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Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum.

Hass-Jacobus BL, Futrell-Griggs M, Abernathy B, Westerman R, Goicoechea JL, Stein J, Klein P, Hurwitz B, Zhou B, Rakhshan F, Sanyal A, Gill N, Lin JY, Walling JG, Luo MZ, Ammiraju JS, Kudrna D, Kim HR, Ware D, Wing RA, San Miguel P, Jackson SA - BMC Genomics (2006)

Bottom Line: When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals.Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa.The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agronomy, Purdue University, West Lafayette, Indiana 47907, USA. barbara.jacobus@alumni.purdue.edu

ABSTRACT

Background: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8-10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome 1 orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40-50 million years ago.

Results: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome 1 in the wild species and provided evidence for at least one rearrangement on chromosome 1 of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa.

Conclusion: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies.

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Distribution of mismatches between rice overgos and Sorghum sequence. The percentage of 36 bp overgo probes having 0 to >6 mismatches to available Sorghum sequence is shown. Sorghum sequences corresponding to regions of rice from which overgo probes were designed were identified using BLAT alignment data available from the Gramene (version 19) database [33]. Overgo sequences were aligned to Sorghum sequence by BLASTN using an open-gap cost of two and a gap-extension cost of one, with low-complexity filtration turned off.
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Figure 3: Distribution of mismatches between rice overgos and Sorghum sequence. The percentage of 36 bp overgo probes having 0 to >6 mismatches to available Sorghum sequence is shown. Sorghum sequences corresponding to regions of rice from which overgo probes were designed were identified using BLAT alignment data available from the Gramene (version 19) database [33]. Overgo sequences were aligned to Sorghum sequence by BLASTN using an open-gap cost of two and a gap-extension cost of one, with low-complexity filtration turned off.

Mentions: To determine the ability of probes to detect sequences of a more distantly related member of the grass family we conducted a pilot study using Sorghum bicolor. Previously, Klein et al. (2003) [16] constructed an integrated genetic and physical map of sorghum and demonstrated extensive colinearity over the lengths of chromosome 3 and rice chromosome 1 [16]. For the current experiment, 3402 physically mapped sorghum BAC clones, spanning ~60 Mb of chromosome 3 (~10X coverage), were hybridized with 288 randomly selected overgo probes. Sixteen probes (5.6%) successfully identified at least one BAC clone, consistent with the trend of decreasing hybridization success rate with increasing evolutionary distance from rice. To better understand the relationship between hybridization success rate and sequence conservation we examined the level of probe-target mismatches in available sorghum sequences. Although available sequence of sorghum is limited, we were able to identify EST and genome survey sequences corresponding to regions of rice that encompass 283 of the 1721 probes, and these were subsequently examined by BLASTN to determine the level of mismatches. As shown in Figure 3, fewer than 4% of probes had zero mismatches, while approximately half had six or greater mismatches. We note that this sample of probes is biased toward greater conservation than the probe set as a whole since it excludes probes targeted to regions of rice for which no homologues exist in sorghum. Among probes for which we were able to obtain mismatch data, the hybridization success rate was 23% (9 out of 39). This was significantly higher (P < 0.001, chi-square test) than the success rate of probes for which no mismatch data was obtainable, which was 3% (7 out of 242). We further found that among the nine positively-hybridizing probes for which we have mismatch data, all but one had six or fewer mismatches, while the success rate among all 20 probes having six or fewer mismatches was 40%, compared to only 5.6% (1 out of 19) for probes having greater than 6 mismatches (data not shown).


Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum.

Hass-Jacobus BL, Futrell-Griggs M, Abernathy B, Westerman R, Goicoechea JL, Stein J, Klein P, Hurwitz B, Zhou B, Rakhshan F, Sanyal A, Gill N, Lin JY, Walling JG, Luo MZ, Ammiraju JS, Kudrna D, Kim HR, Ware D, Wing RA, San Miguel P, Jackson SA - BMC Genomics (2006)

Distribution of mismatches between rice overgos and Sorghum sequence. The percentage of 36 bp overgo probes having 0 to >6 mismatches to available Sorghum sequence is shown. Sorghum sequences corresponding to regions of rice from which overgo probes were designed were identified using BLAT alignment data available from the Gramene (version 19) database [33]. Overgo sequences were aligned to Sorghum sequence by BLASTN using an open-gap cost of two and a gap-extension cost of one, with low-complexity filtration turned off.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1590032&req=5

Figure 3: Distribution of mismatches between rice overgos and Sorghum sequence. The percentage of 36 bp overgo probes having 0 to >6 mismatches to available Sorghum sequence is shown. Sorghum sequences corresponding to regions of rice from which overgo probes were designed were identified using BLAT alignment data available from the Gramene (version 19) database [33]. Overgo sequences were aligned to Sorghum sequence by BLASTN using an open-gap cost of two and a gap-extension cost of one, with low-complexity filtration turned off.
Mentions: To determine the ability of probes to detect sequences of a more distantly related member of the grass family we conducted a pilot study using Sorghum bicolor. Previously, Klein et al. (2003) [16] constructed an integrated genetic and physical map of sorghum and demonstrated extensive colinearity over the lengths of chromosome 3 and rice chromosome 1 [16]. For the current experiment, 3402 physically mapped sorghum BAC clones, spanning ~60 Mb of chromosome 3 (~10X coverage), were hybridized with 288 randomly selected overgo probes. Sixteen probes (5.6%) successfully identified at least one BAC clone, consistent with the trend of decreasing hybridization success rate with increasing evolutionary distance from rice. To better understand the relationship between hybridization success rate and sequence conservation we examined the level of probe-target mismatches in available sorghum sequences. Although available sequence of sorghum is limited, we were able to identify EST and genome survey sequences corresponding to regions of rice that encompass 283 of the 1721 probes, and these were subsequently examined by BLASTN to determine the level of mismatches. As shown in Figure 3, fewer than 4% of probes had zero mismatches, while approximately half had six or greater mismatches. We note that this sample of probes is biased toward greater conservation than the probe set as a whole since it excludes probes targeted to regions of rice for which no homologues exist in sorghum. Among probes for which we were able to obtain mismatch data, the hybridization success rate was 23% (9 out of 39). This was significantly higher (P < 0.001, chi-square test) than the success rate of probes for which no mismatch data was obtainable, which was 3% (7 out of 242). We further found that among the nine positively-hybridizing probes for which we have mismatch data, all but one had six or fewer mismatches, while the success rate among all 20 probes having six or fewer mismatches was 40%, compared to only 5.6% (1 out of 19) for probes having greater than 6 mismatches (data not shown).

Bottom Line: When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals.Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa.The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Agronomy, Purdue University, West Lafayette, Indiana 47907, USA. barbara.jacobus@alumni.purdue.edu

ABSTRACT

Background: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8-10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome 1 orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40-50 million years ago.

Results: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome 1 in the wild species and provided evidence for at least one rearrangement on chromosome 1 of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa.

Conclusion: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies.

Show MeSH