Evolution of the apomixis transmitting chromosome in Pennisetum

Yukio Akiyama 0 1 Shailendra Goel 0 1 Joann A Conner 0 1 Wayne W Hanna 1 Hitomi Yamada-Akiyama 1 Peggy Ozias-Akins 0 1 0 Department of Horticulture, The University of Georgia , 2360 Rainwater Rd., Tifton, GA 31793-5766 , USA 1 Authors' information Current addresses: YA: Livestock and Forage Research Division, Tohoku Agricultural Research Center (TARC), National Agriculture and Food Research Organization (NARO) , Akahira 4, Shimokuriyagawa, Morioka, Iwate 020-0198 , Japan SG: Department of Botany, University of Delhi , Delhi 110007 , India Background: Apomixis is an intriguing trait in plants that results in maternal clones through seed reproduction. Apomixis is an elusive, but potentially revolutionary, trait for plant breeding and hybrid seed production. Recent studies arguing that apomicts are not evolutionary dead ends have generated further interest in the evolution of asexual flowering plants. Results: In the present study, we investigate karyotypic variation in a single chromosome responsible for transmitting apomixis, the Apospory-Specific Genomic Region carrier chromosome, in relation to species phylogeny in the genera Pennisetum and Cenchrus. A 1 kb region from the 3' end of the ndhF gene and a 900 bp region from trnL-F were sequenced from 12 apomictic and eight sexual species in the genus Pennisetum and allied genus Cenchrus. An 800 bp region from the Apospory-Specific Genomic Region also was sequenced from the 12 apomicts. Molecular cytological analysis was conducted in sixteen Pennisetum and two Cenchrus species. Our results indicate that the Apospory-Specific Genomic Region is shared by all apomictic species while it is absent from all sexual species or cytotypes. Contrary to our previous observations in Pennisetum squamulatum and Cenchrus ciliaris, retrotransposon sequences of the Opie-2-like family were not closely associated with the Apospory-Specific Genomic Region in all apomictic species, suggesting that they may have been accumulated after the Apospory-Specific Genomic Region originated. Conclusions: Given that phylogenetic analysis merged Cenchrus and newly investigated Pennisetum species into a single clade containing a terminal cluster of Cenchrus apomicts, the presumed monophyletic origin of Cenchrus is supported. The Apospory-Specific Genomic Region likely preceded speciation in Cenchrus and its lateral transfer through hybridization and subsequent chromosome repatterning may have contributed to further speciation in the two genera. - Background Apomixis is an intriguing trait in plants that allows multiplication of maternal clones through seed reproduction [1]. Besides the potential for apomixis to be a powerful plant breeding tool due to the circumvention of genetic segregation and maintenance of heterosis in hybrid progenies, the trait is also compelling in terms of evolutionary studies. Apomicts have long been regarded as evolutionary dead ends [2] mainly because of their presumed lack of genetic variation in the absence of recombination and intermating, although various studies have shown high levels of chromosomal and morphological variation within agamic complexes [3,4]. More recently, levels of genetic diversity among asexual populations were found to be higher than expected when compared to those in sexually reproducing populations [5-9]. Apomicts can outcross when they produce viable pollen, through the occasional reduced egg, or by fertilization of unreduced eggs, and can thus act as bridges for introgressive hybridization between otherwise reproductively isolated taxa [10-14]. Hybrid lineages can be stabilized by apomixis, allopolyploidy and recombinational speciation [15]. Apomixis does, however, reduce the rate of chromosomal recombination in the female, thereby diminishing the opportunity for unequal crossing over to reduce repetitive element copy number [16], allowing instead an accumulation of transposons in the genome and an increase in genome size, at least in relatively recent lineages [17]. Recombination is further constrained during male meiosis in apomicts in the chromosomal region transmitting the trait to progeny [18,19]. The fundamental importance of recombination and the paradox of sex [20,21] have inspired interest in deciphering the evolution of asexual organisms [14,22]. The Pennisetum/Cenchrus branch of the monophyletic bristle clade of grasses [23] contains a major crop species, sexual pearl millet or Pennisetum glaucum (L.) R. Br., and at least 17 aposporous species [18]. Relationships have been inferred among some of these species using basic chromosome numbers, ITS (the internal transcribed spacers of ribosomal RNA genes) DNA data [24] and sequences from chloroplast genes such as ndhF (F subunit of NADH dehydrogenase) [25], ndhF and trnL-F [26], trnL-F and rpl16 [27]. Chemisquy [26] also used a nuclear gene (knotted) to study the phylogeny in Cenchrus, Pennisetum and related genera. ITS sequences provide limited resolution to estimate genetic similarities of hybrids and their parents due to concerted evolution [28]. Though chloroplast DNA is maternally inherited, and therefore can be criticized for its inability to assess biparental contribution to the genome, it can provide sequences from specific genes or intergenic regions that are phylogenetically informative. The tobacco (Nicotiana tabacum) ndhF gene is 2223 bp in length and has a nucleotide substitution rate [29] which is, for example, two times greater than that of rbcL, a second extensively studied chloroplast gene [30]. More recent studies have also demonstrated that the 3end of ndhF is more variable than the 5 region [31]. For the present study, we chose to sequence two chloroplast gene regions (a 1131-1155 bp fragment from the 3end of ndhF and 811-872 bp region from trnL-F) and a 792-799 bp segment from the ASGR-BBM-like gene, also located within the p208 BAC used in fluorescence in situ hybridization (FISH) analysis. We furthermore report molecular cytogenetic analysis of the genomic region associated with apomixis, the apospory-specific genomic region (ASGR) that was previously identified in P. squamulatum, C. ciliaris and now in 16 Pennisetum and one additional Cenchrus species. The ASGR is conserved between P. squamulatum and C. ciliaris based on high sequence similarity between putative orthologous genes within this region; syntenic relationships between chromosomal sequences identified by BAC probes; shared cytological features of hemizygosity, the heterochromatic nature of the ASGR, and a region of low copy DNA flanked by high copy sequences [32-37]. Nevertheless, there are distinct structural differences in the ASGR-carrier chromosomes of these two species. These previous observations suggested that a conserved ASGR haplotype may occur in different chromosomal contexts among species. We now compare the extent of conservation and variation in the ASGR and ASGR-carrier chromosome in parallel with a Pennisetum and Cenchrus species phylog (...truncated)