinville awaterbury-aLinville.

This series and the ones that follow were designed to eliminate extensive progeny analysis. Pollen carrying aLinville would not be functional in any of these crosses since this complex was present in all styles. Pollen tube growth in the style of one of these combinations would indicate the compatibility of the alpha complex which is combined with aLinville in the pollen parent. Style preparations together with capsule development could be used to demonstrate that acoudersport 111, asmethport, avictorini, and awaterbury each possess a different S allele, which would be expected on the basis of the system established in certain biennis 1 alpha complexes. However, all these crosses were incompatible. Incompatible reactions between pollen and style varied in different crosses, but there was good correlation between different tests of the same cross. Frequently incompatible pollen produced short, twisted tubes confined entirely to the stigma. I n other instances pollen tube growth continued into the style where it stopped in certain crosses at one tenth to four fifths the length of the style.

Series 1 also includes material grown the summer of 1958. At this time the following seven different combinations with aLinville were intercrossed:

acoudersport 1.aLinville acoudersport IIwLinville @Indian RiveraLinville aSmethportaLinville aTonawanda 1.aLinville aTonawanda 1I.aLinville aWilliamsvilleaLinville

Pollen carrying aCoudersport I was compatible with styles of all hybrids listed above except the one in which acoudersport I1 was present. Alpha Coudersport I1 pollen grew in all styles except those containing acoudersport I in the genotype. That is, acoudersport I and acoudersport I1 reciprocally incompatible, but each was compatible with the other complexes involved. On the other hand, pollen carrying dndian River, dmethport, aTonawanda I, aTonawanda 11, o r awilliamsville was incompatible in all crosses made in this series including those made to acoudersport IuLinville and acoudersport 11-aLinville. The failure of pollen carrying one of these complexes to grow through styles containing aCoud- ersport I or acoudersport I1 gives differences in reciprocal crosses. Pollen tube growth did not occur in a number of ihe incompatible crosses but in all cases it was incomplete. The results of these crosses are given in Tables 4 and 5.

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826 MARY E. SCHULTZ

TABLE 4

Crosses between alphaalphas hauing aLinuille as a common complex. Summer 1957

Seed parent

acoudersport 1II.aLinville SI 0 0

aSmethportaLinville 0 SI 0

avictorinialinville 0 SI

aWaterburyaLinville 0 SI

C=Compatible--Pollen tube growth to style base; seed set.

Blank = Incompatible-Pollen tubes fail to penetrate stigma. no seed set. o=Incompatible-Pollen tube growth 1/10 to 4/5 length o?style. no seed set. SI = Self-incompatible-Pollen tubes fail to penetrate stigma; no seed set.

TABLE 5

Crosses between alphaalphas hauing aLinuille as a common complex. Summer 1958

Seed parent

acoudersport I-aLinville SI 0 0 0 0

aCoudersport 1I.aLinville 0 SI 0 0 0

aIndian RiveraLinville C C SI 0

aSmethport.aLinville C C SI 0

aTonawanda I-aLinville C C 0 0 SI 0 0

aTonawanda IIeLinville C C SI 0

aWilliamsville.aLinville C C 0 0 0 SI

C=Compatible-Pollen tube growth to style base; seed set.

Blank = Incompatible-Pollen tubes fail to penetraie stigma; no seed set. o=Incompatible-Pollen tube growth !/IO to 4/5 length of style: no seed set SI = Self-incompatihle-Pollen tubes fail to penetrate stlgma; no seed set.

crosses made to biennis 2 and biennis 3 races with pollen from biennis 1

alpha.aJphas.

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COMPLEX HETEROZYGOTES 827

Inasmuch as acoudersport I11 alone varies in its compatibility relationships with the other complexes when a change is made in genetic background, it would probably be well to note that the acoudersport I11 complex involved in the aLin- ville series was derived from a different pollen parent in the stock culture of that race. Since two different strains of aCoudersport I11 were established by using two different parents in the original crosses, the origin of acoudersport I11 in the aLinville, aBirch Tree 1, and &amp Peary E series is given below.

