Histological and molecular biological analyses of reproductive isolation in interspecific-interploidy crosses Nicotiana suaveolens × Nicotiana tabacum

概要

Reproductive isolation, which is divided into two types, prezygotic and postzygotic barriers, is a mechanism that separates species and plays a crucial role in the evolution of animals and plants (Stebbins 1966; Rieseberg and Blackman 2010). In plants, prezygotic barriers include the inhibition of pollen adhesion to the stigma, pollen germination, pollen tube growth in the style, and pollen tube penetration of the ovule micropyle (Dickinson et al. 2012). Postzygotic barriers, which occur after a successful fertilization, include seed abortion as well as the weakness, inviability, and sterility of the F1 hybrid plants or their offspring (Li et al. 1997; Bushell et al. 2003; Ichitani et al. 2007; Kuboyama et al. 2009; Tezuka et al. 2010; Guo et al. 2016). These barriers hinder plant breeding programs, especially those involving wide hybridizations such as interspecific or intergeneric crosses.
　This study aimed to investigate and overcome the postzygotic barriers in crosses between Nicotiana suaveolens and N. tabacum. In Chapter 1, I investigated what types of reproductive isolation were exhibited using several accessions of N. suaveolens for crosses with N. tabacum. In subsequent chapters, among reproductive isolation observed in Chapter 1, I focused on seed abortion and ovary abscission in interploidy-interspecific crosses. In Chapter 2, I investigated the mechanism of these reproductive barriers using ploidy manipulated lines and histological methods. In Chapter 3, I investigated whether different parental ploidy level is the only factor causing seed abortion and ovary abscission. Finally, in Chapter 4, I carried out exhaustive gene expression analysis of seed abortion in interploidy-interspecific crosses using RNA sequence.

Chapter 1. Nicotiana suaveolens accessions with different ploidy levels exhibit different reproductive isolation mechanisms in interspecific crosses with Nicotiana tabacum
　I investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. I revealed that four N. suaveolens accessions comprised 64 chromosomes (octoploid), while the other four accessions carried 32 chromosomes (tetraploid). Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12–17 days after pollination (DAP). A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, I confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.

Chapter 2. Type II seed abortion causes ovary abscission in interploidy-interspecific crosses between Nicotiana suaveolens and Nicotiana tabacum
　When octoploid Nicotiana suaveolens accession PI 555565 (8x) was used as the seed parent for the cross with tetraploid N. tabacum (4x), enlarged ovaries were dropped at 12-17 DAP and hybrid seeds were never obtained. Meanwhile, seed abortion was observed in another interploidy-interspecific cross between N. suaveolens PI 555561 (8x) as the seed parent and N. tabacum (4x) as the pollen parent (Chapter 1). In this chapter, I investigated whether highly excess maternal genome causes the enlarged ovary abscission. Using the two interploidy-interspecific crosses, comparative analyses for the ovary abscission zone and ovule development were conducted. I revealed that distinct types of abnormalities (Type I and Type II seed abortion) were found during ovule development in the two crosses. Based on the results, I established a hypothesis that ovary abscission is a consequence of Type II seed abortion. Further cross experiments using ploidy manipulated lines added strong supports to this hypothesis: successive increase of maternal ploidy resulted in successively Type I and Type II abnormal ovule developments, and the latter was accompanied with ovary abscission. Ploidy manipulations for balance of parental ploidy levels were sufficient to restore endosperm and embryo development, and prevent the ovary abscission.

Chapter 3. Maternal genome excess causes Type II seed abortion and ovary abscission in intraspecific crosses of Nicotiana suaveolens
　The study in Chapter 2 clearly indicated that unbalanced parental ploidy levels led to different types of seed abnormal development in interploidy-interspecific crosses. Highly excess maternal genome caused Type II seed abortion, which led to the enlarged ovary abscission. However, these experiments were conducted in interspecific crosses, and it was unclear whether the abnormal seed developments were caused only by different parental ploidy levels. To obtain further insight into the relationship between parental ploidy levels and abnormal seed development as well as ovary abscission, I carried out the intraspecific crosses using N. suaveolens accessions with different ploidy levels. In intraspecific crosses, normal seed development was observed in interploidy crosses (8x × 4x). However, Type I and Type II seed abortion were observed in highly maternal-excess interploidy crosses (16x × 4x), and Type II seed abortion led to ovary abscission. These results gave strong evidence to prove that the difference of parental ploidy is sufficient to cause successively Type I and Type II abnormal seed developments, and the latter led to ovary abscission.

Chapter 4. RNA sequencing analysis of seed abortion in interspecific crosses between Nicotiana suaveolens and Niacotian tabacum
　To better understand the mechanisms causing the seed abortion, I conducted RNA sequencing to exhaustively identify differentially expressed genes between abnormal seed development (Type I and Type II seed abortion) and normal seed development. I identified several expression changing genes including plant hormone related genes such as IAA, GID1 and TGA. It is reported that plant hormones are important to seed development (Figueiredo and Köhler 2018, Locascio et al 2014). Thus, my results suggested that abnormal expression patterns of plant hormone related genes were associated with seed abortion in interploidy-interspecific crosses.

　In conclusion, the results obtained in this thesis showed that Type I seed abortion and Type II seed abortion with ovary abscission occur in a phased manner as maternal to paternal genome dosage increases. The expression of plant hormone related genes would be associated with seed abortion. Then, Type II seed abortion triggers several events in ovary abscission zone, such as changes of abscission related gene expression, lignin deposition, and eventually ovary abscission. Furthermore, seed abortion and ovary abscission can be overcome by ploidy manipulations for balance of parental ploidy level. Hence, the outcome of this thesis will contribute towards understanding and overcoming seed abortion and ovary abscission in interspecific and interploidy crosses in plants.