An accurate and efficient method for large-scale SSR genotyping and applications

Abstract

Accurate and efficient genotyping of simple sequence repeats (SSRs) constitutes the basis of SSRs as an effective genetic marker with various applications. However, the existing methods for SSR genotyping suffer from low sensitivity, low accuracy, low efficiency and high cost. In order to fully exploit the potential of SSRs as genetic marker, we developed a novel method for SSR genotyping, named as AmpSeq-SSR, which combines multiplexing polymerase chain reaction (PCR), targeted deep sequencing and comprehensive analysis. AmpSeq-SSR is able to genotype potentially more than a million SSRs at once using the current sequencing techniques. In the current study, we simultaneously genotyped 3105 SSRs in eight rice varieties, which were further validated experimentally. The results showed that the accuracies of AmpSeq-SSR were nearly 100 and 94% with a single base resolution for homozygous and heterozygous samples, respectively. To demonstrate the power of AmpSeq-SSR, we adopted it in two applications. The first was to construct discriminative fingerprints of the rice varieties using 3105 SSRs, which offer much greater discriminative power than the 48 SSRs commonly used for rice. The second was to map Xa21, a gene that confers persistent resistance to rice bacterial blight. We demonstrated that genome-scale fingerprints of an organism can be efficiently constructed and candidate genes, such as Xa21 in rice, can be accurately and efficiently mapped using an innovative strategy consisting of multiplexing PCR, targeted sequencing and computational analysis. While the work we present focused on rice, AmpSeq-SSR can be readily extended to animals and micro-organisms.

Functional Mechanisms of Microsatellite DNA in Eukaryotic Genomes

“…Until recently, significant obstacles to the incorporation of large numbers of repeat loci into GWAS in humans included practical difficulties with large scale genotyping, problems with mapping short sequence reads, and statistical hypothesis testing issues generated by multiple alleles per locus—but these are now being alleviated through theoretical and technological developments, including less expensive, longer read sequencing ( Press etal. 2014 ; Li etal. 2017 ; Shin etal.…”

MultiplexSSR: A pipeline for developing multiplex SSR‐PCR assays from resequencing data

“…The read length is sufficiently long to capture most single sequence repeats, even though the amplification step introduces a large amount of error. Among single sequence repeats, 98.0% are less than 100 bp in length, which is shorter than the single-end length of the read pairs considering that the reads must span the whole repeat region (Gymrek et al, 2012 (Barbian et al, 2018;De Barba et al, 2017;Li et al, 2017;Šarhanová, Pfanzelt, Brandt, Himmelbach, & Blattner, 2018;Vartia et al, 2016;Zhan et al, 2017), which leads to the same dilemma of a higher error rate in dimeric patterns (Weber & Broman, 2000;Yue & Xia, 2014). When there is a large quantity of candidate SSRs, dimeric patterns can be filtered out, even at the expense of losing higher polymorphism in these patterns (Gymrek et al, 2012;Yue & Xia, 2014).…”

Principle and Strategy of DNA Fingerprint Identification of Plant Variety

“…Development of sequencing technology, chip technology, gene editing technology, nucleic acid thermostatic amplification technology and other technologies (Thompson and Milos, 2011;Sander and Joung, 2014;Zhao et al, 2016), will greatly promote the improvement of DNA fingerprint identification scheme and improve its application effect in practice: (1) Combining the third-generation sequencing technology and the more perfect InDel development algorithm (Lv et al, 2016), the mining of the insertion deletion sites of large fragments is faster and more efficient, and such sites are more likely to have better clustering effect, thus making the development of clustering markers in the core locus grouping method easier. With the improvement of second-generation sequencing technology, it is not only more accurate in marker typing (Churbanov et al, 2012;Li et al, 2017), and it is possible to achieve the target detected by SNP, SSR, InDel and other types of markers on a sequencing detection platform at the same time, so that the core locus grouping method is no longer limited to the detection platform in marker selection, but is possible to freely select various types of sequence variation regions. (2) With the development and improvement of high-density DNA chip technology, more loci can be detected with less cost, so that the number of extended loci can be distributed more evenly and densely on the whole gene, to eliminate the deviation caused by fewer loci, and to evaluate the genetic similarity of derived varieties more accurately.…”