Parent 1 Parent 2

aLinville series, 195 7

aBirch Tree 1 series, 1957

aBirch Tree 1 series, 1958

&amp Peary E series, 1958

Five of the six different combinations which were intercrossed in the second part of the cYBirch Tree 1 series are cross-incompatible. They are acoudersport 1II.aBirch Tree 1, aElma I.aBirch Tree 1, aElma V.aBirch Tree 1, aSmethport -aBirch Tree 1, and aVictoriniwBirch Tree 1. Pollen from these genotypes was also unsuccessful when crossed to “ewfound GapaBirch Tree 1. On the other hand, each of the reciprocal crosses, or those in which aNewfound Gap was used as the pollen parent, were fertile due to the growth of aNewfound Gap pollen.

The results of these crosses between alphaalphas containing Birch Tree 1 are given in Tables 6 and 7. Identical genotypes are listed separately where the aBirch Tree 1 complex is derived from different pollen parents, in one case, the race Birch Tree 1 , and in the other, a biennis 1 alphaalpha. The compatibility reaction of the genotypes is consistent in these instances although @Birch Tree 1

was not transmitted as an intact complex by the alpha.alphas.

Crosses composing series 3 are between four alpha.alphas which have the biennis 1 complex &amp Peary E in common. Two genotypes, aVictorini&amp Peary E and aWaterburyaCamp Peary E are cross-incompatible and unsuccessful as pollen parents in crosses made to acoudersport III&amp Peary E and aNewfound GapaCamp Peary E. However, pollen carrying acoudersport I11 or

TABLE 6

Crosses between alphaalphas hauing aBirch Tree I as a common complex. Summer 1957

Seed parent

738 acoudersport IIIaBirch Tree 1 SI 0 0

741 aSmethportaBirch Tree 1 C SI 0 0

740 asmethport-aBirch Tree 1 C SI 0 0

733 aWaterburyaBirch Tree 1 C SI

732 aWaterburyaBirch Tree 1 C SI

C = Compatible-Pollen tube growth to style base, seed set

Blank=Incompatible-Pollen tubes fail to penetrate stigma, no seed set o=Incompatible-Pollen tube growth 1/10 to 4/5 length of style, no seed set

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828 MARY E. SCHULTZ

TABLE 7

Crosses between alphaalphas having aBirch Tree I as a common complex. Summer I958

743 acoudersport 111.

746 acoudersport 111.

220 aElmaI.

776 aElmaV.

222 aElmaV.

777 aNewfound G a p

753 aSmethport.

750 ovictorini.

744 avictorini.

0 0

aBirch Tree 1 SI C

aBirch Tree 1 SI C 0 0

aBirch Tree 1 SI

c

0 0

aBirch Tree 1 SI

aBirch Tree 1

c

SI

aBirch Tree 1 C SI

0 0

C=Compatible-Pollen tube growth to style base; seed set.

Blank= Incompatible-Pollen tubes fail to penetrate stigma; no seed set. o=Incompatible-Pollen tube growth 1/10 to 4/5 length of style; no seed set. S I = Self-incompatible-Pollen tubes fail to penetrate stigma; no seed set.

aNewfound Gap from the latter genotypes is compatible with the styles of aVic- torini&amp Peary E and aWaterbury&amp Peary E. Crosses between aCou- dersport III&amp Peary E and aNewfound GapaCamp Peary E were fertile. That is, mcoudersport I11 pollen is compatible with aNewfound GapaCamp Peary E and aNewfound Gap pollen with acoudersport III&amp Peary E. These results are given in Table 8.

Several additional crosses were made between alphadphas having one complex in common which can conveniently be grouped -together as series

4'.

The first two crosses in this series are between aElma V.aWalkerton and aVictorini. awalkerton. The crosses were incompatible. Alpha Elma V pollen failed to grow in aVictorini.aWalkerton styles and aVictorini pollen failed in cYElma V.aWalker- ton styles. The results are the same as those obtained from crosses between aElma V and avictorini when each was combined with aBirch Tree 1.

