Discovery of MicroRNA169 gene copies in genomes of flowering plants through positional information

Abstract

Expansion and contraction of microRNA (miRNA) families can be studied in sequenced plant genomes through sequence alignments. Here, we focused on miR169 in sorghum because of its implications in drought tolerance and stem-sugar content. We were able to discover many miR169 copies that have escaped standard genome annotation methods. A new miR169 cluster was found on sorghum chromosome 1. This cluster is composed of the previously annotated sbi-MIR169o together with two newly found MIR169 copies, named sbi-MIR169t and sbi-MIR169u. We also found that a miR169 cluster on sorghum chr7 consisting of sbi-MIR169l, sbi-MIR169m, and sbi-MIR169n is contained within a chromosomal inversion of at least 500 kb that occurred in sorghum relative to Brachypodium, rice, foxtail millet, and maize. Surprisingly, synteny of chromosomal segments containing MIR169 copies with linked bHLH and CONSTANS-LIKE genes extended from Brachypodium to dictotyledonous species such as grapevine, soybean, and cassava, indicating a strong conservation of linkages of certain flowering and/or plant height genes and microRNAs, which may explain linkage drag of drought and flowering traits and would have consequences for breeding new varieties. Furthermore, alignment of rice and sorghum orthologous regions revealed the presence of two additional miR169 gene copies (miR169r and miR169s) on sorghum chr7 that formed an antisense miRNA gene pair. Both copies are expressed and target different set of genes. Synteny-based analysis of microRNAs among different plant species should lead to the discovery of new microRNAs in general and contribute to our understanding of their evolution.

Fig. 1.—

Distribution of MIR169 gene copies in the genome of Sorghum bicolor cultivar BTx623. A total of 22 MIR169 gene copies are shown, with 17 copies previously annotated by the sorghum genome-sequencing consortium (shown in black and red) (Paterson et al. 2009) and with five additional MIR169 copies described in this study for the first time (shown in green). The evolutionary trajectory of sorghum MIR169 gene copies arranged in clusters 1, 2, and 3 are described.

Fig. 2.—

Syntenic alignment of rice and sorghum chromosomal segments containing MIR169 gene clusters. Sorghum MIR169 gene clusters on chr2 and chr7 together with their flanking protein coding genes were aligned with rice by orthologous gene pairs. Rice and sorghum chromosomes are represented as horizontal lines, whereas genes along the chromosome are represented as rectangle bars. Known MIR169 gene copies are shown as red bars, whereas new MIR169 gene copies described in this study are shown as green bars. The bHLH and B-box zinc finger and CCT motif (B-box/CCT) genes are represented as yellow bars. All other protein coding genes in the chromosomal regions under study are represented as black bars. Orthologous gene pairs are indicated as lines connecting bars, with red lines indicating orthology between MIR169 gene pairs and yellow lines indicating orthology between bHLH and B-box/CCT gene pairs, respectively. All other orthology between rice and sorghum protein coding genes are indicated as black lines connecting black bars. The physical distance between bHLH and B-box/CCT genes and/or between bHLH or B-Box/CCT genes to the flanking MIR169 copy is indicated. To provide a scale of the chromosomal segments highlighted in the figure, the physical distance between the first and the last gene in the segment is indicated and thus serves as a reference to observe expansion and contraction of genomic regions. An inversion event on sorghum chr7 containing the MIR169 cluster occurred relative to the orthologous regions on sorghum chr2 and rice chr8 and chr9 respectively.

Fig. 3.—

Sequence alignment of sorghum MIR169 cluster on chr1 with orthologous regions from Brachypodium, rice and foxtail millet. The sbi-MIR169o copy in sorghum allowed the identification of the orthologous osa-MIR169r copy in rice and sit-MIR169o copy in foxtail millet, respectively. For the region containing sbi-MIR169o/t/u on chr1, we could not find sufficient conservation of synteny to identify an orthologous region in sorghum, thus a synteny graph is only shown with sorghum chr1. An inversion event on rice chr3 occurred relative to Brachypodium, foxtail millet, and sorghum.

Fig. 4.—

Sequence alignment of sorghum MIR169 cluster on chr7 with orthologous regions from Brachypodium, rice, and foxtail millet. Rice and sorghum MIR169 gene copies were used to identify and annotate five MIR169 genes in foxtail millet (shown in green). The bHLH and B-box/CCT genes were physically adjacent to MIR169 gene copies in the four species examined. The region examined on sorghum chr7 expanded relative to the orthologous region from the other three grasses and was inverted only in sorghum.

