Do s genes or deleterious recessives control late-acting self-incompatibility in Handroanthus heptaphyllus (Bignoniaceae)? A diallel study with four full sib progeny arrays

Background and Aims Genetically controlled self-incompatibility (SI) mechanisms constrain selfing and thus have contributed to the evolutionary diversity of flowering plants. In homomorphic gametophytic SI (GSI) and homomorphic sporophytic SI (SSI), genetic control is usually by a single multi-allelic locus S. Both GSI and SSI prevent self pollen tubes reaching the ovary and so are pre-zygotic in action. In contrast, in taxa with late-acting self-incompatibility (LSI), rejection is often post-zygotic, since self-pollen tubes grow to the ovary where fertilization may occur prior to floral abscission. Alternatively, lack of self fruit set could be due to early-acting inbreeding depression (EID). The aim of our study was to investigate mechanisms underlying lack of selfed fruit set in Handroanthus heptaphyllus in order to assess the likelihood of LSI versus EID. Methods We employed four full sib diallels to study the genetic control of LSI in Handroanthus heptaphyllus using a precociously flowering variant. We also used fluorescence microscopy to study the incidence of ovule penetration by pollen tubes in pistils that abscised following pollination or initiated fruits. Key Results All diallels showed reciprocally cross-incompatible full-sibs (RCI), reciprocally cross compatible full-sibs (RCC), and non-reciprocally compatible full-sibs (NRC) in almost equal proportions. There was no significant difference between the incidence of ovule penetrations in abscised pistils following self- and cross-incompatible pollinations, but those in successful cross pollinations were around twofold greater. Conclusions A genetic model postulating a single S locus with four s alleles, one of which, in the maternal parent, is dominant to the other three, will produce RCI, RCC and NRC situations each at 33 %, consistent with our diallel results. We favour this simple genetic control over an early-acting inbreeding depression (EID) explanation since none of our pollinations, successful or unsuccessful, resulted in partial embryo development, as would be expected under a whole genome EID effect.

Self-sterility May Be Due to Prezygotic Late-acting Self-incompatibility and Early-acting Inbreeding Depression in Chinese Chestnut

Chinese chestnut (Castanea mollissima), which is native to China, has been cultivated as a nontimber forest tree species for 4000 years. This species has been found to display self-sterility, which results in a significantly lower seed set following self-pollination (SP) compared with that following cross-pollination (CP). Self-sterility can be induced by prezygotic or postzygotic late-acting self-incompatibility (LSI) or early-acting inbreeding depression (EID). To elucidate the causes of self-sterility in chestnut, we investigated pollen–pistil interactions, fertilization, and early ovule development following SP and CP by using a paraffin section technique and fluorescence microscopy. The fruit set percentage and seed characteristics also were evaluated among different pollination treatments. The results show that there were no significant differences in pollen tube behavior following SP vs. CP, regardless of the stigmatic or stylar level. Double fertilization was significantly greater following CP (18.09%) than SP (2.58%). The significantly lower percentages of ovule penetration and double fertilization in the selfed vs. crossed ovules support a prezygotic LSI mechanism in C. mollissima. The fruit set resulting from chase-pollination (CHP; 53.85% to 63.64%) was greater than that resulting from SP (12.12% to 14.00%). In addition, the distribution of aborted seed sizes after SP showed a widely clumped pattern. Abortion occurred at different stages during seed development rather than at a uniform stage, which supported the idea that EID was operating in C. mollissima. Levels of self-sterility in the chinese chestnut trees ranged from 88.2% to 90.5%. Thus, partial prezygotic LSI and EID contributed to self-sterility in the C. mollissima ‘Yanshanzaofeng’, with prezygotic LSI rejecting part of the self-pollen in the ovary and EID aborting part of the self-fertilized seeds.

Floral biology and late-acting self-incompatibility in Jacaranda racemosa (Bignoniaceae)

Breeding-system studies have been conducted with 38 of the approximately800 species of Bignoniaceae, and self-incompatibility was found in 31 of these. In species for which the site of self-incompatibility barrier was studied, self-pollinated flowers consistently failed to develop into fruits, even though pollen tubes grew down to the ovary and penetrated most of the ovules. In this study, we have investigated the floral biology and the breeding system in Jacaranda racemosa Chamisso, with hand-pollination experiments and the histology of post-pollination events. Flower anthesis lasted 1–3 days, and although the frequency of flower visitation was extremely low, natural pollination seemed to be effected mainly by medium-sized bees. Because the conspicuous staminodium favours eventual pollination by small bees, a possible role of the staminodium in the increase of potential pollinators is suggested. Hand-pollinations indicated that J. racemosa is a self-sterile species. Histological analysis of post-pollination events indicated the occurrence of a kind of late-acting self-incompatibility in which the processes of ovule penetration, fertilisation and endosperm initiation were slower in selfed than in crossed pistils. Until the time of self-pollinated pistil abscission, no signs of endosperm malfunction or proembryo development were observed in selfed pistils. Therefore, inbreeding depression is an unlikely explanation for self-sterility in J. racemosa.