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COMPLEX HETEROZYGOTES 829 Crosses between aWilliamsvilleaLinville and aWilliamsvilleaNewfound Gap were fertile. Since awilliamsville is common to both genotypes, the success of these crosses is due to the growth of aLinville pollen in aWilliamsville.aNew- found Gap styles and the compatibility of "ewfound Gap pollen with aWil- liamsvillewlinville.

The crosses included in series

4

are listed in Table 9. The results of these crosses are all consistent with those reported previously. It is evident that a l i n - ville and aNewfound Gap function successfully in outcrosses whereas aElma V, aIndian River, and aVictorini do not.

Crosses between alphaalpha combinations hauing unlike complexes: A number of crosses were made between alpha.alphas in which both complexes in the pollen parent were different from the two combined in the seed parent. That is, the crosses were between biennis 2 and biennis 3 combinations which contained no complex in common. These crosses, all of which gave a good seed set, are listed below. The seed parent is given first.

1. aIndian RiveraNewfound Gap x aSmethportaLinville 2. aIndian RiveraNewfound Gap x aVictorini.aLinville

TABLE 8

Crosses between alphaalphas having aCamp Peary E as a common complex. Summer 1958

Seed parent

acoudersport IIIaCamp Peary E SI 0 0 C

aVictorinivxCamp Peary E C SI C

crWaterburyaCamp Peary E C SI C

aNewfaund GapaCamp Peary E C SI

C=Compatible-Pollen tube growth to style base. seed set.

Blank= Incompatible-Pollen tubes fail io penetr:te stigma; no seed set. o=Incompatible-Pollen tube growth 1/10 to 4/5 length of style; no seed set. SI = Self-incompatible-Pollen tubes fail to penetrate stigma: no seed set.

TABLE 9

Various crosses between alphaalphas having one complex in common

Cross' Results

aElma VaWalkerton x aVictorinieWalkerton i cyVictoriniaWalkerton x aElma VwWalkerton i

I I

aIndian RiveraNewfound Gap x aVictorinieNewfound Gap aVictoriniaNewfound Gap x @Indian RiveraNewfound Gap

aWilliamsvilleaLinville x aWilliamsvilleaNewfound Gap C

aWilliamsville.aNewfound Gap x aWilliamsvillecuLinville C

* Seed parent grvep first.

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830 MARY E. SCHULTZ

3 . aIndian RiveraNewfound Gap x aWaterburyaLinville 4. aIndian River.aNewfound Gap x nCoudersport 1II.aLinville

5.

aSmethportwLinville

x

d n d i a n RiveraNewfound Gap 6. aVictorini.aLinville x @Indian RiveraNewfound Gap 7. aWaterbury.aLinville

x

&Indian RiveraNewfound Gap 8. acoudersport 1II.aLinville x dndian RiveraNewfound Gap 9. aSmethportaLinville x aWilliamsville.aNewfound Gap I O . d n d i a n RiverwLinville x aWilliamsvilleotNewfound Gap 1 1. aTonawanda 1,aLinville x aWilliamsville~aNewf~und Gap 12. aTonawanda IIwLinville x aWilliamsvilleaNewfound Gap

Progenies of the first three crosses listed were analyzed during the summer of 1958. On the basis of the system of S alleles found in the alpha complexes of

biennis 1, four compatibility classes were expected among the progeny of each cross. However, the progenies from the three crosses belonged to a single compatibility class. Phenotypically these plants were aIndian River.aLinville and their chromosome configurations confirmed this genotype. Only one complex, aLinville, was transmitted through the pollen, and aIndian River functioned exclusively as the egg complex. The failure of aSmethport, avictorini, and awaterbury as pollen complexes in these crosses is consistent with the data from previous crosses.

