Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare × Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). In addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare × Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.

Sweetpotato (Ipomoea batatas L.) is an outcrossing hexaploid species with a large number of chromosomes (2n = 6x = 90). Although sweetpotato is one of the world’s most important crops, genetic analysis of the species has been hindered by its genetic complexity combined with the lack of a whole genome sequence. In the present study, we constructed a genetic linkage map based on retrotransposon insertion polymorphisms using a mapping population derived from a cross between ‘Purple Sweet Lord’ (PSL) and ‘90IDN-47’ cultivars. High-throughput sequencing and subsequent data analyses identified many Rtsp-1 retrotransposon insertion sites, and their allele dosages (simplex, duplex, triplex, or double-simplex) were determined based on segregation ratios in the mapping population. Using a pseudo-testcross strategy, 43 and 47 linkage groups were generated for PSL and 90IDN-47, respectively. Interestingly, most of these insertions (~90%) were present in a simplex manner, indicating their utility for linkage map construction in polyploid species. Additionally, our approach led to savings of time and labor for genotyping. Although the number of markers herein was insufficient for map-based cloning, our trial analysis exhibited the utility of retrotransposon-based markers for linkage map construction in sweetpotato.

The southern root-knot nematode, Meloidogyne incognita, is a pest that decreases yield and the quality of sweetpotato [Ipomoea batatas (L.) Lam.]. There is a demand to produce resistant cultivars and develop DNA markers to select this trait. However, sweetpotato is hexaploid, highly heterozygous, and has an enormous genome (∼3 Gb), which makes genetic linkage analysis difficult. In this study, a high-density linkage map was constructed based on retrotransposon insertion polymorphism, simple sequence repeat, and single nucleotide polymorphism markers. The markers were developed using F1 progeny between J-Red, which exhibits resistance to multiple races of M. incognita, and Choshu, which is susceptible to multiple races of such pest. Quantitative trait locus (QTL) analysis and a genome-wide association study detected highly effective QTLs for resistance against three races, namely, SP1, SP4, and SP6-1, in the Ib01-6 J-Red linkage group. A polymerase chain reaction marker that can identify genotypes based on single nucleotide polymorphisms located in this QTL region can discriminate resistance from susceptibility in the F1 progeny at a rate of 70%. Thus, this marker could be helpful in selecting sweetpotato cultivars that are resistant to multiple races of M. incognita.

Next-generation sequencing (NGS)-based genotyping methods can generate numerous genetic markers in a single experiment and have contributed to plant genetic mapping. However, for high precision genetic analysis, the complicated genetic segregation mode in polyploid organisms requires high-coverage NGS data and elaborate analytical algorithms. In the present study, we propose a simple strategy for the genetic mapping of polyploids using low-coverage NGS data. The validity of the strategy was investigated using simulated data. Previous studies indicated that accurate allele dosage estimation from low-coverage NGS data (read depth < 40) is difficult. Therefore, we used allele dosage probabilities calculated from read counts in association analyses to detect loci associated with phenotypic variations. The allele dosage probabilities showed significant detection power, although higher allele dosage estimation accuracy resulted in higher detection power. On the contrary, differences in the segregation patterns between the marker and causal genes resulted in a drastic decrease in detection power even if the marker and casual genes were in complete linkage and the allele dosage estimation was accurate. These results indicated that the use of a larger number of markers is advantageous, even if the accuracy of allele dosage estimation is low. Finally, we applied the strategy for the genetic mapping of autohexaploid sweet potato (Ipomoea batatas) populations to detect loci associated with agronomic traits. Our strategy could constitute a cost-effective approach for preliminary experiments done performed to large-scale studies.

In many crop species, DNA fingerprinting is required for the precise identification of cultivars to protect the rights of breeders. Many families of retrotransposons have multiple copies throughout the eukaryotic genome and their integrated copies are inherited genetically. Thus, their insertion polymorphisms among cultivars are useful for DNA fingerprinting. In this study, we conducted a DNA fingerprinting based on the insertion polymorphisms of active retrotransposon families (Rtsp-1 and LIb) in sweet potato. Using 38 cultivars, we identified 2,024 insertion sites in the two families with an Illumina MiSeq sequencing platform. Of these insertion sites, 91.4% appeared to be polymorphic among the cultivars and 376 cultivar-specific insertion sites were identified, which were converted directly into cultivar-specific sequence-characterized amplified region (SCAR) markers. A phylogenetic tree was constructed using these insertion sites, which corresponded well with known pedigree information, thereby indicating their suitability for genetic diversity studies. Thus, the genome-wide comparative analysis of active retrotransposon insertion sites using the bench-top MiSeq sequencing platform is highly effective for DNA fingerprinting without any requirement for whole genome sequence information. This approach may facilitate the development of practical polymerase chain reaction-based cultivar diagnostic system and could also be applied to the determination of genetic relationships.