Developing Gene-Tagged Molecular Markers For Evaluation Of Genetic Association Of Apple V-ATPase Genes With Fruit Acidity

Apple(Malus domestica)is one of the most economically important fruit crops in the world.Apple fruit flavor is a derivative of fruit quality,a very important trait significantly influenced by organic acid.Malic acid constitutes approximately 90%of total organic acids in apple fruit.A number of genes are involved in the regulation of malic acid accumulation in fruit,including organic acid transporter genes.The Ma gene has been identified as major gene controlling apple acidity.Besides the Ma gene,genes encoding V-ATPase(VHA)are also known to be critical transporters regulating malic acid accumulation in fruit.Despite their critical role in malic acid accumulation,little information is available on how the transporter genes influence fruit acidity.It is unclear if the MdVHA genes have a significant influence on malic acid accumulation in apple.In this study,we identified MdVHA genes in apple based on the apple draft genome of ’Golden delicious’ doubled-haploid tree(GDDH13 v1.1).A total of 15 VHA genes were found located on 12 out of the 17 apple chromosomes(Chr),and their expression profiles were determined using qRT-PCR in fruit of two apple cultivars,which contained high and low malic acid content,respectively.Phylogenetic analysis showed that the MdVHA genes are grouped into five clades.qRT-PCR analysis revealed 9 out of the 15 MdVHA genes were highly expressed throughout fruit development.To determine if these nine MdVHA genes have an effect on fruit malic acid content,gene-tagged molecular markers were developed for each MdVHA gene,and subsequently subjected to screen 98 apple germplasm.As a result,each molecular marker yielded two or more polymorphic bands and each band was recorded as an allele.Statistical analysis showed that only one gene MdVHA3(MD05G1124700)is significantly associated with malic acid content in mature fruit.The MdVHA3 gene is located on Chr5.This study not only provides an efficient way to investigate gene functions using gene-tagged molecular markers,but is also helpful for genetic improvement of fruit acidity in apple breeding programs in the future.