One-locus sporophytic S-gene system with traces of gameto- phytic pollen control in arvense ssp. stricturn ()"

ARNE LUNDQVIST

Department of Genetics, University of Lund, Lund, Sweden

LUNDQVIST,A. 1990. One-locus sporophytic S-gene system with traces of gametophytic pollen control in Cerastium arvense ssp. srrirtum (Caryophyllaceae). - Hereditas 113: 203-21 5. Lund, Sweden. ISSN 0018-0661. Received August 6, 1990. Accepted September 21, 1990

Cerastium arvense ssp. stricturn (2n=36), obtained from the Swiss alps, was investigated by fluorescence microscopy of pollen tube growth in 52 population , 77 F,-plants in four F,-families, and 5 1 backcross plants from two backcrosses F, x parent. All plants were self-incompatible with pollen tubes arrested on the stigmatic surface. One disomic S-locus with sporophytic pollen control and independent action of the S-alleles was indicated by the four groups segregating in all four F,-families. The data are interpreted to show that any of the two S-substances produced in the pollen and style is sufficient alone to cause incompatibility, and that a fraction of the pollen grains react as if one of the two imprints were absent. It is suggested that, by occasional successive cell wall formation at microsporogenesis, S-gene transcription, even if initiated before S-gene segregation in the first anaphase of the pollen meiocyte, may be insufficient to prevent the S-gene of the dyad cell to put its imprint alone on the two microspores formed in the second meiotic division. This gametophytic imprint on the pollen grain may form a necessary substitute to a system of dominance relationships in the pollen, cross-compatibility being promoted without loss of self- incompatibility. Estimates of the number of S-alleles in the sample of population plants ranged from 7 to 19; the weighted mean was 10. - The presence of an S-gene lacking dominance interaction supports the opinion that the orders and Ranunculales form a phyletic lineage.

Arne Lundqvist, Department of Genetics, University of Lund, Solvegatan 29, S-223 62 Lund. Su,eden

