Developing genome-wide microsatellite markers of bamboo and their applications on molecular marker assisted taxonomy for accessions in the genus Phyllostachys

Abstract

Morphology-based taxonomy via exiguously reproductive organ has severely limitation on bamboo taxonomy, mainly owing to infrequent and unpredictable flowering events of bamboo. Here, we present the first genome-wide analysis and application of microsatellites based on the genome of moso bamboo (Phyllostachys edulis) to assist bamboo taxonomy. Of identified 127,593 microsatellite repeat-motifs, the primers of 1,451 microsatellites were designed and 1,098 markers were physically mapped on the genome of moso bamboo. A total of 917 markers were successfully validated in 9 accessions with ~39.8% polymorphic potential. Retrieved from validated microsatellite markers, 23 markers were selected for polymorphic analysis among 78 accessions and 64 alleles were detected with an average of 2.78 alleles per primers. The cluster result indicated the majority of the accessions were consistent with their current taxonomic classification, confirming the suitability and effectiveness of the developed microsatellite markers. The variations of microsatellite marker in different species were confirmed by sequencing and in silico comparative genome mapping were investigated. Lastly, a bamboo microsatellites database (http://www.bamboogdb.org/ssr) was implemented to browse and search large information of bamboo microsatellites. Consequently, our results of microsatellite marker development are valuable for assisting bamboo taxonomy and investigating genomic studies in bamboo and related grass species.

Figure 1. Distribution of microsatellite repeat motif, length and different genomic regions.

(a) Microsatellite length distribution. The x-axis represents the nucleotide length of microsatellites. The y-axis indicates the number of microsatellites with different length in the six selected plant species. (b) The x-axis indicates the proportion of microsatellites with various repeat motifs. The y-axis represents different repeat motif in the six selected plant species. P2: di-nucleotide repeats; P3: tri-nucleotide repeats; P4: tetra-nucleotide repeats; P5: penta-nucleotide repeats; P6: hexa-nucleotide repeats; compound: compound microsatellite. (c) Total number of each repeat motif. The x-axis indicates repeat motif with di- to hexa- nucleotides. The y-axis represents the number of microsatellites with various repeat motifs. The different plant species were marked with different color.

Figure 2. Phylogenetic analysis of 78 bamboo accessions in genus Phyllostachys based on microsatellite data.

No. 1–78 represent different bamboo accessions, detailed in Supplementary table S3.

Figure 3. Multiple sequence alignment of PhEMS-855 demonstrating the presence of microsatellite repeat motif in Phyllostachys edulis and other species of genus Phyllostachys.

The analysis indicates multiple point mutations and insertion/deletions happened in a large number of repeat motifs among different species.

Figure 4. Genome relationship of moso bamboo with other plant species.

Based on the experimentally physical mapping, syntenic relationship of moso bamboo genome with (a1) Zea mays, (b1) Oryza sativa, (c1) Sorghum bicolor and (d1) Brachypodium distachyon chromosomes using 984 physically mapped moso bamboo microsatellite markers. Maximum syntenic relationships of the genome of moso bamboo with Oryza sativa chromosomes based on experimental microsatellite markers were apparent. Besides, based on the bioinformatics comparative mapping, syntenic relationship of moso bamboo genome with (a2) Zea mays, (b2) Oryza sativa, (c2) Sorghum bicolor and (d2) Brachypodium distachyon chromosomes using 101,683 mapped moso bamboo microsatellite markers. Similarly, maximum syntenic relationships of the genome of moso bamboo with Oryza sativa chromosomes based on predicted microsatellite markers were apparent as well. Zm1-10 used to be short for the chromosome 1–10 of Zea mays; Os1–12 used to be short for the chromosome 1–12 of Oryza sativa; Sb1–10 used to be short for the chromosome 1–10 of Sorghum bicolor; Bd1-1-5 used to be short for the chromosome 1–5 of Brachypodium distachyon; PSG1–6 used to be short for the group of scaffold 1–6 of Phyllostachys edulis.

Figure 5. Screenshot showing the pages of browse and results in bamboo microsatellite database.

(a) The Boolean search was provided in the searching page of moso bamboo microsatellites. (b) The results page was included, such as microsatellite ID, microsatellite type, microsatellite position, microsatellite primers and so on. (c) The physical location of microsatellites in moso bamboo genome was displayed in GBrowse tool.