Bruno Rafael Alves Rodrigues, Charles S Gasser, Samy Pimenta, Marlon Cristian Toledo Pereira, Silvia Nietsche.

Plant Reprod.; 2023 May 9. doi: 10.1007/s00497-023-00464-9. Online ahead of print.

Abstract

Inheritance of the presence/absence of seeds in Annona squamosa is mediated by a single fully recessive gene and is caused by a deletion of the INNER NO OUTER (INO) locus. For some fruits, seedless varieties are desirable for consumption and processing. In the sugar apple tree (Annona squamosa L.), the seedless trait in the Thai seedless (Ts) and Brazilian seedless (Bs) accessions was associated with defective ovules and an apparent deletion of the INNER NO OUTER (INO) ovule development gene locus. Segregation analysis of F₂ and backcross descendants of crosses of Bs to fertile wild-type varieties in this species with a multi-year generation time showed that seedlessness was recessive and controlled by a single locus. Comparison of whole genome sequence of a wild-type plant and a third accession, Hawaiian seedless (Hs), identified a 16 kilobase deletion including INO in this line. Ts and Bs lines were shown to have an identical deletion, indicating a common origin from a single deletion event. Analysis of microsatellite markers could not preclude the possibility that all three seedless accessions are vegetatively propagated clones. The sequence of the deletion site enabled a codominant assay for the wild-type and mutant genes allowing observation of complete cosegregation of the seedless/defective ovule phenotype with the INO deletion, showing maximal separation of less than 3.5 cM. The observed deletion is the only significant difference between the wild-type and Hs line over 587 kilobases, likely encompassing much more than 3.5 cM, showing that the deletion is the cause of seedless trait. The codominant markers and obtained progenies will be useful for introgression of the seedless trait into elite sugar apple lines and into other Annonas through interspecific crossings.

See https://pubmed.ncbi.nlm.nih.gov/37160783/

Figure 5: PCR of INO deletion region. A AsINODel primers designed to amplify a fragment spanning the deletion junction were used on Hs and Ts DNA and the products were electrophoresed on an agarose gel. The Hs DNA produced the expected 457 bp fragment, and a comigrating fragment was produced from Ts. B A combination of the AsINODel and LMINO1/2 primer pairs was used in single reactions to amplify DNA from Ts, wild-type and a mixture of wild-type and Ts (“Mix”). Only the expected 457 and 350 bp fragments were amplified from the Ts and wild-type DNA, respectively. Both bands were amplified from the mixture demonstrating effective detection of a heterozygous state. Del. and WT indicate deletion-specific and wild-type-specific PCR products, respectively

Fruit of A. squamosa. Longitudinal sections of fruit from A M₂ wild-type and B the mutant Brazilian seedless (Bs) showing seed rudiments (arrows), and C split fruit from Hawaiian seedless (Hs). Backgrounds of images were normalized to white for clarity