Objective 1- Genotype the 1,200 inbred lines by means of high-density SNP markers. Genotyping-By-Sequencing (GBS), a more general term that includes RAD-seq, has become the state of the art technique in recent years for genotyping efforts of crops (21). A first pipeline in terms of lab work and further bioinformatics analyses, amenable to maize genotyping, has been first proposed by Elshire et al., in 2011 (21). Their seminal work represented the foundation for the study of Romay et al., 2013 (5), that led to the comprehensive genotyping of the USA national maize inbred seed bank. Through our objective stated above we aim at becoming the first European country that have extensively genotyped its germplasm, and to the best of our knowledge the second in the world.
Objective 2- Use bioinformatics tools to group the inbred lines into heterotic groups. The main advantage in SNP discovery through RAD-seq and its later use in grouping the inbred lines in heterotic groups is the availability of the reference B73 genome (6). This tremendously simplifies SNP discovery through mapping to the reference genome. Consequently, bioinformatics pipelines are already available to streamline the analysis. We plan to use one such pipeline that has been adapted especially for maize research, namely the latest version of TASSEL (v. 5.0), developed by the Buckler Lab (22).
Objective 3- Predict crosses among members of different heterotic groups that will lead to hybrids characterized by high levels of heterosis. It has been shown that one could maximize heterosis in maize by crossing two genetically distant lines that come from similar environmental conditions (23). We have already produced six hybrids during our ZEAHYBR project using 90 inbred lines as a starting point (13). Having 1,200 genotyped inbred lines will exponentially increase our predictive power in terms of generating better hybrids. We plan to continue our collaboration with our research collaborator from the ZEAHYBR project, i.e., the Agricultural Research Station Turda, who has played one of the leading roles in Romania in terms of generating new maize hybrids, since 1956, when it had been established.
Objective 4– Rare alleles. In our previous study (13)we show that an admixed population existed in the investigated biological material that could not be anchored to the international standard-lines used in maize breeding. The majority of inbred lines grouped in the admixed population have been created from the local Romanian germplasm. Since maize has been growing here for centuries, being introduced by the Turks (12), it most probably developed into local populations that were later used in creating those inbred lines. Probing the 1,200 inbred lines with a high density of SNP markers will allow us to identify rare alleles that are specific to Romanian germplasm and that could be used to enrich international maize breeding programs. Furthermore, rare alleles have the potential to explain phenotypes like those of the two lines mentioned above (i.e., TC221 and T381), which show high protein-, fat- and fiber-content.