Genetic And Epigenetic Alterations In Long Terminal Repeat Retrotransposons Of Rice Caused By Introgerssion From Zizania Latifolia Griseb

Hybridization between different species plays an important role in plant genome evolution, as well as is a widely used approach for crop improvement. Wide hybridization constitutes a "genomic shock", which can cause genetic and epigenetic alterations in the recipient genome. In this study, three rice-Zizania latifolia introgression lines, RZ1, RZ2 and RZ35, which derived from introgressive hybridization beteen rice (cv. Matsumae) and Z. latifolia by a novel approach called "repeated pollination", were studied by 13 LTR low-copy, and potentially active rice LTR (long terminal repeat) retrotransposons. Copy number estimation indicated that the approximate copy numbers of the LTRs of all 13 retrotransposons are more than twice than those of the corresponding reverse-transcriptase (RT) regions, indicating presence of solo LTRs for all the elements in both rice and Z. latifolia. Southern blotting using genomic DNAs digested by EcoRI or HindIII indicated that extensive de novo genomic rearrangements in 10 of the 13 LTR retrotransposons occurred in one or more of three introgression lines. For these retrotransposons, largely concordant rearrangements occurred at both RT and LTR regions in eight elements, and alterations occurred only in the RT regions for the rest two elements. Southern blotting using genomic DNAs digested by a pair of isoschizomers, HpaII and MspI, which are differentially sensitive to either or both of the cytosines at the CCGG sites, showed that apparent alterations in DNA methylation in 12 of the 13 LTR retrotransposons occured in one or more introgression lines. For these retrotransposons, largely concordant methylation alterations occurred at both RT and LTR regions in 11 elements, and alterations occurred only in the RT regions for the rest 1 elements. A marked proportion of the genetic and epigenetic changes were shared by two or all three introgression lines that were derived from a single F1 plant. This suggests that most of the alterations occurred at early developmental stages of the F1 somatic cells, which then gave rise to germline cells, and hence, enabled inheritance of the changes to later generations.RT-PCR analysis indicated that Osr7 is transcribed in Matsumae and all three introgression lines, while Osr42 is transcribed in Matsumae and introgression line RZ35.Bisulfite sequencing analysis further proved the methylation alterations in introgression lines, and also indicated that the expression of Osr42 was associated with its cytosine methylation.Sequence analysis of the retrotransposon-flanking regions indictaed that in the13 flanking sequences of the of 5 LTR retrotransposons, most of them have higher copy numbers, indicating that LTR retrotransposns are mainly resided in heterochromatic regions, though some in gene regions. RT-PCR analysis of 9 low copy flanking genes showed that differential expression between Matsumae and the introgression lines are apparent in 3 genes, but the differences showed no correlation with the presence or absence of the retrotransposons.Possible causes and potential implications of the introgression-induced genomic rearrangements and DNA methylation alterations in LTR retrotransposons are discussed in the context of plant genome evolution and breeding.