Allelotyping of S-RNase Gene and Promoter Region of MdMYB10 Gene in some Genotypes of Apple (Malus spp.)

علوم باغبانی

Article 2, Volume 35, Issue 4 - Serial Number 52, January 2022, Pages 469-477   

Document Type: Research Article

DOI: 10.22067/jhs.2021.60821.0

Authors

M. Vakili-Gartavol; N. Mahna^*

Department of Horticultural Sciences, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

Abstract

Introduction: Red-fleshed apples (Malus spp.) are one of the rarest apple genotypes in the world and the accumulation of a high amount of anthocyanin, is the main cause of the redness of their fruit flesh. Anthocyanins are among important flavonoids and due to antioxidative activity, scavenge reactive oxygen species, and hence, are considered as one of the health-promoting nutraceuticals in the human diet. The amount of anthocyanins depends upon the expression of the transcription factors that are regulating their metabolic pathway. Among these transcription factors are the members of the MYB gene family. MdMYB10, belonging to this gene family in apple, has been shown to have a significant role in controlling the amount of anthocyanin production and redness in fruit flesh. The expression of MdMYB10 and consequently, the production of MdMYB10 proteins has positive feedback on its own expression. This happens due to a 23 bp microsatellite tandemly repeated 5 times in its promoter region (called allele R₆) which is a target sequence for MdMYB10 acting as a positive regulator. This structure invokes the overexpression of MdMYB10 which in turn increases the expression of anthocyanin producing enzymes and finally the amount of anthocyanin in all organs of the apple plant including fruit flesh. The apple Malus pumila var. Niedzwetzkyana and its derivatives have been reported to have such a structure in the promoter region of the MdMYB10 gene. The length of the R₆ allele is 496 bp, while the R₁ allele is only 392 bp long.
However, in some cases, a locus linked to the S₃ allele of the S-RNase gene has been proposed to be responsible for the redness of the fruit flesh in some genotypes. It has been reported that even the offspring of these plants have had red-fleshed fruits. 
Materials and Methods: To study the mechanism of the redness of the fruit flesh in some local genotypes, genomic DNA was extracted using the CTAB method from the leaf samples obtained from 9 red- and white-fleshed apple genotypes including Red Delicious, Golden Delicious, Miandoab, Makamik (Khalatpoushan), Bud 9, Varzighan, and Ivand. Then the allelotype of the promoter region of the MdMYB10 gene as well as the existence of S₃ allele at S-RNase locus was investigated using polymerase chain reaction. For amplification of the target sequences, MdMYB10 and S₃ specific primers were exploited and 1% agarose gel electrophoresis of the amplified fragments was used for observing and scoring the bands. All steps were repeated seven times.
Results and Discussion: The results in this research showed that the white-fleshed genotypes (Red Delicious, Golden Delicious, and Granny Smith) were lacking any R₆ allele at the promoter region of the MdMYB10 gene and were R₁R₁ homozygotes, while the red-fleshed genotypes (Miandoab, Makamik (Khalatpoushan), Bud 9, and Varzighan) had at least one R₆ allele at the mentioned promoter region as well as a S₃ allele in the self-incompatibility locus S-RNase. These results were in accordance with the previous reports. Therefore, these samples could be traced back to Malus pumila var. Niedzwetzkyana. Evaluating the S-RNase locus in these genotypes illustrated that Granny Smith (as positive control), Golden Delicious (as positive control), Makamik (Khalatpoushan), Miandoab, Varzeghan, Bud 9 and tissue culture sample, showing a band around 500 bp (smaller) had S₃ allele, while for Ivand and Red Delicious (as negative control) no S₃ band was obtained. For the tissue culture sample which was R₁R₁ at the promoter region and S₃ at S-RNase locus, it was postulated that flesh-redness may be due to the locus linked to the S₃ allele. We also got an unknown R band for the Ivand genotype when analyzing for the MdMYB10 promoter region. The sequencing of in the future studies, may help to unravel the mechanism by which shoot-redness happens in this genotype.   
Conclusion: The development of highly potent and novel cultivars for the fast-evolving market is indispensable in the plant breeding field. In this way, breeding apple plant, as an important temperate fruit with a long postharvest life, for redness of fruit flesh can be considered as a noticeable case. We could confirm in this research that in the endemic, red-fleshed apples, R₆ may be responsible for their high anthocyanin production. However, the S₃-RNase-linked locus should also be considered in marker-assisted breeding methods for this trait. Therefore, these red-fleshed genotypes are highly recommended to be employed in the national breeding programs for increasing the anthocyanin content of apple fruits.

Keywords

Anthocyanin; MdMYB10 gene promoter; Red-fleshed apples; S-RNase

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