Genotyping of polyploid plants using quantitative PCR: application in the breeding of white-fleshed triploid loquats (Eriobotrya japonica)

Haiyan Wang,Zhongyi Xie,Guolu Liang,Qigao Guo,Jingnan Luo,Jiangbo Dang,Di Wu,Shuang Yan

doi:10.1186/s13007-021-00792-9

Abstract

BackgroundPloidy manipulation is effective in seedless loquat breeding, in which flesh color is a key agronomic and economic trait. Few techniques are currently available for detecting the genotypes of polyploids in plants, but this ability is essential for most genetic research and molecular breeding.ResultsWe developed a system for genotyping by quantitative PCR (qPCR) that allowed flesh color genotyping in multiple tetraploid and triploid loquat varieties (lines). The analysis of 13 different ratios of DNA mixtures between two homozygous diploids (AA and aa) showed that the proportion of allele A has a high correlation (R2 = 0.9992) with parameter b [b = a1/(a1 + a2)], which is derived from the two normalized allele signals (a1 and a2) provided by qPCR. Cluster analysis and variance analysis from simulating triploid and tetraploid hybrids provided completely correct allelic configurations. Four genotypes (AAA, AAa, Aaa, aaa) were found in triploid loquats, and four (AAAA, AAAa, AAaa, Aaaa; absence of aaaa homozygotes) were found in tetraploid loquats. DNA markers analysis showed that the segregation of flesh color in all F1 hybrids conformed to Mendel's law. When tetraploid B431 was the female parent, more white-fleshed triploids occurred among the progeny.ConclusionsqPCR can detect the flesh color genotypes of loquat polyploids and provides an alternative method for analyzing polyploid genotype and breeding, dose effects and allele-specific expression.

Highlights

Ploidy manipulation is effective in seedless loquat breeding, in which flesh color is a key agronomic and economic trait
The flesh color-specific marker was not easy to observe in some red-fleshed heterozygous materials (‘DY1’, ‘JH1’, ‘Pell’ and ‘H30-6’), which affected the accuracy of the identification of unknown materials
This study shows that quantitative PCR (qPCR) technology can be used for polyploid genotyping and can effectively distinguish different heterozygotes

Summary

Introduction

Ploidy manipulation is effective in seedless loquat breeding, in which flesh color is a key agronomic and economic trait. Few techniques are currently available for detecting the genotypes of polyploids in plants, but this ability is essential for most genetic research and molecular breeding. Ancient whole-genome duplication (WGD) events resulting in polyploidy in various plants have promoted the innovative development of. Wang et al Plant Methods (2021) 17:93 brought about by the broad changes in ploidy provide abundant raw materials for the breeding system, allowing researchers to develop different breeding plans according to different breeding goals. Polyploid breeding is carried out in many crops and horticultural plants, such as wheat [8], watermelon [9], banana [10] and citrus [11]. Recent research suggests that the apple tribe (apple, pear and loquat) shared a WGD from a common ancestor with nine chromosomes [14], and found the frequency of large-scale fragment rearrangements [15]. The loquat chromosomes LG1/LG2, LG7/LG8 and LG11/LG13 showed high homologous relationships, but no high density and distributed genetic linkage map has been constructed [15, 16]

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