Molecular karyotypes of loquat (Eriobotrya japonica) aneuploids can be detected by using SSR markers combined with quantitative PCR irrespective of heterozygosity

Abstract

Background: Aneuploidy, a condition caused by an imbalance between the relative dosages of chromosomes, generally produces a novel phenotype specific to the molecular karyotype. Few techniques are currently available for detecting the molecular karyotypes of aneuploids in plants. Results: Based on this imbalance in chromosome dosage, a new approach (referred to as 'SSR-qPCR') combining simple sequence repeat (SSR) markers and quantitative real-time PCR (qPCR) has been developed and utilized to detect some common aneuploids irrespective of heterozygosity. We screened 17 specific SSR markers covering all loquat linkage groups and redesigned 6 pairs of primers for SSR markers that can detect loquat chromosome aneuploidies. The SSR-qPCR detection results obtained for hybrid progeny and open-pollination progeny of triploid loquat showed diagnostic accuracies of 88.9% and 62.5%, respectively, compared with the chromosome preparation results. Conclusion: SSR-qPCR can detect loquat aneuploids and be used to construct the entire molecular karyotypes of aneuploid individuals. Therefore, this method offers a novel alternative for the detection of chromosome aneuploidies.

Genome-Wide Identification, Characterization and Expression Profiling of Aluminum-Activated Malate Transporters in Eriobotrya japonica Lindl.

“…Current investigation shows that the ALMT gene family expansion in loquat are likely to be WGD-or segmental duplication-driven as verified through synteny analysis (Figure 6, Table 3). Loquat is a diploid species with a basic chromosome number of n = 17 [23,52,53]. Out of 24 EjALMT genes, 10 genes were found to be located on chromosomes 6-9, one on chromosome 3, four on chromosome 4, two on chromosome 12, another four on chromosome 14 and rest of the two genes on chromosome 17.…”

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

“…Loquat leaflets of 49 lines (varieties) and F 1 hybrids were collected and brought back to the laboratory, where they were stored in the refrigerator at − 80 °C. The genomic DNA (gDNA) extraction method referred to Wen Guo's improved CTAB method [ 42 ]. DNA quality was detected by 1.0% agarose gel electrophoresis, and high-quality gDNA was diluted to 50 ng/μL for PCR and qPCR analyses.…”

“…Flesh color genotyping was carried out by qPCR with gDNA (analytikjena qTOWER 3 ), and a DNA sequence with a constant copy number in the genome was used as an internal control. The SSR marker CH03g12 shows no polymorphism in loquat, and its copy number only changes with ploidy [ 42 ]. CH03g12, actin [ 60 ] and H4-1 were used for the screening of loquats with different ploidies to identify primers with high consistency for generating the reference DNA sequence.…”

“…When two or more male parent-specific bands appeared in the F 1 hybrids, they were considered to be true hybrids. The PCR amplification and electrophoresis of SSRs were run according to the literature with slight modifications [ 42 , 81 ].…”

“…Hence, for polyploid genotyping, a more economical and convenient technique must be found. Quantitative real-time polymerase chain reaction (qPCR) is often used to detect gene expression [ 40 ], copy number variation [ 41 ] and aneuploidy [ 42 ], and has high application potential for genotyping and other analyses.…”