Crosses of alphaalphas to synthesized alphabetas: Crosses were made using biennis 2 and biennis 3 alpha.alphas as the pollen parent and synthesized alpha. betas as the female parent. Synthesized alpha.betas differ from the naturally occurring ones in that they combine complexes from different geographical loca- tions. Since the beta complexes do not carry S alleles, the seed parents used in these crosses contain a single S allele in the diploid tissue of the style, the homol- ogous locus being occupied by an Sf gene. These crosses were designed to determine whether there was any indication of sporophytic control in the style of the compatibility reactions. That is, if there were any changes in the compatibility relationships between these alpha complexes from those determined in alpha.alpha intercrosses, this would be good evidence of gene interaction between the S alleles of the alpha.alpha seed parent. The different reaction then would be due to the individual action of the S allele in the stylar tissue of synthesized alpha.beta. The details of these crosses are given in Table I O .

Cross 1 in Table 10 shows that pollen carrying aNewfound Gap is compatible with styles containing aIndian River. Alpha Newfound Gap pollen is eliminated in this cross by the presence of that complex in the genotype of the seed parent. Crosses 2 and 3 indicate that d n d i a n River pollen is incompatible with styles containing aNewfound Gap. Pollen carrying aNewfound Gap was not expected to grow since it is present in the seed parents. These compatibility reactions between aIndian River and aNewfound Gap are the same as those previously established in the alpha.alpha intercrosses.

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COMPLEX HETEROZYGOTES 831 TABLE 10

Crosses using synthesized alphabetas as seed parents and biennis 2 and biennis 3 alphaalphas as pollen parents

1.

2.

3.

4. 5. 6. 7. 8. 9. 10.

Cross Results

aIndian RiverpElma V x aIndian RiveraNewfound Gap @Newfound GappElma V

x

aIndian RiveraNewfound Gap

a h d i a n RiverpSmethport x aIndian RiverTLinville

aVictorini.pWaterbury

x

avictorinielinville C aSmethport.pWaterbury x aSmethport.aLinville C aElma V.PWaterbury x aVictorinieLinville C

aElma V.pCoudersport 111 x aIndian RiveraNewfound Gap aElma V.PSmethport x aIndian RiveraNewfound Gap aElma V.PWaterbury x aIndian RiveraNewfound Gap

C

I

I C aNewfound GappMountain Lake x dndian RiveraNewfound Gap

C C

C

C=Compatible-Pollen tube growth to style base, seed set. I=Incompatible-Pollen tubes fail to penetrate stigma; no seed set.

data from the progeny analysis of alpha.alpha intercrosses. Crosses 5 and 6 indicate aLinville is compatible with avictorini (cross 5) and aSmethport (cross 6). These compatibility relationships were previously established through progeny analysis of race intercrosses.

I n crosses 7 through 10 progeny analysis is necessary to determine the compatibility reaction of the alpha complexes combined in the pollen parent with aElma V. Unfortunately, this was not possible. Seed of cross 7 failed to germinate and all attempts to grow progeny of the latter crosses failed at the seedling stage. Apparently all three progenies were composed of the same lethal combination which was probably aElma VwNewfound Gap.

DISCUSSION A N D CONCLUSIONS

Incompatibility characteristics of the alpha complexes: It has been shown that the alpha complexes of the phylogenetic groups biennis 2 and biennis 3 are uniformly self-incompatible. In view of the data obtained from this study it is possible to characterize the mechanism operating in these complexes as follows: 1. Pollen tube growth is inhibited in a style in which both tube and style 2. One group of alpha complexes are cross-incompatible (Class A ) .

3. The second group of alpha complexes are cross-compatible (Class B)

.

4. Crosses between Class A and Class B alpha complexes give reciprocal differences.

5. The compatibility relationships between the alpha complexes insofar as tested are completely consistent.

The first feature above is characteristic of the gametophytic oppositional system described in Nicotiana sanderae by EAST and MANGELSDORF (1925). This system involves a single gene S which exists in a large series of allelic forms. The pollen reaction is controlled by the gametophyte, and the two alleles present in the style have individual action with no interaction. Pollen tubes carrying a specific S allele are inhibited in styles containing the same S allele.