In flowering plants with male and female organs common in systems with sporophytic control of the produced on the same individual, self-fertilization pollen. may be prevented by systems for self-recognition Related species, up to the level of genera within and -rejection, male and female tissues (in general the same family or closely related families, have the pollen grain and the pistil) reacting incompat- adopted the same type of self-incompatibility con- ibly when they are specified by identical S-genes. trol. The gametophytic type of pollen control is The self-incompatibility promotes crossbreeding, much more widespread, the scattered sporophytic hybridity, and genetic recombination. system among homomorphic angiosperms being In homomorphic plants, the S phenotype of the known from only 6 families: Compositae (GERSTEL pollen may be determined early, by the genotype 1950; HUGHESand BABCOCK1950), Cruciferae of the pollen-producing (sporophytic sys- BATEMAN1954, 1955), Sterculiaceae (KNIGHTand tems), or later, by the genotype of the pollen grain ROGERS1955; COPE1962; JACOB1980), Convolvu- itself (gametophytic systems). This difference in laceae (MARTIN1968), Betulaceae (THOMPSON time of gene action can imprint the pollen with 1979), and Caryophyllaceae (LUNDQVIST1979). higher number of specificities in the sporophytic PANDEY(1960) was the first to argue that the sporo- case. Dominance interaction between S-alleles phytic pollen control is derived from the gameto- serves to lower this number of specificities and to phytic S-gene system. The S-genes are likely to be make the sporophytic system more efficient through very ancient constituents of the breeding systems the reduced degree of cross-incompatibility. There- in angiosperms (WHITEHOUSE1950); hence, their fore. dominance interaction between S-alleles is study may provide an insight not only into the age and evolution of the incompatibility systems them- selves but also into taxonomical affinity and split- * Dedicated to the memory of Dr Kamla Kant Pandey: Keen researcher, generous friend! ting. From such points of view the order Caryophyl- forms with n=36 are located to the plains of central Idles (Centrospermae) is attractive Its taxonomical and western Europe (SOLLNER19.54). The subspe- pomon is \oniewhat disputed. DAHLGRFP.(1975) cies stricturn, with n=18, is distributed in the central considers the order to occupy an isolated position Alps. where this perennial herb is growing on dry, within the angiosperms. whereav TAKHT~JA~( 1969) stony, calcareous or primitive soil on the subalpine con\ider\ the order to be "in all probability" derived or alpine level (FRIEDRICH 1969). directly from the order Ranunculales An evolution- Sollner (1954). investigating 17 different proven- arj line of descent down to a common dicotyledon- ances of ssp. strictunz, observed, without exception, ous ancestral bms (vm WETTSTEI\1935) may be 2n=36, with perfectly normal bivalent formation in traced for a hind of S-gene lacking dominance and the first meiotic division. He observed secondary competition allelic interaction and forming the com- chromosome association in the second meiotic divi- plementary S-gene teams that operate with gameto- sion as an indication of polyploidy from the basic phytic pollen control in monocots and in the number x=9. Admitting that not a single Cerastium dicotj ledonous tamilie\ Ranunculaceae and with n=9 has so far been observed, even among Chenopodiaceae, the latter belonging to the order localized and truly primitive forms in the Alps, he Car!ophyllales (LL\rqLisT et al. 1973. LL\DQLIST points out, in favour of x=9, cases of n=45 within 1975) the genus Cerastiuni, the presence of four SAT- Within the fdmilj Cdrjophyllaceae polyploidy is chromosomes in C. semidecandrum (2n=36), and treyuent. the family ma). theretore. offer difficult the presence of n=9 in some representatives of the material tor genetical in\ e5tigationc into selt-in- related genera Safiiria and Spergula. Plants of C. compatibilities No clear indications of self-incom- ari-erisc with 2n=54 having a very irregular meiosis, patibilit\ operating u ithin the fmiily are reported resembling a triploid meiosis, he considers the two in FK\ xt I i '\ ( 1957)tables on mode of reproduction '>-chromosome genomes putatively forming n=18 to in higher plant\ Xot until CCIBOROGHO'~(1973. have undergone a deep-going differentiation. 1974. 1977) exten\ive sttidie\ ot the genus Ct2ru\- fi/ini I\ 'I\ it revealed that dl1 the t'iu studied u ithin E.vper.iniental nlUtei.it71 the form complex ot Ccr ~itiiinitri iwic uere self- incompatible. not on15 uith regard to tomi\ M ith Seeds of C~rastiirniuriseiise ssp. stricturn (Haenke) ?11=3h but ,tiso to tho\e uith ?11--71 Gaudin, collected in October 1973 at Col du Susten LI \IXJ\ I\ I ( 1970)publi\hed 'I preliniinq anal) - 1950 m. Canton de Berne. Switzerland, were kindly \I\ (if the genetics of selt-iiicomp~tibilit~in one ot sent to me by Prof. Claude Favarger, lnstitut de the h=16toms reported bq [Jgborogho. C'eiu~tiiim Botanique de I'liniversite Neuchiitel, Switzerland. OI I rii( I_ \sp (fr I( tiini (Hxnkc) G'iudiii One- Put to gemiinntion in petri dishes on wet filter IOLLI\ \poioph! tic control ot the pollen ~'1sindi- paper or in pots with soil, the seeds gcmiinated Lateti 5poraphk tic control ot elf-incompatibilities readily. Totally 52 potted population plants were ued uith tnnuclente pollen (RKIM B \kt~ wintered outdoors and, next spring, brought to the i9) stigiii,is of dij thpt (HF\I cw-H ~KKI- greenhou\e for te5ti with strict self-pollination. pol- io\ e' '11 i 0751, and I,tch ot bre,ihdcinn b\ polj- lination with pollen from another tlowcr on the ploicti (HONXKI) 1947. (ii ~\iiI 1950) tr'iitj thd \ame plsnt. and pollination by another plant. Four '111 Lh'ticicteriicthe C c/~i\nuruni'iti'i id On the other F,-Eaniilies. with altogether 77 plants, and. later, hd%i the putdtl\e LI( rel,ttion\hip betutxn the t~obachcrosx families from F, x P. with 51 plants tmiil\ <'tii\oph>Il'tcex aid the ,iip,ir beet tmiil> totally. uere produced for further studies. For prac- ('~ciic~pottia"o,i,. u ith 11.. tour-lucu\ 5-gene temi tical reasoiis. in spite of the flowering being favour- .ind ~ii~pi~niei~t~ii~g,inietoph\tli pollen control ably influenced by sxposure to winter cold OUI- opm\ intaemig 'i\pects tbdt hd~cmde the e\peri- doors. the \\ hole material, consisting of the 52 lll\~:ltLl IllJtcI 1'1' ucll A ("11 of '1 more ilecp-~oirlg population plants and the F,- and backcross fami- ..'Ll

