Degenerate oligonucleotide primer MIG-seq: an effective PCR-based method for high-throughput genotyping.

Next-generation sequencing (NGS) library construction often involves using restriction enzymes to decrease genome complexity, enabling versatile polymorphism detection in plants. However, plant leaves frequently contain impurities, such as polyphenols, necessitating DNA purification before enzymatic reactions. To overcome this problem, we developed a PCR-based method for expeditious NGS library preparation, offering flexibility in number of detected polymorphisms. By substituting a segment of the simple sequence repeat sequence in the MIG-seq primer set (MIG-seq being a PCR method enabling library construction with low-quality DNA) with degenerate oligonucleotides, we introduced variability in detectable polymorphisms across various crops. This innovation, named degenerate oligonucleotide primer MIG-seq (dpMIG-seq), enabled a streamlined protocol for constructing dpMIG-seq libraries from unpurified DNA, which was implemented stably in several crop species, including fruit trees. Furthermore, dpMIG-seq facilitated efficient lineage selection in wheat and enabled linkage map construction and quantitative trait loci analysis in tomato, rice, and soybean without necessitating DNA concentration adjustments. These findings underscore the potential of the dpMIG-seq protocol for advancing genetic analyses across diverse plant species.

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.).

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.

The early flowering trait of an emmer wheat accession (Triticum turgidum L. ssp. dicoccum) is associated with the cis-element of the Vrn-A3 locus.

KEY MESSAGE: We identified a novel allele of the Vrn-A3 gene that is associated with an early flowering trait in wheat. This trait is caused by a cis-element GATA box in Vrn-A3. To identify novel flowering genes in wheat, we investigated days from germination to heading (DGH) in tetraploid wheat accessions. We found that the tetraploid variety Triticum turgidum L. ssp. dicoccum (TN26) harbors unknown genes that surpass the earliness effect of the early flowering allele Ppd-A1a harbored by TN28 (T. turgidum L. ssp. turgidum conv. pyramidale). Using recombinant inbred lines resulting from a cross between TN26 and TN28, we performed a quantitative trait locus (QTL) analysis for DGH. We identified a QTL for earliness in TN26 on chromosome 7AS, the chromosome on which Vrn-A3 is located. By sequence analysis for the Vrn-A3 locus in both TN26 and TN28, we identified a 7-bp insertion that included a cis-element GATA box sequence at the promoter region of the Vrn-A3 locus of TN26. Based on an expression analysis using sister lines for Vrn-A3, we suggest that the early flowering trait of TN26 was caused by the GATA box in Vrn-A3. In addition, we identified tetraploid wheat as a useful genetic resource for wheat breeding.

The parthenocarpic gene Pat-k is generated by a natural mutation of SlAGL6 affecting fruit development in tomato (Solanum lycopersicum L.).

BACKGROUND: Parthenocarpy is a desired trait in tomato because it can overcome problems with fruit setting under unfavorable environmental conditions. A parthenocarpic tomato cultivar, 'MPK-1', with a parthenocarpic gene, Pat-k, exhibits stable parthenocarpy that produces few seeds. Because 'MPK-1' produces few seeds, seedlings are propagated inefficiently via cuttings. It was reported that Pat-k is located on chromosome 1. However, the gene had not been isolated and the relationship between the parthenocarpy and low seed set in 'MPK-1' remained unclear. In this study, we isolated Pat-k to clarify the relationship between parthenocarpy and low seed set in 'MPK-1'. RESULTS: Using quantitative trait locus (QTL) analysis for parthenocarpy and seed production, we detected a major QTL for each trait on nearly the same region of the Pat-k locus on chromosome 1. To isolate Pat-k, we performed fine mapping using an F4 population following the cross between a non-parthenocarpic cultivar, 'Micro-Tom' and 'MPK-1'. The results showed that Pat-k was located in the 529 kb interval between two markers, where 60 genes exist. By using data from a whole genome re-sequencing and genome sequence analysis of 'MPK-1', we could identify that the SlAGAMOUS-LIKE 6 (SlAGL6) gene of 'MPK-1' was mutated by a retrotransposon insertion. The transcript level of SlAGL6 was significantly lower in ovaries of 'MPK-1' than a non-parthenocarpic cultivar. From these results, we could conclude that Pat-k is SlAGL6, and its down-regulation in 'MPK-1' causes parthenocarpy and low seed set. In addition, we observed abnormal micropyles only in plants homozygous for the 'MPK-1' allele at the Pat-k/SlAGL6 locus. This result suggests that Pat-k/SlAGL6 is also related to ovule formation and that the low seed set in 'MPK-1' is likely caused by abnormal ovule formation through down-regulation of Pat-k/SlAGL6. CONCLUSIONS: Pat-k is identical to SlAGL6, and its down-regulation causes parthenocarpy and low seed set in 'MPK-1'. Moreover, down-regulation of Pat-k/SlAGL6 could cause abnormal ovule formation, leading to a reduction in the number of seeds.