Fig. 5.—

Sequence alignment of sorghum MIR169 cluster on chr2 with orthologous regions from Brachypodium, rice, and foxtail millet. MIR169 gene copies were deleted from Brachypodium chr4 but the flanking genes remained. The MIR169 gene cluster in rice was composed of two copies, whereas in sorghum and foxtail millet, the cluster comprised three copies. The bHLH gene was present in all four grasses and was physically adjacent to MIR169 gene copies in rice, sorghum, and foxtail millet. Sorghum MIR169 gene copies were used to identify and annotate the orthologous copies on foxtail millet scaffold 2 (shown in green).

Fig. 6.—

Gains and losses of MIR169 gene copies during grass evolution. (A) Phylogenetic distribution of MIR169 gene copies in ancestral and current species with gain and losses of MIR169 copy number during grass evolution. Numbers in squares represent the number of MIR169 gene copies for a given cluster in each species. Numbers along each line represent gains (+) and losses (−) of MIR169 gene copies. The estimated divergence time for each species is given at each node in the tree according to Paterson et al. (2009), Brachypodium-Sequencing-Initiative (2010), Bennetzen et al. (2012) and Zhang et al. (2012). The gain in MIR169 copy number of sorghum relative to Brachypodium is depicted. Note: WGD in maize is used as a term to represent the allotetraploidy event that took place. NJ phylogenetic trees with bootstrap support are shown depicting the relationships of MIR169 stem-loop sequences from the grass species shown in (A). (B) NJ phylogenetic tree with Brachypodium (bdi) and rice (osa) MIR169 stem-loop sequences orthologous to sorghum MIR169 copies on chromosome 7. (C) NJ phylogenetic tree with rice (osa) and foxtail millet (sit) MIR169 stem-loop sequences (top) and rice, foxtail millet, sorghum (sbi), and maize (zma) MIR169 stem-loop sequences (bottom) orthologous to MIR169 copies on sorghum chromosome 2. (D) NJ phylogenetic tree depicting the relationship of foxtail millet and maize MIR169 copies orthologous to sorghum MIR169 copies on chromosome 1 (top), and Brachypodium, rice, foxtail millet, and maize MIR169 copies orthologous to sorghum MIR169 copies on chromosome 1 (bottom).

Fig. 7.—

Sequence alignment of sorghum MIR169 cluster on chr7 with orthologous regions from Brachypodium, soybean, and cassava. There is conservation of synteny between monocot species Brachypodium and sorghum and dicot species soybean and cassava when chromosomal segments containing MIR169 gene copies and their flanking genes are aligned. Conservation of synteny allowed the identification of new MIR169 gene copies on soybean chromosome 6 (gma-MIR169w) and cassava scaffold 01701 (mes-MIR169w), respectively. Physical association on the chromosome between MIR169 and the flanking bHLH gene was retained in soybean and cassava as well. Notice the inversion on soybean chr6.

Fig. 8.—

Sequence alignment of sorghum MIR169 cluster on chr2 with orthologous regions from Brachypodium, soybean, and cassava. The alignment of sorghum MIR169 cluster on chr2 with soybean chr8 and cassava scaffold 09876 allowed the identification of two new MIR169 gene copies in soybean (gma-MIR169x and gma-MIR169y) and one new copy in cassava (mes-MIR169y), respectively. The physical association of MIR169 gene copies with the bHLH was retained in soybean and cassava. An inversion occurred on soybean chr8.

Fig. 9.—

Conservation of synteny between sorghum and grapevine chromosomal segments containing MIR169 gene copies. Sorghum segments containing MIR169 gene clusters from chr2 and chr7 were aligned to the grapevine genome based on orthologous gene pairs. Because grapevine is a hexopaleo-polyploid, we found a 2:3 chromosomal relationship between sorghum and grapevine. Collinearity allowed the identification of a new MIR169 copy (vvi-MIR169z) in grapevine chr14. Different grapevine chromosomes are represented in colors, whereas sorghum chromosomes are in black. Relative to sorghum chr2, grapevine had an inversion event on chr14 and chr17. The association of MIR169 with its flanking COL gene was maintained on grapevine chr14 and chr1, whereas the association of MIR169 with the bHLH gene was maintained on chr1.