Table 1. Mean numbers of pollen tubes and mean seed-set per pollinated flower after selfings, intra-, and intergroup pollinations involving the four groups distinguished within F,-family 32x33

Mean no. of No. comb. for pollen seed pollen seed Type of pollination tubes seet tubes set - Selfings 0.6 1.0 24 6 Crosses within groups 0.7 0.5 108 11 Crosses between groups outside compatible diagonal 3.5 2.7 213 29 inside compatible diagonal 28.7 20.7 112 10

minations turned out to be substantial, mainly be- newly opened anthers from the particular pollen cause of the lots of subterranean runners produced donor being cautiously rubbed against the stigmatic by the plants. In spite of extensive trimming, barri- part of the styles, and after another 24 hours in the ers put around the plants, and removal of putatively isolation cage the pollinated flowers were fixed for foreign elements from a clone, some contamina- further treatment according to the schedule of PREIL tions were made probable; they will be commented (1970). Removed from the ovary all five styles of upon in the Results section. Contamination from the flower were investigated on the same slide. stray seed, on the other hand, may be deemed a Fluorescence microscopy showed pollen tubes to trivial risk. be arrested on the stigmatic surface after self-polli- nation, whereas compatible tubes grew down the whole length of the style. After cross-pollination up Technical details to 50-60 such long pollen tubes reaching the ovary Studies of seed-set. - These studies were con- could be found within the individual style, corre- ducted on a limited scale in the population plants sponding to a total of some 150-300 tubes for the and in one of the F1-families. Individual buds were five styles. Besides, small to moderate numbers of isolated in pergamine bags a couple of days before long pollen tubes were frequently observed, the anthesis and then pollinated with their own pollen grades 0, 1, 2, 3, 4, and + corresponding to totals or cross-pollinated with pollen from unisolated of 0, 1-5, 610, 11-15, 16<50, and >I50 long flowers. Seed-set was assessed from ripe capsules. pollen tubes formed. Observations available from more than one season have been represented by Studies of pollen tube formation. - Staining pollen their means, with obviously deviant observations tubes in the style by means of aniline blue fluoro- being excluded. chrome and observing their fluorescence in the UV- microscope (MARTIN1959) turned out to be highly efficient in the Cerastium material with its five fi- lamentary styles protruding from the ovary. The Results main body of the present genetical analyses is based on this technique. Self seed set in the 52 population plants (165 iso- For the extensive lots of test pollinations re- lated flowers) indicated general self-sterility, 40 quired, bagging and pollination on flowers in situ plants setting no seed, 10 plants, up to 3 seeds. the were not manageable; a semi-in-vitro technique was remaining 2 plants setting 0-8 and G14 seeds, re- found to serve well. The flowers being protandric, spectively. Cross-pollinations (8 1 flowers) yielded their ten closed anthers can be removed easily when from 0 up to >40 seeds per capsule. the five styles are still undeveloped. Individual flowers were collected at this stage, their stems put F1-families in test tubes, 50x15 mm, in water, and grouped into sets of up to 10 such tubes, with the numbers of the Pilot studies with fluorescence microscopy of pol- respective plant donors attached, and emasculated. len tube formation in one of the four F,-families After 1-2 days in a plastid isolation cage, when the (P32xP33) indicated strict parallelism between seed styles had developed to some 34mm length, the set and formation of long pollen tubes (Table 1); tube set of flowers were pollinated, individual hence the self-sterility is due to an incompatibility un r d .- 2266 :1 13 16 P?3 P3.4

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3 .. 7. ,--~ <5: :.:;;cs - .: >I 50 tutt>cor 21: Ivalent to t.!le dcnsity cf pollination Scheme 1. F,-famil> I. P33xP34. Frequencies of long pollen tubes formed when 77- FI-sibswere selfed. intercrossed, and hackcrossed to the two parents Line below figure denotes 0-value obsen~ed Line on top of figure denotes + value observed Heieditoc /I3 i/YYO) s GENE ACTIOU IN C~KAS~I~M 207

Scheme 2. F,-family 2, P32xP33. Frequencies of long pollen tubes formed when 19 F,-sibs were selfed, intercrossed, and backcrossed to the two parents Line below figure denotes O-value observed Line on top of figure denotes + value observed

reaction, incompatible pollen being arrested on the trix. Seed set and pollen tube formation in crosses stigmatic surface. Four groups of plants, a-d, could among groups outside this diagonal did only be distinguished among the F1-sibs. Within groups, slightly exceed the data from selfing (Table l), as cross-pollinations yielded a pattern of pollen tube was also found for pollen tube formation in combi- formation and seed set similar to that obtained from nations between the F1 and the parental plant (see self-pollinations (Table 1). The four groups formed Scheme 2). two both-way compatible pairs (a and d, b and c), Similar patterns of pollen tube behaviour after forming a “compatible diagonal” in the mating ma- selfings and pollinations within and among four cd