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832 MA RY E. SCHULTZ

The gametophytic incompatibility system has already been demonstrated in Oenothera organemis (EMERSON 1938) and 0. rhombipetala (HECHT 1944). More recently it has been found in 0. deltoides, 0. pallida, and 0. missouriensis

(CROWE 1955). STEINER found this system operating in the alpha complexes of certain biennis 1 races of 0. biennis. He showed that the alpha complexes studied are self-incompatible due to a n inhibition of pollen tube growth and, on the basis of their cross-compatibility, postulated that each complex carries a different S

allele.

Self-incompatible, cross-compatible complexes: This study has shown that the alpha complexes of five of the 15 biennis 2 and biennis 3 races contain an incompatibility mechanism that has the characteristics of the gametophytic system. These complexes are self-incompatible due to pollen tube inhibition, but with one exception they are successful as pollen complexes in crosses “inter se” as well as in crosses to combinations of the other ten alpha complexes. The one exception is the cross-incompatibility of nCoudersport I and acoudersport 11,

which is probably due to the presence of the same S allele in each of the two complexes. These alpha complexes whose genetic behavior can be explained on the basis of the gametophytic oppositional system are categorized as Class A.

They are as follows:

biennis 3

doudersport I aLinville

acoudersport I1 aNewfound Gap

aCoudersport 111, strain 1

Two different strains of acoudersport 111 were established in the original crosses by the use of different parent plants in the stock culture of the race. The behavior of acoudersport 111, strain 1, is characteristic of the Class A complexes. Strain 2 is included with the second group of alpha complexes, which are referred to collectively as Class B.

Self-incompatible and cross-incompatible complexes: The alpha complexes composing Class B were found cross-incompatible, as well as self-incompatible. Perhaps the first question proposed by the failure of these Class B alpha complexes as pollen complexes is whether they carry gametophytic lethals resulting in inviable pollen grains. The answer to this question is found in the data obtained from style preparations. These observations revealed that the failure of pollen carrying one of these complexes is frequently due to incomplete pollen tube growth. That is, pollen carrying one of these complexes has, at least in certain crosses, germinated and developed tubes which extend from one fifth to three fourths the length of the style. The growth is usually terminated by the tubes bursting, but sometimes by losing their original orientation and turning

either to the side or upward. The Class B complexes are the following:

biennis 2 biennis 3

LvElma I aTonawanda I oiSmethport

aElma V aTonawanda I1 acoudersport 111,

avictorini aWilliamsville strain 2

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COMPLEX HETEROZYGOTES 833 It is evident, then, that the self- and cross-incompatibility of these alpha complexes is due to pollen tube inhibition. The cross-incompatibility can be explained in terms of the gametophytic oppositional system if it is assumed that each of these complexes carries an identical S allele. However, this assumption would not account for the reciprocal differences obtained from crosses between the alpha complexes of Class A and Class B. That is, if it is assumed that all Class B complexes contain the same S allele, which may be designated SI,, then the incompatibility of

s,,

pollen with styles containing a Class A alpha complex would lead to the conclusion that all Class A complexes also contain the S,, allele. This could not be possible since they are intercompatible, indicating that each Class A complex, except acoudersport I and acoudersport 11, carries a different S allele, and none of these could be SI,, since they are compatible as pollen complexes with Class

B

complexes which are assumed to carry S,,.

Reciprocal differences, such as those obtained from crosses between Class A and Class

B

alpha complexes are characteristic of the sporophytic oppositional system. This system of incompatibility was first recognized by HUGHES and

BABCOCK in Crepis foetida (1950) and by GERSTEL in Parthenium argentatum (1950). In the light of these studies it is now realized that an incompatibility system of this type was described earlier in Cardamine praetensis by CORRENS

(1913). Although now the sporophytic system is known to occur in a number of species and is shown to be characteristic of the self-incompatible genera of the Compositae and Cruciferae, it has not yet been found in the oenotheras, or for that matter, in any member of the Onagraceae. However, the presence of a sporophytic oppositional system in the alpha complexes of biennis 2 and biennis 3 may be indicated by the reciprocal differences.

