Inheritance of seedlessness and the molecular characterization of the INO gene in Annonaceae

Abstract The inheritance of the seedless fruit characteristic of Annona squamosa has not yet been explained. Molecular techniques may aid breeding programs, mainly in the assisted selection of the target gene. The INO gene may be related to seed development in these fruits. The objective of the present paper was to investigate the inheritance of seedlessness in the 'Brazilian seedless' sugar apple and INO gene conservation in Annona squamosa and Annona cherimola x Annona squamosa genotypes by assessing their homology with the INO database genes. The F1 generation was obtained by crossing the mutant 'Brazilian seedless' (male genitor) (P1) with the wild-type A. squamosa with seeds (M1 and M2, female genitors). The INO gene was studied in mutant and wild-type A. squamosa (P1, M1, M2 and M3) and in the Gefner atemoya (A. cherimola x A. squamosa) (M4) cultivar. The DNA was extracted from young leaves, and four sets of specific primers flanking the INO gene were amplified. The seedless characteristic was identified as stenospermatic in the fruits of parental P1, suggesting monogenic inheritance with complete dominance. High sequence similarity of the INO gene amplifications in the sugar apple accessions (M1, M2, M3) and the atemoya cultivar Gefner (M4) reinforces the hypothesis of their conservation.


Introduction
Annona fruits are tasty, with a sweet, creamy flesh and fragrant flavor when fully ripe (Pareek et al., 2011) in addition to presenting bioactive components with medicinal potential (Vilar et al., 2008(Vilar et al., , 2011Quílez et al., 2018). However, the development of seedless varieties of each progeny of the two crosses (P 1 x M 1 and P 1 x M 2 ) with the hexadecyltrimethylammonium bromide (CTAB) buffer method (Doyle and Doyle, 1990) associated with the purification of polysaccharides (Cheung et al., 1993).
DNA samples were submitted to amplification reactions (PCR) with specific primers PCR-LMINO 01/02, LMINO 03/04, LMINO 05/06 and LMINO 07/08 (Lora et al., 2011). The sequences available in GenBank for the A. thaliana INO gene were amplified to confirm its presence or absence in the accessions. A specific primer from the ACC gene (1 aminocyclopropane-1-carboxylate synthase) was also amplified as a control.
PCR reactions were performed on Techene TC-412 thermocyclers with a program of 35 cycles with the following temperatures and periods: initial denaturation at 94 °C for three minutes, 35 cycles at 94 °C for 30 seconds, annealing temperature of each primer for 30 seconds and extension at 72 °C for 40 seconds, and final extension at 72 °C for 4 minutes. The final volume per sample was 25 μL with 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 3.0 mM MgCl 2, 0.2 mM dNTPs, 0.4 mM each primer, 25 ng of genomic DNA, 1.0 unit of Taq DNA polymerase (Invitrogen) and ultra-pure water to adjust the volume to 25 μL.
The presence or absence of the band corresponding to each pair of primers used was detected in the amplified products in the visualization of the direct gel bands per parent (M 1 , M 2 , M 3 and M 4 ) and F 1 progenies (P 1 x M 1 and P 1 x M 2 ). Products generated in the parents were purified with the Wizard ® SV Gel and PCR Clean-Up System kit, and the fragments sequenced by Ludwing Biotechnology. The sequences were compared with those from the NCBI (National Center for Biotechnology Information) database as the BLAST (Basic Local Alignment Search Tool) program (Altschul et al., 1997) for nucleotides after sample sequencing.
A dendrogram was constructed from the GenBank sequence data from the INO gene transcription factor to evaluate the similarity between them and the treatments (accessions of seeded A. squamosa and the cultivar of A. cherimola x A. squamosa).

Results and Discussion
The fruits of all individuals from the M 1 and M 2 accessions and F 1 progenies (P 1 x M 1 and P 1 x M 2 ) had seeds. The seedless characteristic was observed only in the fruits of the male parent P 1, cultivar Brazilian seedless ( Figure 1). These preliminary data suggest a monogenic inheritance with complete dominance-type allelic interaction. The seedlessness of the Annona Brazilian seedless variety was due to the formation of a stenospermal fruit with partially formed seeds due to their abortion after fertilization, as reported for Vitis vinifera and Annona squamosa (Bouquet and Danglot, 1996;Lora et al., 2011;Mendes et al., 2012;Santos et al., 2014). A monogenic inheritance with complete cultivar, originated from a natural mutation, is the only seedless A. squamosa cultivar developed in Brazil, but it has an irregular shape and low yield and commercial value ).
An aspermic A. squamosa variety designated 'Thai seedless' produces normal size, seedless fruits following pollination. The stenospermy in this mutant is due to the suppression of the INO gene, leading to a loss of the outer integument of the ovule, which affects seed development (Lora et al., 2011). The first reports regarding the aspermic condition of the 'Brazilian seedless' variety appeared starting from 2014, together with the stenospermy type, which may be related to the suppression of the INO gene .
The transfer of the seedless characteristic from A. squamosa 'Brazilian seedless' to atemoya genotypes with potential for commercialization could improve fruit tree crops. This makes it necessary to understand the inheritance of the characteristic and INO gene conservation in Annonaceae as the mechanism of stenospermy and the occurrence of seedlessness in this family.
The objectives of the present study were to investigate (1) the inheritance of the seedless characteristic in A. squamosa and (2) study the conservation of the INO gene in genotypes of sugar apple and atemoya plants with seeds by measuring their homology with the INO genes deposited in a database.