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Scheme 3. F,-tamiIy 3, P24xP9b Frequencies of long pollen tubes formed when 15 F1-sibs were \elfed. intercro~sed.and bachcrossed to the two parents Line beloa tigure denotes O-\alue obsened Line on top of tigure denotes + \due observed

groups appeared in the mating matrices for the three and d13. In family 2 (P32xP33) plant a15 has be- other F, -families investigated (P33xP34. P23xP9b. haved somewhat aberrantly. For family 3 and P33xP37) (see Schemes I, 3, and 3). Table 2 (P24xP9b) there are indications that plant d9 is con- is a summary of the grades of pollen tube formation taminated. Besides, the original P9 turned out to be in selfings and cross combinations within and a mixture of two clones, 9a being the contaminant. among the four groups a4distinguished within the In family 3 (P34xP37) plant c10 has behaved aber- fcwr F-families: figures underlined in a matrix have rantly and is most probably contaminated. been tabulated as 0-values, whereas figures with line on top correspond to plus values in the table. Genetical interpretation Fl-families I and 2 have, by far, been those most extensively and carefully investigated, with several Fi data of relevance for the genetical interpretation retest\ of combinations where results were deemed are summarized in Table 3. Self-incompatibility ambiguous or dubious. For family 1 (P33xP34) prevails. Clearly, there are four groups formed. with there are indications that, in any case, plant a13 is intra-incompatibility and forming two both-way contaminated. less probably so for plants b9, b22. compatible pairs. Intercrosses in other combinations Hriedrfoc 1 1.1 (19901 S-GENE ACTION IN CERASWM 209

b C c:

Scheme 4. FI-family 4, P34XP37. Frequencies of long pollen tubes formed when 21 F1-sibs were selfed, intercrossed, and backcrossed to the two parents Line below figure denotes 0-value observed Line on top of figure denotes + value observed among the four groups are more successful than yield similar results. But the pattern of pollen beha- selfings and intragroup cross-pollinations, but with viour in group intercrosses outside the “compatible quite a considerable majority of incompatible pollen diagonal” of the mating matrix forms the conclusive grains. argument against this genetical model, the vast ma- All four F1-families were derived from crosses jority of the pollen grains reacting incompatibly. with full seed set and no trace of incompatibility. Sporophytic control with independent action (co- Assuming 1 disomic S-locus, a cross S1,2xS3.4 dominance) of the S-alleles in the heterozygote ap- would explain the four groups appearing in the FI. pears a more adequate interpretation. This hypothe- Garnetophytic control of the pollen would explain sis is strongly supported by the fact that the number a mating matrix where reciprocal combinations of long pollen tubes is low also when F,-plants are ruhk 2 Grdde\ of long pollen tube\ tormed in selfings and iros5 cornhination, within dnd among the four group\ a4distingui5hed withm the tour F,-famiIies

P plant5 Grades of long pollen tubes formed crocied Type of pollination 01?34+ Total 33 x 34 Selfingj I4 7 I -_77 Crosses bithin groups 68 35 4 3 110 Crosses between groups a x b (and recipr.) 10 12 8 3 9 42 a x c (and recipr.) 916 11 3 30 a x d (and recipr.) 1 I 40 42 b x c (and recipr.) 1 69 70 b x d (and recipr. i 23 34 24 11 6 98 c x d (and recipr. ) 3s 29 2 2 2 70 Rackcrosses to parents P33 x F, (and recipr.) 19 12 5 3 4 43 P34 x F, (and recipr.) 919 9 I4 42 ?2x 31 Selfings I4 5 19 Crossey within groups 64 19 I 84 Crmses between groups a x b (and recipr.) 22 25 3 50 a x c (and recipr. I 2693 20 ax d (and recipr.) I11 66 69 b x c (and recipr.) 20 20 b x d (and recipr. ) 49 17 l 2 1 70 c x d (and recipr.) 716 3 2 28 Backcrosse5 to parents P32 x F, (and recipr.) 523 7 2 1 38 P33 xF, (and recipr.) II 20 4 2 1 38 24 x 9b Selfings 95 14 Crosses within groups 33 13 I 47 Crosses between groups axh (and recipr.) 22 16 I 39 a x c (and recipr.) 251 8 a x d (and recipr.) 1411 31 38 b x c (and recipr. j 10 10 b x d (and recipr.) 8 27 4 1 40 c x d (and recipr.) 422 8 Backcrosses to parents P24 x F, (and recipr.) 6135 1 25 P9b x F, (and recipr.) 677221 25