The sporophytic system, like the gametophytic, involves a single gene, S, which has multiple alleles. It differs from the gametophytic system in having the pollen reaction controlled by the sporophyte; that is, interaction between the S alleles may occur in the pollen parent in the form of dominance or compe- tition which results in great variability in incompatibility relationships. Addi- tional complexity occurs in the compatibility patterns if the stylar reaction is also controlled sporophytically.

On the basis of the sporophytic system the incompatible cross asmethport. aLinville x J n d i a n RiveraLinville would be interpreted as follows: aLinville S allele is dominant to the &Indian River S allele in the pollen mother cell; therefore d n d i a n River pollen is functionally aLinville and is inhibited in otSmeth- portorlinville styles due to the presence of the aLinville S allele in the stylar tissue. The reciprocal would be incompatible due to the dominance of the aLin- ville S allele over aSmethport in the pollen mother cell.

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834 MARY E. SCHULTZ

when dmethportalinville is used as the pollen parent because aLinville S allele is dominant over dmethport S allele.

In a broader view, it would be assumed that aLinville is dominant over each of the Class B complexes tested and has no effect on the Class A complexes. This same dominance pattern then would exist not only between aBirch Tree 1 and the Class A and Class B complexes but also between these complexes and &amp Peary E. That is, aLinville, aBirch Tree 1 and &amp Peary E, S alleles which are known to be different, are nevertheless all dominant to the Class B complexes and have no effect on the Class A complexes. It seems highly improbable that three randomly chosen alpha complexes and three different S alleles would interact with the S allele in each of the alpha complexes studied to give such uniform results. The consistency in the compatibility relationships between the alpha complexes is probably the strongest evidence against a sporophytic system. Evidence against sporophytic control in the stylar reaction was obtained in crosses of alpha.alphas to naturally occurring and synthesized alpha.betas. Since the beta complexes do not carry S alleles, only one allele is present in these seed parents. The results of crosses using races, i.e., naturally occurring alpha.betas, as seed parents and four different alpha.alpha combinations as pollen parents showed exclusive transmission of aLinville through the pollen. Alpha Couder- sport 111, cusmethport, avictorini, and awaterbury failed as pollen complexes

(Table 2 ) . The same results were Qbtained in alpha.alpha intercrosses.

The reciprocal differences in the compatibilities between Class A and Class B complexes found in alpha.alpha intercrosses also occurred in crosses of alpha. alphas to synthesized alpha.betas. The results of the second cross listed in Table 10 show that d n d i a n River pollen is incompatible with “ewfound GappElma

V

styles. Since pElma V contains a Sr gene the stylar reaction is produced here, evidently, by a single S allele which would be that of “ewfound Gap. The first cross in that same table was fertile, indicating that aNewfound Gap pollen is compatible with styles containing d n d i a n RiverPElma V. In this case the lack of inhibition of aNewfound Gap pollen tubes could not be attributed to gene interaction since there is only one S allele in the stylar tissue.

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COMPLEX HETEROZYGOTES 835

RiveraVictorini had been outcrossed, aVictorini would have been successful as a pollen complex, aiid two strains would then be established as was done in acoudersport 111. One strain behaves as a Class A complex and the other, Class B. The only other races which transmitted the alpha complex through the pollen in these crosses are Linville and Newfound Gap. Both were also successful as pollen complexes in the alpha.alpha intercrosses, upon which basis they are categorized as Class A complexes. Except for avictorini, none of the Class B complexes were transmitted through the pollen in the crosses between races.

A gametophytic system with two incompatibility loci: I n view of the foregoing, it seems unlikely that the incompatibility mechanism operating in the alpha complexes is the sporophytic type. It has features which identify it most nearly with the gametophytic oppositional system. Since it also possesses characteristics not yet known for this system, the writer would like to propose a gametophytic scheme that would include these as well.

