Clade-Specific Plastid Inheritance Patterns Including Frequent Biparental Inheritance in Passiflora Interspecific Crosses

Abstract

Plastid inheritance in angiosperms is presumed to be largely maternal, with the potential to inherit plastids biparentally estimated for about 20% of species. In Passiflora, maternal, paternal and biparental inheritance has been reported; however, these studies were limited in the number of crosses and progeny examined. To improve the understanding of plastid transmission in Passiflora, the progeny of 45 interspecific crosses were analyzed in the three subgenera: Passiflora, Decaloba and Astrophea. Plastid types were assessed following restriction digestion of PCR amplified plastid DNA in hybrid embryos, cotyledons and leaves at different developmental stages. Clade-specific patterns of inheritance were detected such that hybrid progeny from subgenera Passiflora and Astrophea predominantly inherited paternal plastids with occasional incidences of maternal inheritance, whereas subgenus Decaloba showed predominantly maternal and biparental inheritance. Biparental plastid inheritance was also detected in some hybrids from subgenus Passiflora. Heteroplasmy due to biparental inheritance was restricted to hybrid cotyledons and first leaves with a single parental plastid type detectable in mature plants. This indicates that in Passiflora, plastid retention at later stages of plant development may not reflect the plastid inheritance patterns in embryos. Passiflora exhibits diverse patterns of plastid inheritance, providing an excellent system to investigate underlying mechanisms in angiosperms.

Keywords: Passiflora; heteroplasmy; interspecific cross; plastid; plastid segregation/sorting-out; restriction digestion; uniparental and biparental inheritance.

Figure 1

Passiflora hybrid seeds under dissecting microscope at 10× magnification. (A) P. menispermifolia (8039) × P. menispermifolia (Sirena) progeny for comparison with interspecific hybrid in B that includes same maternal parent. (B) P. menispermifolia (8039) × P. miersii from subgenus Passiflora. (C). P. rufa × P. auriculata from subgenus Decaloba. (D) P. sphaerocarpa × P. pittieri from subgenus Astrophea. Left to right, seeds soaked in water for 24 h, seed without seed coat and embryo only.

Figure 2

Agarose gels of PCR amplified target regions following digestion with restriction endonucleases from Passiflora hybrid embryos. (A) The intergenic region (rpl32-trnL) amplified from P. menispermifolia and P. miersii and their hybrid (1044) with estimated fragment sizes after NdeI digestion. (B) Lanes 2 and 4 are amplicons of rpl32-trnL for P. miersii and P. menispermifolia and lanes 3 and 5 are products after digestion with NdeI at 37 °C for 12 h. (C). Lanes 2–11 are NdeI digestion (at 37 °C for 12 h) products of amplified rpl32-trnL for 10 embryos of hybrid 1044. Lane 12 is undigested PCR product for embryo 1 of hybrid 1044 as a reference. (D) The partial coding region within rpoC1 amplified from P. rufa and P. auriculata and their hybrid progeny (2005) with estimated fragment sizes after XhoI digestion. (E) Lanes 2 and 4 are amplicons of rpoC1 for P. auriculata and P. rufa and lanes 3 and 5 are products after digestion with XhoI at 37 °C for 12 h. (F) Lanes 2–11 are XhoI digestion (at 37 °C for 12 h) products of amplified rpoC1 for 10 embryos of 2005. Lane 12 is undigested PCR product for embryo 1 of hybrid 2005 as a reference. Lane 1 in all gel images (B,C,E,F) contains one kb DNA ladder (NEB). DNA bands were separated in 1.5% agarose gel stained with RedSafe^TM (INtRON Biotechnology). Abbreviations: P—Paternal, M—Maternal and B—Biparental.

Figure 3

Agarose gels of PCR amplified target regions following digestion with restriction endonucleases from Passiflora hybrid seedlings and older leaves. (A) The intergenic region (psbA-matK) amplified from P. lancetillensis and P. microstipula and their hybrids (2031) with estimated fragment sizes after SpeI digestion. (B) Lanes 2 and 4 are amplicons of psbA-matK from P. microstipula and P. lancetillensis and lanes 3 and 5 are products after SpeI digestion at 37 °C for 12 h. Lanes 6–11 are SpeI digested (37 °C for 12 h) PCR products amplified from hybrid seedlings (2031). For each seedling, the digested PCR products are shown for cotyledon (coty) and the first leaf (1st). Lane 12, undigested PCR product of 2031 seedling 1 (cotyledon) as a reference. (C) Lanes 2–5, SpeI digestion products using mature leaves for four hybrid (2031) individuals (Ind.) that were not included in the seedlings analyses. Lane 6, undigested PCR product for 2031 Ind.1. (D) The partial coding region within rpoB amplified from P. misera 9023 and P. misera 9355 and their hybrid progeny (2027) with estimated fragment sizes after EarI digestion. (E) Lanes 2 and 4 are amplicons of the rpoB fragment for P. misera 9355 and P. misera 9023 and lanes 3 and 5 are products after EarI digestion at 37 °C for 12 h. (F) Lanes 2–11, EarI digested (37 °C for 12 h) PCR product for five hybrid seedlings (2027) of P. misera 9023 × P. misera 9355. For each seedling, the digested products are shown for cotyledon (coty) and the first leaf (1st^‘). Lane 12, undigested PCR product amplified from 2027 seedling 1 (cotyledon). (G) Lanes 2–5, EarI digested PCR products for two additional hybrid seedlings of 2027, in the same order as F. (H) Lanes 2–8, EarI digested PCR products for seedling 6 of 2027 at different developmental stages, coty—cotyledons, 1st—first leaf, 2nd—second leaf, 3rd—third leaf, 4th—fourth leaf, 5th—fifth leaf, 20th—20th leaf. Lane 9, undigested PCR product of 2027 seedling 6 (20th leaf) as a reference. Lane 1 in all gel images (B,C,E,H) contains one kb DNA ladder (NEB). DNA bands were separated in 2% agarose gel and visualized with RedSafe^TM (INtRON Biotechnology). Abbreviations: P—Paternal, M—Maternal and B—Biparental.

Figure 4

Passiflora hybrid seedling phenotypes. (A,B) P. organensis x P. biflora hybrids. The hybrids displayed green cotyledons with white (A) or pale green (B) leaves; all hybrids died within a few weeks. (C,D) P. rufa × P. auriculata hybrids. The hybrids produce white/pale cotyledons (C) or hypocotyl (D) and die before developing leaves. (E) P. microstipula × P. lancetillensis hybrid. Variegation was observed in cotyledons. (F) P. lancetillensis × P. microstipula. Variegation was observed in cotyledons as well as few early leaves in the seedling. (G,H) P. misera (9023) × P. misera (9335) hybrids. Variegation was noted in cotyledons and occasionally in first leaf in some seedlings. The variegation phenotype often coordinates with vegetative segregation of biparentally inherited plastids detected through molecular analysis. Agarose gel images for seedlings 1 and 6 in Figure 3F,H are associated with seedling images G and H in this figure, respectively.