34 x 37 Selfings 16 2 2 20 Crosses within groups 4818 6 12 1 76 Crosses between groups a x b (and recipr.) 414 7 214 3 44 a x c (and recipr.) 14534 17 a x d (and recipr.) 1 53 54 b x c (and recipr.) 24 24 b x d (and recipr.) 4519 133 1 12 c x d (and recipr.) 235275 24 Backcrosses to parents P34 x F, (and recipr.) 717 2 3 4 33 P37 x F1 1113 6

crossed to their parent plants. But even here, the than after self-pollinations and cross-pollinations experimental data from group intercrosses outside within groups. The difference is not strong for F1- the “compatible diagonal” are not strictly consistent family 32x33, more considerably so for family with expectation. It is obvious from Table 2 that 33x34. Some variation among different seasons has long pollen tubes are more frequently formed in been noticed. these group intercrosses, and also in backcrosses, Weak dominance allelic relationships, strongly Table 3. F, data of relevance for the genetical interpretarion Table 4. Frequencies of long pollen tubes formed on F, I:IxP33 backcross offspring when the panel of test pollinators involved the Postulates four S-genes of the original cross S, x S3.4

P33 P34 (Sl 2 x S3.4) + Fl-family 1 (SU S1 4. 3. SZ,) No self-compatibility x SZ Backcross 1:l x P33 (Sl.3x S1.z) -+ BC family I1 4 groups No female sterile plants BC Tester set of pollinators/S-genes No one-way compatibility BC Within-group pollinations similar to selfing plant 1:l 1:2 1:4 1:8 P33 P34 plant 4 between-group combinations highly successful ~__~-____ no. 1.3 1.4 2.3 2.4 1.2 3.4 genotype 8 between-group combinations slightly exceeding selfing P x F, (and reciprocally) slightly exceeding selfing 11:16 1 1 + + 1 + 1.1 11:18 0 2 + + 3 + 11:44 2 1 + + 4 + 11:26 10+ 0 I.3 modifiable, and with the normal state approaching independent action would appear to be the best in- 11:11 4 4 1 1 0 + 1.2 11:22 4 2 1 1 0 + terpretation. Since modifiable dominance relation- 11:24 4 4 oo+ ships should be expected to promote pseudo-self- 11:32 2 1 0 2 0 + fertility, independent S-gene action in the pistil 11:33 4110+ should be expected. 11:37 3 4 0 0 0 + 11:45 1 1 1 1 1 + Various models involving a tetrasomic S-gene 11:47 1 4 0 0 0 + mechanism were tried extensively, but had to be 11:4Y 2 4 0 1 0 + discarded. 11:l 1 + 0 0 0 0 2.3 11:6 0 + 0 1 0 1 11:12 0 + 0 0 0 1 Backcross families 11:2Y 1 + 0 2 0 1 11:31 1 + 0 0 0 0 Plants 1 and 1.5 of F1 family 1 (P33xP34) were 11:38 0 + 0 0 0 1 selected for a study of backcross offspring. Polli- 11:40 0 + 0 1 0 1 nated by P33, plant 1 : 1 yielded between 31 and 35 11:41 0 + 0 1 0 0 seeds in seven capsules, plant 1: 15, 1.5 and 31 seeds 11:43 +01 11:46 I + 0 0 0 0 in two capsules, which corresponds to grades 4 and 11:so 1 + 0 1 0 0 34,respectively, of long pollen tube formation; the 11:51 1 + 0 4 1 3 outcome were backcross families 11 and 12, the 1153 + 3 + 2 + 4 Contaminant'? former from 100 seeds sown. The two selected FI-plants turned out both to Pollen tube grades: 0 =no long tubes formed I = I - 5 tubes belong to group a (S1,3, the cross P33xP34 being 2= 6 -10 rubes symbolized by S1.2xS3.4).Accordingly, the geno- 3 = 11-15 tubes 4 = itasn tubes types S1,l, S1.3,S1,2, and S2.3 should be expected to += >isn tubes occur in the backcross offspring from the cross S1,3 (female) x S1,2(male). A panel of test pollinators involving the two P plants and one representative interesting, consistent pattern of relatively high and for each of the four groups a4of the F1-family 1 low grades of long pollen tube formation (Tables 4 was applied to the two backcross families. Mainly and 5). Thus, there were generally high grades when because of some inbreeding deterioration, only 26 plants S1,2were pollinated by the S1.3 and S1,4test- and 25 plants, respectively, were available to be test ers, whereas plants S2.3 pollinated by the S1.2tester pollinated in the season of 1981 (Tables 4 and 5). showed low grades. Table 6 studies, for the four S- The