Characterization of miniature inverted repeat transposable elements inserted in the CitRWP gene controlling nucellar embryony and development of molecular markers for reliable genotyping of CitRWP in Citrus species

Abstract

Nucellar embryony, a form of apomixis, is one of the most important traits in citrus breeding. Transposition of a miniature inverted repeat transposable element (MITE) in the promoter of CitRWP gene has been previously reported as the causal mutation. However, features of the MITE such as sequences of terminal inverted repeats (TIRs) and target site duplications (TSDs) remain unclear. An MITE family, DTM10, showing the highest homology was identified from 110 citrus MITE families. At least 80 DTM10 elements were identified from each of 10 genome sequences of Citrinae species. Analysis of 114 sweet orange elements showed that DTM10 contained 56-bp TIRs and mostly 9-bp TSDs. Phylogenetic analyses of DTM10 elements identified from five Citrinae species showed that MITEs in CitRWP might have transposed in a common ancestor of Citrus species. Two inverted MITEs were identified from three mutant alleles analyzed. In addition, three tandem MITEs were identified in the mutant allele of Fortunella CitRWP. A strong DNA secondary structure formed between MITEs was predicted. Due to a strong stem-loop structure, a phenomenon of polymerase jumping was observed during PCR amplification. To develop reliable molecular markers, sequences of mutant and wild-type CitRWP alleles were obtained from diverse citrus accessions. Phylogenetic analysis of CitRWP alleles showed that mutant alleles might have originated from a common ancestor of Citrus species. Based on MITE insertion and a single nucleotide polymorphism in the genic region, three complementary molecular markers were developed. Genotyping results of 241 citrus accessions were found to be identical among three molecular markers.