Software Development Of SSR Genotyping Across A Genetic Mapping Population And Construction Of SSR Genetic Maps In Populus

The genus Populus is an important industrial timber and ecological tree species in China.The conventional poplar breeding mainly focuses on phenotypic selection,which has a long time cycle with poor stability and is easily affected by various environments.With advances in next-generation sequencing（NGS）technology,single nucleotide polymorphisms（SNP）have been widely used for constructing genetic linkage maps,quantitative trait locus（QTL）mapping and molecular marker-assisted breeding in forest trees.Compared with SNP markers,simple sequence repeats（SSR）markers have high polymorphism and generality across species.However,there is no software package available for genotyping SSR markers from NGS data for genetic mapping.In this study,we first developed a software package to extract high-quality SSR genotype data across a large number of individuals in a genetic mapping population using NGS data.We then used this software to successfully genotype SSR markers in an F₁ hybrid population of P.deltoides and P.simonii with restriction-site associated DNA sequencing（RAD-seq）data,and finally constructed SSR genetic linkage maps of the two parents.The main results are as following:1.We have developed a software package called SSRGT,which is freely available at https://github.com/zhangjinpeng Github/SSRGT.The software algorithm was implemented by four steps:（1）identifying SSR site information in a reference sequence provided by users;（2）aligning paired-end sequencing reads of each individual in a mapping population to the reference sequence,obtaining a Variant call format（VCF）file from the high-quality mapping result;（3）calling SSR genotypes from the VCF files for each parents and progeny according to the SSR site information file generated in the first step;and（4）filtering the SSR genotype data according to the Mendelian segregation ratio and the allowed percentage of missing genotypes.All the steps can be run with one command or each step can be run separately.2.We performed a statistical analysis of polymorphic SSR markers in P.deltoides and P.simonii using whole-genome resequencing data.The results showed that there were 28,862polymorphic SSR loci between the two species.These polymorphic SSR loci can be divided into4 regions by genome annotation,including intergenic,coding,untranslated and intron regions.Among them,the largest number of SSRs was observed in intergenic regions,accounting for59.58%of the total polymorphic SSR loci,followed by intron regions（29.05%）,untranslated regions（6.02%）and coding regions（5.35%）.The most frequent SSRs for coding regions were found to be trinucleotide repeat,up to 1264.Further functional annotation of SSRs in coding regions revealed that genes containing SSRs were mainly involved in basal metabolism and signaling,and some of the others belonged to WD40 transcription factors,AP2 gene family,Myb gene family or WRKY gene family.3.We used our newly developed software SSRGT to successfully obtain a large number of high-quality SSR markers for both parents and their 106 progeny in an F₁ hybrid population of P.deltoides and P.simonii,leading to two parental SSR genetic linkage maps constructed.The maternal genetic linkage map of P.deltoides contained 336 SSR markers and 21 linkage groups with a total map distance of 4740.87 c M,while the paternal genetic linkage map of P.simonii contained 219 SSR markers and 23 linkage groups with a total map distance of 2690.99 c M.By performing polyacrylamide gel electrophoresis on 12 progeny at five SSR marker loci,we found that 54 out of 59 successfully amplified SSR genotypes（91.5%）were consistent with the genotyping result calculated from the software SSRGT.In summary,we developed a new software to extract SSR genotypes with NGS data across many individuals in a mapping population for genetic mapping.It can be applied to the traditional backcross and F₂ populations in inbred lines as well as to an F₁ hybrid population in outbred species.The construction of the two parental SSR linkage maps demonstrated a new strategy for genetic mapping by using NGS data,which will accelerate the research process of population genetic variation analysis,genetic linkage map construction,QTL mapping and molecular marker-assisted breeding in forest trees.