two backcross families showed surprisingly alleles involved, the influence of matched and non- similar genotypic patterns (Tables 4 and 5). All four matched S-alleles on the frequencies with which genotypes expected were recovered, but S1.l and long pollen tubes were formed. When two alleles S1,3were in a clear minority, being represented by were matched together, the incompatibility was totally 6 plants. The S1,2and S2.3genotypes were strongest. When a single allele was matched, there about equally frequently represented among, to- was an increased grade of pollen tube formation in tally, 41 plants. Of the four plants with question all three cases, Sl being the weakest inhibitor marks, 2 may represent the S1,2and S2.3genotypes; against a breakthrough. Conversely, one may look the remaining 2 are difficult to explain. upon S2, S3, and S4 in non-matched position to be Moreover, the two backcross families showed an somewhat dominant to S1. The relationships among Tuble 5. Frequencies of long pollen tubes formed on F, 1: ISxP33 Table 6. Influence of matched and non-matched S-allele, on the backcross offspring when the panel of test pollinators involved the frequencies of long pollen tubes formed when backcross offspring four S-genes of the original cross S I ?x S3 SI I, s1.2, SI 3, and S23 were pollinated by the panel of hix testers SI 2, S, 3, SI4, S2.3, S? 4. and S3 (cf. Tables 4 and 5) P33 x P34 (S,2 x S3 4) + FI-family 1 (S,3, S, 4r S2 3, S24) Backcross 1:15 x P33 (S, xS, ?) + BC family 12 S-alleles Grades of long pollen tubes formed Tester set of pollinators/S-genes BC BC Matched Non- 0 I 2 3 4 Total matched plant 1:15 1:2 1:14 1:8 P33 P34 plant ______~ no. 1.3 1.4 2.3 2.4 1.2 3.4 genotype SI s2 2 12s s3 146 1921 12:28 2 2 + + 1 + 1.1 s4 5 7 1 10 23 12:lS 0 3 +41 1.3 s2 s1 16 6 22 12:s 44010+ 1.2 s3 12 s 17 12:6 4200c+ S4 19 17 4 1 41 12:lO 1 000+ 12:17 2 2 0 0 0 + s3 s, 11 11 22 12:19 4 2 0 0 0 + s2 1 I 12:20 2 2 0 1 1 + s4 10 10 2 2 24 12:21 4 4 0 1 0 + 12:23 4 4 1 0 0 + s, +sz - 16 2 18 12:30 4 4 0 0 0 + s, +& - 1 1 S2+& - 23 23 12:l 0+0011 2.3 12:2 0+0101 12:4 1+0012 12:8 0+0103 frequency of fully compatible combinations may 12:9 1+0211 serve as a measure of the number of different S- 12:Il 1 + 0 0 1 0 12:13 0 + 0 0 0 1 alleles in the population. 12:14 1 + 0 0 1 0 At genotypic equilibrium for n S-alleles, with the 12:18 0 + 0 1 0 1 heterozygous genotypes equally frequent, a particu- 12:31 0 + 0 2 0 2 12:32 1 + 0 1 0 0 lar gene pair, when tested to n(n-1)/2 different gene pairs possible in the population, will meet the fol- 12:3 1++00+ ? 12:12 4 + 0 0 0 + 1.2? lowing probabilities: 12:27 1 1 0 1 0 1 2.3? double identity = 1 For pollen tube grades, see Table 4 single identity = 2(n-2) no identity = (n-2) (n-3)/2 the three “dominant” alleles are less clear. It is, Total = n(n-1)/2 however, provocative that, in non-matched position, Seven plants out of the sample of 52 population even the SIallele was able to slightly increase the plants grown from the original Swiss seed sample grade of long pollen tube formation. were used as pollen donors onto the majority of the Finally, among the gametes forming the 47 geno- remaining plants. Their ratios of fully compatible typically controlled plants, there were 22 Sl and 25 combinations and corresponding estimates of S-al- S3 eggs, whereas there were 6 Sl and 41 S2 sperms. lele numbers are entered in Table 7. Plants 9b, 32, Thus, among the fertilizing pollen tubes those that and 34 were mutually incompatible and reacted did not carry an S-gene matched in the pistil were identically as pollen donors onto the population in a startling majority. Does this indicate that a plants; they are likely to have the same S-genes. gametophytically controlled imprint is possible in The estimates range from 7 to 19 alleles in this exceptional cases, an imprint that governs the pollen restricted population sample; the weighted mean is grain’s reaction to the pistil? 10.