This scheme postulates an additional incompatibility locus in both Class A and Class B complexes, which is occupied, functionally, by a single allelic form. This locus will be referred to as the T I locus to distinguish it from the SI locus, which is occupied by a different allele in each Class A complex here studied except acoudersport I and acoudersport 11. The SI locus in Class B complexes is occupied by a self-fertility gene, Sf, which may have arisen by the mutation SI+S,. Furthermore, the S I locus is epistatic to the T I locus in the pollen but not in the style. The T I locus is therefore not functional in pollen carrying a Class A alpha complex. These loci have individual action with no interaction in the style. The SI locus functions here to inhibit pollen carrying a specific Class A complex, whereas, the T I locus functions to inhibit pollen carrying any Class B complex. According to this scheme, the incompatibility loci of the alpha complexes included in this study may be represented as follows:

acoudersport I acoudersport I1

acoudersport 111, strain 1

aElma I aElma V aVictorini awaterbury

acoudersport 111, strain 2

Class A biennis 3

SI,

T r aLinville

SI&

TI aNewfound Gap

Si,

TI

Class B biennis 2

Sf TI aTonawanda I

sf

Ti

Sf T I aTonawanda I1 Sf T I

Sf T I d n d i a n River S f TI

Sf T I aWilliamsville Sf TI

Sf T I asmethport Sf

Ti

biennis 3

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836 M A R Y E. SCHULTZ

1. aSmethportaLinville x aWaterburywLinville (Sf T I ) (SI, T I ) (Sr T I ) (SI$ T I )

This cross was incompatible. Alpha Linville pollen is functionally S,, and is eliminated by the presence of S I , in the style. Pollen carrying awaterbury is inhibited by the presence of T , in both tube and style.

2. aCoudersport I.aLinville x aIndian RiveraLinville

This was also an incompatible cross. Alpha Linville pollen tubes are inhibited because the S I , allele they carry is present in the style, and aIndian River pollen fails because T I is present in both tube and style.

(Si4

Ti)

(Si9 T , ) ( S , Tr) (SI, Tr)

3. @Indian RiveraLinville x aCoudersport IwLinville

This cross is the reciprocal of the one immediately preceding. It was found compatible due to the growth of acoudersport I pollen. With the S, locus epistatic over the T I , this pollen would be functionally SI,, an allele not present in the seed parent.

Although the T I locus functions in outcrosses as though it were occupied by one gene with no alleles, the differential growth of pollen tubes may indicate that it also exists in a series of allelic forms. That is, pollen tubes carrying aVic- torini are confined to the stigma in the self-pollination of aVictorini.aBirch Tree 1. However, if it is used in an outcross to aElma V.aBirch Tree 1, the tubes de- velop to approximately three fourths the length of the style. In aElma I-aBirch Tree 1 styles aVictorini pollen tube growth stops at approximately one half the length of the style; in aCoudersport II1,aBirch Tree 1, approximately one third. Inhibition does occur in such outcrosses, but the time of the reaction is frequently later than in a self-pollination. If this variation in the time of reaction is interpreted to be the result of different T alleles in each of the alpha complexes, then the difference between these alleles would be of a smaller magnitude than that which must exist between the S alleles. On the basis of the theory advanced by LEWIS (1 952) that the incompatibility is due to a reaction between an antigen in the pollen with an antibody in the style, it might be that the T alleles possess overlapping antigenic properties.

It is not difficult to account for the origin of the second incompatibility locus in the complex-heterozygotes of Oenothera. It may be a duplication of the SI locus which has arisen by unequal segmental interchange. Intrachromosomal duplication and deficiency are characteristic of many translocation heterozygotes.

CATCHESIDE (1 947) has shown them to occur frequently in Oenothera blandina.

It has already been suggested that the self-fertility gene in the Class B alpha complexes may have arisen by a mutation SI to S f . Such a mutation has been found in Oenothera organensis (LEWIS 1951) and it seems possible that it occurred spontaneously in aCoudersport I11 to produce strain 2 which has the characteristics of a Class B complex.

The gametophytic oppositional system with two incompatibility loci is proposed, therefore, as the pollen lethal in the ring-forming oenotheras. Both loci function to eliminate the structurally homozygous alpha.alphas in self-pollina-

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COMPLEX HETEROZYGOTES 837

tions, but the T , locus operates to eliminate them also in cross-pollinations. The role of eliminating pollen carrying the alpha complex in cross-pollinations as well as self-pollinations would give the T I locus a selective advantage over the

SI

locus since it would be more effective in preserving the permanent heterozygosity of these forms.

