Potential SNPs related to microspore culture inRaphanus sativusbased on a single-marker analysis

Abstract

Radish (Raphanus sativus) is an economically important crop grown for its edible roots and leaves. It is a self-incompatible, outcrossing species, making the production of homozygous lines and the development of breeding populations difficult. However, this can be overcome with haploids production techniques using isolated microspores, providing the rapid production of homozygous lines for breeding. Thus, it would be useful to identify radishes with a high regeneration rate from microspore culture. In the current study, 96 radish cultivars or germplasms were evaluated for high regeneration rates. Also, a single-marker analysis (SMA) was applied to detect single nucleotide polymorphisms (SNPs) potentially associated with this trait using genotype-by-sequencing (GBS) technology. The regeneration rate from microspore culture of 96 lines showed a wide range, from 0% to 269.5%. From the SMA, 52 markers were detected at a p value of 0.001 and a total of 11 physically nearby genes with high levels of similarity in various species were identified as candidates for high regeneration rates. This result could be used for clarifying the genetic basis underlying these traits and developing molecular markers associated with regeneration rates and would be beneficial for generating homozygous inbred lines.

Quantitative Trait Loci (QTLs) Associated with Microspore Culture in Raphanus sativus L. (Radish)

“…The experiments showed that over 50% of the population did not regenerate, and only a few have high regeneration rates. These regeneration rates are consistent with previous findings [23,36].…”

“…A sequencing library was prepared according to the GBS protocol in [23] using two restriction endonucleases, Nsil-HF and MseI, which were successfully used for constructing GBS libraries in our previous report [24]. For the construction of the GBS library, a set of 64 barcoded adapters was generated from complementary oligonucleotides with the Nsil-HF (New England Biolabs, Ipswich, MA) overhang sequence and unique barcode of length 5-10 bp and they have been tested using two parental lines to determine the optimum conditions for the GBS analysis.…”

“…Unlike the costly and time-consuming re-sequencing, GBS analysis is a technique that uses a reduced representation library (RRL) to partially sequence a genome of interest using restriction enzymes [37]. Since GBS does not sequence the entire genome, it can reduce the complexity of the chromosome to allow a complex genome analysis, and large numbers of SNPs can be found for a variety of plant genetic studies, including genetic mapping and population analysis [23,38]. Creating a linkage map plays a very important role in detecting the location of loci in the chromosome, such as QTL analysis.…”

“…However, most of these or other plants are the results of QTL analyses involved in regeneration rates via anther culture [49,50] or tissue culture [51][52][53][54]. The QTL analysis of the regeneration rate in Raphanus sativus has not been reported to date, but a single marker analysis to find candidate genes involved in the efficiency of microspore culture has recently been reported [23]. There have been many reports on experiments on heat treatment, plant growth regulators, temperature controls, and light conditions to find suitable culture conditions for microspore culture for the regeneration of plants in Brassicaceae [55][56][57][58][59][60].…”

Expanded functionality, increased accuracy, and enhanced speed in the de novo genotyping-by-sequencing pipeline GBS-SNP-CROP

“…As a reference-optional (or de novo ) pipeline, GBS-SNP-CROP has proven useful to breeders of under-researched crop species for which the lack of a reference genome presented a barrier to the efficient use of GBS data (Cheng et al , 2017; Hale et al , 2018; Melo et al , 2017; Sogbohossou et al , 2018; Wang et al , 2017). The pipeline has facilitated studies of genetic diversity and population structure for natural populations of both plants (Arredondo et al , 2018; Bartaula et al , 2018; Sunseri et al , 2018) and animals (Drury et al , 2017; Xu et al , 2017) and has successfully supported molecular breeding analyses in a variety of crop and non-crop plant species (Adhikari et al , 2018; Chung et al , 2018; Holloway et al , 2018; Munjal et al , 2017). In presenting GBS-SNP-CROP v.4.0, this note describes the expanded functionality and enhanced performance of the pipeline relative to its original version.…”