Outcrossing in interspecific hybrids between Eucalyptus spathulata and E. platypus

Outcrossing was investigated in interspecific hybrids between self-fertile Eucalyptus platypus Blakely and partially self-sterile E. spathulata Hook., which shows both pre- and post-zygotic timing. Four hybrid trees were used for the study, two with E. spathulata and two with E. platypus as female parent. Each hybrid had a similar number of locules to each other and to the E. platypus parent, and an intermediate number of ovules per flower compared with the parent species. Controlled hand-pollinations were carried out, in which both self- and cross-pollen from the other hybrid tree with the same female parent species was applied to flowers on each of the four trees, and observations were made 10days, 4weeks and 8weeks after pollination and at seed maturity. In all hybrids, mean seeds per capsule was consistently higher following cross-pollination than following self-pollination. All hybrids showed a reduction in pollen tube number between the top and base of the style when examined by fluorescence microscopy. One tree had significantly fewer cross- than self-pollen tubes at the base of the style, but a similar number of ovules was penetrated by pollen tubes following both treatments. In the other three, there was no difference between cross- and self-pollination in pollen tubes in the style. In three of the four trees there was no difference in ovule penetration following self- or cross-pollination, but in the other, more crossed than selfed ovules were penetrated. Light-microscopy observation of ovules indicated that ovule abortion following fertilisation accounted for the reduced numbers of seeds following self-pollination and to a lesser extent following cross-pollination. All four hybrid trees, irrespective of female parent, were partially self-sterile and resembled the partially self-sterile E. spathulata rather than the self-fertile E. platypus. While the timing of outcrossing control of E. spathulata was both pre- and post-zygotic, only one hybrid was similar, with the other three showing post-zygotic control.

Self-incompatibility in Eucalyptus globulus ssp. globulus (Myrtaceae)

Controlled pollinations with self- and cross-pollen were applied to individual flowers of five mature Eucalyptus globulus Labill. ssp. globulus trees to investigate the site of action of the self-incompatibility mechanism. Growth of pollen tubes in styles at 2 weeks after pollination and ovule penetration by pollen tubes at 2 and 4 weeks after pollination were investigated by fluorescence microscopy. Some pollinated flowers were left to develop to seed maturity, then harvested to quantify the level of self-incompatibility of each tree. Trees ranged from 76 to 100% self-incompatible. There was no significant difference in the number of pollen tubes in the style between treatments although variation was present between trees. The number of pollen tubes present was similar to the number of ovules present within flowers. Penetration of ovules by pollen tubes over all five trees combined revealed no difference between treatments at 2 weeks after pollination; however, there was slightly greater penetration by cross-pollen tubes at 4 weeks after pollination. This difference was not large enough to account for the near complete lack of selfed-seed production, suggesting late pre- or post-zygotic arrest of selfed ovules.

Embryology of Eucalyptus Spathulata and E. platypus (Myrtaceae) Following Selfing, Crossing and Reciprocal Interspecific Pollination

Embryology of Eucalyptus spathulata Hook. and E. platypus Hook. (subgenus Symphyomyrtus, section Bisectaria) was investigated by bright field microscopy to determine the cause of ovule failure at 2 months following selfing, crossing and reciprocal interspecific pollination. Eucalyptus spathulata retained more capsules and produced more seeds following cross- than self-pollination, whereas there was no difference between selfing and crossing for E. platypus. Both species produced seeds following interspecific pollination, but germination was low. Highest ovule penetration by a pollen tube, and ovule growth at 2 months, was observed following crossing of E. spathulata and following interspecific pollination of E. platypus. Most self-pollinated ovules of both species were degenerating and were either unfertilised or had undivided zygotes and free nuclear endosperm. Muticellular pro-embryos were more common in cross-pollinated and interspecific pollinated ovules than following selfing. It was concluded that ovule degeneration of E. spathulata and E. platypus following selfing resulted from low levels of pollen tube penetration and fertilisation, and that in those ovules which were fertilised, the zygote generally failed to divide. Degeneration following cross-pollination of E. spathulata also resulted from failure of the zygote to divide, and in E. platypus additional reasons were low levels of pollen tube penetration and fertilisation, and lack of cellularisation of the endosperm. Ovule degeneration of both species following interspecific pollination included failure of the zygote to divide, but in E. spathulata it also resulted from slower embryo development and reduced cellularisation of the endosperm.