Extensive de Novo genomic variation in rice induced by introgression from wild rice (Zizania latifolia Griseb.)

Abstract

To study the possible impact of alien introgression on a recipient plant genome, we examined >6000 unbiased genomic loci of three stable rice recombinant inbred lines (RILs) derived from intergeneric hybridization between rice (cv. Matsumae) and a wild relative (Zizania latifolia Griseb.) followed by successive selfing. Results from amplified fragment length polymorphism (AFLP) analysis showed that, whereas the introgressed Zizania DNA comprised <0.1% of the genome content in the RILs, extensive and genome-wide de novo variations occurred in up to 30% of the analyzed loci for all three lines studied. The AFLP-detected changes were validated by DNA gel-blot hybridization and/or sequence analysis of genomic loci corresponding to a subset of the differentiating AFLP fragments. A BLAST analysis revealed that the genomic variations occurred in diverse sequences, including protein-coding genes, transposable elements, and sequences of unknown functions. Pairwise sequence comparison of selected loci between a RIL and its rice parent showed that the variations represented either base substitutions or small insertion/deletions. Genome variations were detected in all 12 rice chromosomes, although their distribution was uneven both among and within chromosomes. Taken together, our results imply that even cryptic alien introgression can be highly mutagenic to a recipient plant genome.

Figure 1.—

Examples of genomic variation in the three rice RILs (RZ1, RZ2, and RZ35) as compared with their rice parent (cv. Matsuame) detected by AFLP analysis with primer combinations (from left to right) EcoRI + AAG/MseI + CAA, EcoRI + AAG/MseI + CAG, and EcoRI + AAG/MseI + CAT, respectively. The arrows, arrowheads, and circles respectively refer to parental bands disappeared in one or more RILs, novel bands appeared in one or more RILs, and bands putatively from the donor species Z. latifolia.

Figure 2.—

Examples of validation of the genomic variations in the RILs by DNA gel-blot analysis. Hybridization of each of four isolated AFLP-derived bands to a blot containing EcoRI- or HindIII-digested genomic DNAs from the rice parental cultivar Matsumae (lane 1), the RILs RZ1 (lane 2), RZ2 (lane 3), and RZ35 (lane 4), and the wild species Z. latifolia (lane 5). a–d are, respectively, AF206, AF34, AF480, and AF153. Arrows and circles respectively refer to rice parental bands disappeared in one or more of the RILs and novel bands appeared in one or more of the RILs.

Figure 3.—

Examples of homogeneity within (a) the rice parent cultivar Matsumae and (b) the RILs. Hybridization of probe AF206 on HindIII-digested genomic DNA isolated from eight random individual plants respectively from Matsumae and RZ35. Similar monomorphic hybridization patterns were observed for Matsumae and the three RILs in all 17 selected AFLP fragments were used as probes.

Figure 4.—

Examples of genomic stability in two rice lines (RZ36 and RZ60), siblings to the three RILs (RZ1, RZ2, and RZ35) but with no introgressed DNA from Z. latifolia, as revealed by DNA gel-blot analysis. Hybridization of probe AF206 on HindIII-digested genomic DNA isolated from seven random individual plants (lanes 2–8) from each of RZ36 and RZ60 revealed monomorphic patterns as the rice parent Matsumae (lane 1). Similar results were obtained for all 17 selected AFLP fragments that showed conspicuous genomic variations in one or more of the three RILs (e.g., Figure 2).

Figure 5.—

A dendrogram derived from unweighted pair group method with arithmetic mean (UPGMA) cluster analysis using the Jaccard's coefficient of similarity calculated on the AFLP markers to show the overall genomic differentiation of the RILs from their rice parental cultivar Matsumae.

Figure 6.—

Distribution of the genomic variations affecting different types of sequences (depicted as different colors) within and among the 12 rice chromosomes in the RILs (from left to right: RZ1, RZ2, and RZ35). Predicted functions of the mapped sequences are based on BLASTX analysis at the NCBI website (http://www.ncbi.nlm.nih.gov/), whereas chromosomal position of the variations is based on BLASTN analysis at the Gramene (Ware et al. 2002) website (http://www.gramene.org/).

Figure 7.—

Diagram of the number of genomic variations that are shared by all three RILs (173); by two RILs, RZ1 and RZ2 (173 + 107); by RZ1 and RZ35 (173 + 77); and by RZ2 and RZ35 (173 + 189), as well as genomic variations unique to each RIL: RZ1 (226), RZ2 (185), and RZ35 (274).