STEINER (1957) had previously established the presence of the SI locus in certain alpha complexes of biennis 1 which are transmitted through the pollen in outcrosses. He stated, however, “In races which in outcrosses rarely or never transmit the alpha complex through the pollen, alpha pollen must be eliminated in some other way, since incompatibility alleles would normally be inoperative in outcrosses.” According to the above hypothesis, one can predict that the biennis

1 races which do not transmit alpha pollen in outcrosses are the Class

B

type, and that the gametophytic system described for the alpha complexes of biennis 2 and biennis 3 also applies to those of biennis 1 .

S U M M A R Y

1. Alpha.alpha hybrids were produced which combine complexes of different races belonging to the phylogenetic groups biennis 2 and biennis 3 of Euoeno- thera. These alpha.alphas were uniformly self-incompatible.

2. Alphaalpha intercrosses were made to determine the compatibility reactions of the alpha complexes in outcrosses. The alpha complexes of certain races were transmitted through the pollen in outcrosses while those of other races were not.

3. Compatibility tests indicate that incompatibility alleles are characteristic of the alpha complexes i n biennis

2

and biennis 3, as well as biennis 1. Their presence in these groups supports the hypothesis that incompatibility alleles function as the pollen lethal in the self-pollinating complex-heterozygotes of Oenothera.

4. A gametophytic incompatibility system is postulated which involves two incompatibility loci, S, and T I , and epistasis in the pollen.

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838 MARY E. SCHULTZ ACKNOWLEDGMENTS

This study was made under the direction of PROFESSOR ERICH E. STEINER. The author is very grateful to DR. STEINER for giving freely of his time and helpful criticism of this work. Sincere thanks are extended to MR. W. F. KLEINSCHMIDT

and the staff at the University of Michigan Botanical Gardens for their sug- gestions and help in growing the plants used in this investigation. She wishes to acknowledge with gratitude the support given to this work by the faculty of the Department of Botany in the form of a graduate assistantship and teaching fellowship. She is also grateful to DR. R. E. CLELAND and DR. H. T. STINSON for providing seed of some of the races used in this study. A National Science Foun- dation grant (G-3298) to DR. STEINER supported this study during the summers of 1957 and 1958.

LITERATURE CITED

CATCHESIDE, D. G.. 1947 CLELAND, R. E., 1950 CORRENS, C., 1913

CROWE, L. K., 1955 293-322.

EAST, E. M., and A. J. MANGELSDORF, 1925

plants. Proc. Natl. Acad. Sci. U. S. 11: 166171. EMERSON, S. H., 1938

23: 190-202.

GERSTEL, D. U., 1950 482-506.

HECHT, A., 1944

HUGHES, M. B., and E. B. BABCOCK, 1950

LEWIS, D., 1951

A duplication and a deficiency in Oenothera. J. Genet. 48: 99-110. Studies in Oenothera cytogenetics and phylogeny. Indiana Univ. Publ. 16.

Selbsterilitiit und Individualstoffe. Bid. Zentr. 33 : 389-423.

The evolution of incompatibility in species of Oenothera. Heredity 9:

A new interpretation of the behaviour of self-sterile

The genetics of self-incompatibility in Oenothera organensis. Genetics

Self-incompatibility studies in Guayule. 11. Inheritance. Genetics 35:

Induced tetraploids of a self-sterile Oenothera. Genetics 29: 69-73.

Self-incompatibility in Crepis foeti& (L.) subsp.

Structure of the incompatibility gene. 111. Types of spontaneous and induced

Serological reactions of pollen incompatibility substances. Proc. Roy. Soc. London B

Das Problem der Unbefruchtbarkeit. Natunv. Wochenschr. N. F. 20: 440-446.

New aspects of the balanced lethal mechanism in Oenothera. Genetics 41:

Further evidence of a n incompatibility allele system in the complex-heterozygotes of