Number of S-alleles in the population It is assumed that, in the present S-gene system, full Discussion compatibility between two plants indicates that no The one-locus sporophytic incompatibility control S-allele is shared, whereas plants sharing one or in Cerustium arvense ssp. stricturn is associated both alleles will be clearly incompatible. Thus, the with trinucleate pollen, dry stigmas, pollen reaction Tuhk 7. Number of S-alleles of the population, estimated from allele matched showed their clearly highest grades the frequencies of fully compatible combinations between 7 males of long pollen tube formation when SI was the and 43 females matched allele. This could be taken to indicate an Pollen Plants pollinated effect of dominance exerted by the non-matched - No. alleles S2, S3, and Sq. On the other hand, even Sl donor Total Fully compatible alleles no. Number Ratio estimated in non-matched position slightly increased the grade of long pollen tube formation, which could indicate 9b 41 20 0.487 7 some effect of mutual weakening. And against an 32 40 19 0.475 7 34 37 18 0.486 7 interpretation with mutual weakening speaks the fact that the incompatibility was complete, or nearly 10 39 23 0.590 9 25 41 19 0.463 7 so, when both alleles of the pollen donor were 9a 42 32 0.762 16 matched in the pistil, as in self-pollinations and in 24 39 31 0.795 19 intragroup cross-pollinations. The data may be in- terpreted to show that the S-heterozygote produces both S-substances in the pollen and style, that any on the stigmatic surface, and persistency in poly- of these substances alone is sufficient to cause in- ploids; thus far this self-incompatibility system ap- compatibility, and that for some reason a fraction pears quite typical. Untypical is the striking ten- of the pollen grains react as if one of the two im- dency to independent action of the S-alleles prints were absent. involved; clear-cut dominance does not occur in any SAMPSON(1960) has suggested a model for S- of the four investigated F1-families. Quite unex- gene interaction, where a certain quantity of the pected is the observation that a pollen grain may particular incompatibility substance is required for have a functional advantage when its S-gene is not incompatibility to result, assuming different pro- shared by the pistil, the fertilizing pollen grains in duction rates and durations characterizing different the cross S1,3 (female) x S1,2 (male) being almost S-alleles and putting limits to their effectiveness, exclusively Sz. and with external conditions or modifying genes To be sure, a total of only 6 different S-alleles swinging the balance through degrees of activity to may have been introduced into the four F1-families. inactivity. Considered in the light of this model, the It is a strange coincidence that among the six plants present data will be interpreted to indicate (1) that sampled as parents for the Fl’s from among the transcription producing the S-specific messengers original 52 population plants, three (9b, 32, and 34) may not start simultaneously for the two alleles; (2) should carry the same two S-alleles, whereas plants that the S-locus at least sometimes shows first divi- 24 and 33 have, at least, one of their S-alleles in sion segregation; and (3) that in exceptional cases common. Still, even at this restricted number of S- there will be successive cytokinesis in the micro- gene pair constellations studied, the absence of full sporogenesis, the cell wall formation restricting S- dominance has to be judged a significant trait. specificity determination to within the individual It was a fortunate choice that from the very begin- microspore, as suggested by PANDEY(1958, 1960). ning of the pollen tube investigations, the exact Thus, by the successive cell wall formation, S-gene numbers of long pollen tubes produced were transcription, even if initiated before S-gene segre- counted in combinations judged incompatible, and gation in the first anaphase of the pollen mother put in relation to an approximative evaluation of the cell, may be insufficient to prevent the S-gene of lot of pollen grains applied (rich, medium, sparse). the dyad cell to put its imprint alone on the two The gap between combinations judged fully com- microspores formed in the second meiotic division. patible, and assigned a + sign, and the remaining Hence, the trace of gametophytic pollen control ob- ones is the more accentuated, since in the former served in the backcross families. case the individual filamentary style can evidently We are confronted here with an incompatibility lead only about 60 pollen