Plant materials and the inheritance of the seedless characteristic in A. squamosa
The inheritance of the seedless fruit characteristic was evaluated in two accessions of seeded A. squamosa (M 1 and M 2 ) and one seedless cultivar, the Brazilian seedless (P 1 ). The INO gene conservation was evaluated in another seeded sugar apple accession (M 3 ) and in the genotype of the cultivar Gefner atemoya (M 4 ) of the Annonaceae Germplasm Collection at UNIMONTES, Campus Janaúba (15º47'50"S, 43°18'31"W, 516 m), Minas Gerais, Brazil.
The F 1 generation was obtained with the male genitor of the Brazilian seedless cultivar (P 1 ) and the female of two accessions of A. squamosa with seeds (M 1 and M 2 ).
The A. squamosa tree seedlings (M 1 , M 2 and P 1 ) were produced from cuttings in a nursery and the F 1 generation with the seeds harvested from the fruits from the artificial hybridizations. The plant materials were planted at UNIMONTES in August 2009, with five meters between rows and three meters between plants. The presence or absence of seeds in fruits from the F 1 generations of the two crosses (P 1 x M 1 and P 1 x M 2 ) and their respective parents (P 1 , M 1 and M 2 ) were evaluated in the fifth, sixth and seventh years after planting the F 1 seedlings in 2014, 2015 and 2016, respectively.
Genotyping to verify INO gene conservation-The DNA was extracted from young leaves of the three seed A. squamosa accessions (M 1 , M 2 and M 3 ), the seedless A. squamosa (Brazilian Seedless, P 1 ), the A. cherimola x A. squamosa atemoya cultivar (Gefner, M 4 ) and the 30 F 1 dominance-type allelic interaction of this trait in 78 F 1 A. squamosa plants (crossing wild-type A. cherimola with another seedless mutant) was also reported (Lora et al., 2011).
The similarity between the M 1 , M 2 , M 3 and M 4 sample sequences and those deposited in the GenBank (accession: GU828033.1) for the transcription factor of the INO gene in A. squamosa was high due to the presence of the INO gene and, consequently, of seeds in its fruits (Souza et al., 2010;Varoquaux et al., 2000). Similarity in 100% of the GenBank sequences, added as a standard with one of the sequences sampled, reinforces the hypothesis of INO gene conservation in seeded A. squamosa accessions (M 1 , M 2 , M 3 ) and in the Gefner cultivar of A. cherimola x A. squamosa (Nachtigal et al., 2005) The INO gene was amplified with bands in the accessions of the seeded sugar apple, the atemoya cultivar Gefner and in all progeny of the F 1 population. This gene did not present a band in the Brazilian seedless A. squamosa variety (P 1 ) (Figure 2). Sequence homology, generated by the amplification of the M 1 , M 2 , M 3 and M 4 accessions, was high, with 79 to 100% similarity to the INO gene transcription factor in A. squamosa (Figure 3). The amplification of the INO gene, from the specific primers for seeded A. squamosa, from the A. cherimola x A. squamosa Gefner and from the F 1 accessions is due to its conservation in these genotypes as reported for Annonaceae (Souza et al., 2010). On the other hand, the absence of amplification in the Brazilian seedless variety indicates the deletion/discontinuation of this gene, as reported for the 'Thai seedless' variety of A. squamosa (Lora et al., 2011).
The high homology of the sequences generated by the M 1 , M 2 , M 3 and M 4 accession amplification indicates that seed presence in A. squamosa and probably in atemoya is due to the INO gene. This gene is conserved in species and accessions of the Annona genus, forming seeds in external teguments that may be common to all angiosperm groups (Mcabee et al., 2005;Yamada et al., 2003). The presence of the INO gene and the origin of the external integument appear to have evolved concomitantly by genetically regulating ovule development in association with the evolution of the specific structure of the angiosperms (Mcabee et al., 2005;Lora et al., 2011). The INO expression pattern in Annonaceae is consistent with the conservation of the function of this gene in Arabidopsis, and the absence of the external integument in A. squamosa without seeds (Thai seedless) is associated with the suppression of this gene (Villanueva et al., 1999) The similarities of the transcription factor of the INO gene in wild A. squamosa was observed between the sequences of the 16 samples obtained and the one deposited in the GenBank (accession: GU828033.1) (Figure 3). The INO gene transcription in wild-type A. squamosa varieties was not observed in the seedless mutants (Lora et al., 2011). Seedlessness due to INO gene disruption may allow assisted  selection by molecular markers using these primers in subsequent generations and backcrosses with the cultivar Gefner to detect seed absence in plants at the seedling stage, consequently reducing costs and time to obtain a new Annonaceae variety (Arif et al., 2010). The origin of the Thai and Brazilian seedless varieties is not well understood. The 'Brazilian seedless' was first identified in Brazil in 1940 by the agronomist José Chaves da Cunha and its origin has not yet been fully determined but it may have come from a natural mutation or from other parts of the world. Both varieties present the deletion of the INO locus but this does not prove that they belong to the same line and additional studies would need to be performed in order to resolve this question.

Conclusions
The inheritance of the seeded characteristic is possibly monogenic with complete dominance-type allelic interaction, with the INO gene conserved in seeded A. squamosa cultivars and high sequence homology of the transcription factor of this gene in A. squamosa.