Quantitative real-time PCR to determine allele number for the astringency locus by analysis of a linked marker in Diospyros kaki Thunb

Abstract

Persimmon (Diospyros kaki Thunb.) is a polyploidy fruit tree species of economic importance to East Asia. Natural astringency loss is an important trait in persimmon breeding programs. Quantitative real-time PCR was used to determine the number of AST/ast alleles for fruit astringency in persimmon (D. kaki Thunb.). To this end, the cultivar Jiro was transformed with one or two copies of a gene encoding NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH), which was used as a standard for measuring the allele number of a sequenced marker tightly linked to the recessive ast locus for nonastringency. Primers for markers linked to the AST or ast allele were then used to measure the AST to ast ratio directly in the progeny of a full-sib cross. From determination of the AST to ast ratio and the results of the S6PDH copy number, the number of AST and ast alleles at the AST/ast locus was estimated. This research supported the hypothesis that D. kaki is a hexaploid with six AST and/or ast alleles. In addition to the determination of the allelic status of the AST locus, the application of real-time PCR for confirmation of the ploidy level and allelic composition of target genes in autopolyploids or allopolyploids was demonstrated.

Relationships among Asian persimmon cultivars, astringent and non-astringent types

“…PCNA-type cultivars appear to have originated from a mutation causing loss of the ability to accumulate tannins in the fruit at an early stage of fruit development; this is a qualitative trait Akagi et al 2009Akagi et al , 2010Akagi et al , 2012. The first PCNA-type cultivar is thought to have been derived from PCA cultivars .…”

Seasonal Abscisic Acid Signal and a Basic Leucine Zipper Transcription Factor, DkbZIP5, Regulate Proanthocyanidin Biosynthesis in Persimmon Fruit

“…Expression analysis was performed by quantitative real-time (qRT)-PCR analysis. Primer pairs designed for amplification were based on the conserved sequences of target genes as used in previous reports (Akagi et al, 2009a(Akagi et al, , 2009c(Akagi et al, , 2010). An aliquot of 1:5 diluted pool cDNA was used as a template for PCR.…”

“…This mutant nonastringent (NA) phenotype is also called the pollination constant NA type (Yonemori et al, 2000). It has been suggested that a single gene known as the ASTRINGENCY (AST) locus controls the allelotype of normal A-and mutant NA-type fruits, and the NA genotype expression requires six homozygous recessive alleles (ast) at the AST locus (Yamada and Sato, 2002;Akagi et al, 2009c). However, the AST locus has not been identified, and the genetic bases for the A/NA phenotypes have been partially elucidated.…”

Development of Molecular Markers Linked to the Allele Associated with the Non-astringent Trait of the Chinese Persimmon (Diospyros kaki Thunb.)

“…Identifying a locus showing the polyploidy inheritance mode on a genetic map is difficult; however, genomewide mapping has recently been performed using AFLP markers in the hexaploid sweet potato (Ipomoea batatas) ( Cervantes-Flore et al, 2008a, b;Kriegner et al, 2003). Furthermore, Akagi et al (2009Akagi et al ( , 2010 determined the allelic status in the AST locus, which contains six AST/ ast alleles, using quantitative real-time PCR analysis in the hexaploid persimmon. In the future, the three AFLP markers isolated in this study and the above-mentioned new techniques for genetic analysis of polyploid plants would contribute to identification of the CPCNA locus in Chinese PCNA persimmon.…”