Discussion

Genus-wide Transferable markers design based on core genome 

A set of 'universal' genetic markers that work for close related species is desired in many areas of studies. In molecular assisted breeding, universal markers can be used in distant hybridization and alien gene introgression  \cite{Chagné2004,Brondani2006,Diaz2011} . In molecular ecology and evolutionary studies, universal markers allow comparison genetic characters among related species\cite{Singh_2012,Bernardes_2018}.  In some clade/genus that is enriched in economically important species, universal markers that transferable across species will largely decrease the time and effort in developing unique markers for each species\cite{Kuleung2004,Pan_2018}. The transferability for the microsatellite markers and SSR marker ranges from 27% to 77% in the different taxonomic groups of plants and animals \cite{Barbará2007}. However, the transferability of high-throughput genetic markers are as low as 2% when transferring markers across species \cite{Vezzulli2008}\cite{Chagné2012}.  In this study, we developed a pipeline for develop universal markers that work for the whole Vitis genus with diverged 20 Mya. Using rhAnpSeq platform, 93% of markers return data for all four population we test. And also around 82% of markers are polymorphic in all the population. And the genetic map built for these four population are consistent, which indicating that the consistency of marker order and segregation pattern. Although for each population there are 10% to 20%  with unexpected Mendelian segregation ratio, these markers are population specific. We compare the distance between distorted markers and a random sampling of the same number of markers from the entire set, the distance between distorted markers is significantly smaller than the random expectation ( Mann-Whitney test, p<1e-13). This reduced distance between distorted markers indicating that they are clustered on the chromosome, which indicating the distorted markers are linked. In addition, when we combine individuals from these four population to form a meta-population, only XX% of markers are distorted. This further suggested that the majority of the markers are informative in constructing a genetic map, a small portion of them might fail due to different genetic background. We also found that in two population that the sex loci were located,  the markers that explain the biggest phenotypic variation are the same, which indicated that not only the random markers are transferable, the functional markers are also transferable. In one word, we validated that the markers designed based on the genus-wide core genome and genome polymorphism are transferable at different levels, which includes the amplification level, polymorphism level, segregation level, and marker-trait association level. 

The key to develop transferable markers is the construction of the genus-wide core genome considering the colinearty. Previously markers that designed based on resequecing a large number of samples has limited transferability. By DNA resequencing, rich information of small genetic variantion can be accessed, however large and complex structure variation is often missing. The long collinear blocks conserved within the genus are suggestive of strong selection against  structure variation in this region, which increase the probability to identify markers that has consistent occurrence in the genome and also consistent segregation pattern. Out result indicate that in order to identify inter species transferable markers, core genome with collinearity should be considered. [core transcriptome 70% design rate]. Many marker design approach has considered the polymophism of the markers. In our study, we used the genus-wide polymophism to detect regions with moderate diversity and also used the polymorphism to design highly conserved probes, which contain no or very little variations across the whole genus, to ensure the binding specificity between the probe and the target sites. 

The advantage of rhAmpSeq genotyping platform for highly diverse and heterozygous species 

In our previous study, we have found that Ampseq genotyping platform outperforms GBS or other NGS based genotyping platform for highly diverse and heterozygous species, due to limited missing data, increased coverage and accuracy at heterozygote sites, and elevated transferability among distinct species. Different from SNP array or KASP which target one specific polymorphism site, another advantage of AmpSeq genotyping platform is that it allows the identification of novel alleles and a short haploblock because the entire amplified region (from 90 bp to 250bp long) are sequenced through NGS. Therefore, for a pair of individuals with a genetic diversity greater than 1 SNP per 250 bp, the amplified region should contain at least one SNP to distinguish the two individual, which increase the information content of the markers and make it suitable not only for distant but also for close related bi-parental population. This high coverage and unbiased sequencing of amplicons make this platform is applicable in population genetics and ecology studies. Comparing to AmpSeq, the rhAmpSeq technology add an RNA base and a blocker in the primer, and this RNA-base and the blocker can only be cleaved by RNase H2 enzyme only when the match is perfect between the primer and the templates \cite{Dobosy2011}. This step increases the specificity in the genotyping and also increases the throughput to 5000 probes in one tube (rhAmpSeq, IDT ).