tubes from among a con- system where the S-genes are in a state of transition siderably higher number of pollen grains applied, between gametophytic and sporophytic control of and, on the other hand, grade 4 corresponding to the pollen, a system quite different from the G, S- about 3-7 long pollen tubes produced from about system reported from some cruciferous species (LE- 100 pollen grains applied per style in a rich pollina- WIS et al. 1988; ZUBERIand LEWIS1988). One reason tion. for seeking homologies between sporophytic and The interaction between the S-alleles is not easy gametophytic self-incompatibility systems is to to judge. On the one hand, crosses with a single clarify evolutionary relationships. PANDEY(1960, 1970) argued forcefully that the gametophytic type and for the order Ranunculales, lends support to of system arose first and was later replaced by the TAKHTAJAK'S( 1969) opinion that the two orders sporophytic type in some groups, by a "slight shift form a phyletic lineage. For the order Papaverales, in the time of S transcription alone without altering which is closely related to Ranunculales, observa- the time of S protein synthesis" (PAKDEY1979). tions reported by OJALAet al. (1990) that Papaver And we owe to him the attention called to the im- orirritnle (2n=28) and P. pseudo-orientale (2n=42) portance of successive cytokinesis to form the have reduced self seed-set, indicating self-incompa- border between gametophytic and sporophytic pol- tibility. may signify that the one-locus gameto- len control (PAXDEY19.58, 1960). phytic incompatibility established for the genus Pa- A sporophytic incompatibility system based on pa\'ei. (LAWRENCE1975; LAWRENCEet al. 1978) is .S-genes with independent action may be deemed based on the same kind of S-gene. Sequence data less likely to evolve because of increased cross- for S-genes may shed decisive light on the question incompatibility (PANDEY1977). Nevertheless. this whether the S-gene lacking dominance interaction system is likely to be the one functioning success- and occurring in monocots (LUNDQVIST197.5) and fully within a large range of forms of Cerasfium in the presently discussed dicotyledonous phyletic UI'IY~C.even on the polyploid level (UGBOROGHO lineage can be traced down into a common dicotyle- 1977). As stressed by LEWIS(1976). at independent donous ancestral basis. action with less than four alleles in a sporophytic syqteni, all pollinations would be incompatible. and ,-\c~~rio~r~/eil~n.moltc.- I am most grateful to Professor Claude Fa- even to obtain SO % of random pollinations compat- varger. lnstitut de Botanique. Uiiiver4te de Neuchltel. for supply- ing me with the seeds of Cerusriuni U~I'PIISCasp. stricrrtm. Thanks ible aould require 8 alleles. In the present small are due to the late Mr Gunnar Welander. Mr Erik Dahlrnan, Egon population. some 10 alleler were made likely. It Ernanuelton. and Bengt Jacohssoii for able management of the may wll be that, in a sporophytic system with inde- plant,. For skilful and patient technical assistance with the cross- pendent action. occasional successive cell wall for- pollinations. I am most grateful to Mrs Lena Ghatnekar and Carin Lassen. Mrs Ghatnekar has conducted, in a most commendable mation at microsporogenesis with ensuing gameto- nay. the preparations and evaluations of the lots of slides for phytic imprint on the pollen may form a necessary fluorescence microscopy: I am greatly indebted to her. substitute to a system of dominance relationships Financial suppon for the present investigations by $rant no. B in the pollen, cross-compatibility being increased ?411-005 from the Suedish Natural Science Research Council is gratefull) acknowledged. without loss of self-incompatibility. In this way. a young jporophytic S-gene system evolving from a gametophytic basis may be permitted gradually to evolve dominance interaction between S-alleles. References The observations from the present sporophytic S- B.\TEvA\. A. J. 1954. Self-incompatibillty rysteins in angio- gene control of the pollen have offered strong indi- \pennr. 11. Iheris un~aru.-Heredity 8: 305-332 cations of incompatibility substances being synthes- B&rEv4\. A. J. 1955. Self-incompatibility systems in angio- sperms. JII. Cmciferae. - Heredit! 9: 53-68 ized in the young spore under the instruction of BRFNHAKEK.J. L. 1957. Pollen cytology and self-incompatibility diplopha5e messengers carried through meiosis. \y\tem\ in plants. - .I Hered. 48: 271--277 This conclusion should, in no way. be considered BREWHAKER.J. L. 1959. 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