Exploring a possible role of floral ANK-TPR proteins in the apomixis genetic control

STEIN, JULIANA; ORTIZ, JUAN PABLO AMELIO; SIENA, LORENA; AZZARO, CELESTE; PESSINO, SILVINA

Bahía Blanca

Workshop; V Ciclo de seminarios sobre avances en la caracterización genética y molecular de la apomixis; 2016

CERZOS-CONICET

Apomixis is a natural asexual mode of reproduction by seeds, which generates progenies consisting of exact genetic replicas of the mother plant. The harnessing of apomixis represents an enormous potential benefit to agriculture, since its introgression into sexual crops would greatly expand the current breeding and seed production capacity. In grass species, this trait is controlled by one or few genetic determinants. Particularly in Paspalum notatum, apomixis segregates a simple dominant locus (i. e. apospory controlling locus or ACL) with a distorted segregation ratio. Comparative mapping analyses revealed that a segment of rice chromosome 12 long arm is consistently syntenic to the Paspalum ACL in at least four species of the genus. This chromosome segment, of about 5.8 cM, likely includes key apomixis governing genes. One of the candidates mapping in this area is LOC_Os12g40770 (OS12G0599900), which encodes an ankyrin (ANK)-containing protein of 423 aa. ANK repeats are present in proteins involved in diverse biological processes such as cell cycle regulation, growth and development and were associated with apomixis in Penissetum ciliare and Poa pratensis. OS12G0599900 encodes a protein with six ANK and two tetratricopeptide (TPR) repeats at the N- and C-terminal regions, respectively. This type of ankyrin (ANK-TPR) is represented by a single member in Arabidopsis (AT3G04710), which encodes a carboxylate clamp-tetratricopeptide repeat (TPR) protein with potential to interact with Hsp90/Hsp70. In rice, the ANK-TPR are represented by 22 members. The objective of this work was to investigate a possible contribution of the Paspalum OS12G0599900 orthologs to the genetic control of apomixis. The OS12G0599900 cDNA sequence was used as query in BLASTx and BLASTp searches onto floral 454/Roche transcriptome databases of apomictic and sexual P. notatum. Ten homologous alleles/splice variants (isotigs), corresponding to six different genes (isogroups), were detected in the apomictic database. The lengths of the transcripts varied from 539 to 1971 bp, while the e-values ranged from e-131 to e-18. In the sexual library eight homologous alleles/splice variants, belonging to seven different genes were detected. The lengths of sequences varied between 733 and 1795 bp. E-values ranged from e-130 to 3e-38. Protein prediction analysis showed that only five out of the 18 isotigs (apoisotigs 11446, 11445 and 20350 and sexisotig 17384, 20570) encoded proteins of the ANK-TPR family (the rest showed homology in the ANK domain only). Four of these sequences (apoisotigs 11446 and 11445 and sexisotig 17384, 20570) mapped in silico within the region of rice chromosome 12 associated with apomixis (by applying the criterion of 65% identity over at least 60% of the length of the sequences and E-values < 0.005). Therefore, they qualified to be Paspalum OS12G0599900 candidate orthologs. We conducted experiments to identify which sequences map onto the ACL by using a segregating family for apomixis of 15 individuals, derived from the sexual tetraploid mother plant (Q4188) and an apomictic pollen donor (Q4117). The reproductive mode of each F1 plant was determined by cytoembryological observation of cleared ovules at anthesis and by assaying the SCAR marker PNSA2, which generates a band of 180 bp fully linked to the trait. Both parental plants, a sexual bulk (SB) and an apomictic bulk (AB) (5 individuals each) were used for testing the linkage to the ACL. PCR amplification of apoisotig11445 with three allele-specific primer combinations showed polymorphic patterns between the parental plants, but co-segregation with the ACL was not detected. Amplification of apoisotig 11446 showed a monomorphic band, which made segregation analysis impracticable. Amplification of apoisotig 20350 rendered several segregating bands, but none of them mapped at the ACL. The sexisotig 17384 didn´t amplified with the primers designed, and thus new pair of primers should be analyzed. Results obtained so far indicated that at least 6-7 genes (isogroups) encoding for proteins with ANK repeats are being expressed during the P. notatum reproductive development. At least five sequences corresponded to the ANK-TPR family and could represent putative orthologs to the rice gene mapping onto the ACL syntenic region. However, genetic linkage of these sequences with the trait could not be experimentally confirmed yet. Shortly, wide-genome sequencing will be used to investigate the existence of an OS12G0599900-like ankyrin sequence within the Paspalum ACL and its expression in flowers (the latter through comparisons with the 454/Roche transcriptome databases). Moreover, qRT-PCR experiments will be carried out to analyze the ANK-TPR expression pattern during sexual and apomictic reproductive development.