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Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome.

Ríos G, Naranjo MA, Iglesias DJ, Ruiz-Rivero O, Geraud M, Usach A, Talón M - BMC Genomics (2008)

Bottom Line: Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues.In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina.The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias, Carretera Moncada-Náquera km 4,5, 46113 Moncada (Valencia), Spain. rios_gab@gva.es

ABSTRACT

Background: Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background.

Results: Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues.

Conclusion: In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement.

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Chromosome mapping of poplar, Arabidopsis and grapevine homologues of the 39E7 deleted genes. The TBLASTX hits for the 78 39E7 candidates, identified as stated in figure 2, are represented on linkage groups (LG) from Populus trichocarpa, Arabidopsis thaliana and Vitis vinifera. The number of hits contained in each cluster is shown.
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Figure 3: Chromosome mapping of poplar, Arabidopsis and grapevine homologues of the 39E7 deleted genes. The TBLASTX hits for the 78 39E7 candidates, identified as stated in figure 2, are represented on linkage groups (LG) from Populus trichocarpa, Arabidopsis thaliana and Vitis vinifera. The number of hits contained in each cluster is shown.

Mentions: Microsynteny comparisons with homologous stretches from the sequenced genomes of Arabidopsis thaliana, Populus trichocarpa and Vitis vinifera [27-29] were performed in order to elucidate hypothetical clustering of Citrus deleted genes in the genome. TBLASTX, which searches for translations of a crude genome similar to a translated query, was utilized with an E-value cut-off of 10-5. The homologous regions produced by the best TBLASTX hit of each of the Citrus candidate genes were located on the chromosome maps of Arabidopsis, poplar and grapevine. Homologues of Citrus genes were then grouped into clusters in each species when the distance between them was shorter than 250 kb. The second and third TBLASTX best alignments were similarly placed in the respective maps when they were included in an existing cluster. In this case, a binding line was drawn linking the second and third hits to the best hit of the same Citrus query. Thus, two chromosomal maps, one for each mutant, in the three species was obtained. Figure 2 represents in detail chromosome mappings of the 39B3 mutation, which was subjected to further analyses. The results indicate that the Populus mapping exhibited rather lower complexity than the Arabidopsis and grapevine ones since it included fewer chromosomes and only 3 clusters although the number of 39B3 candidate genes represented in the map was identical (21) for the three genomes. Note that the number of represented hits in these mappings is higher than 21 due to the inclusion of second or third homologues. In Populus, most of the candidate genes mapped to two different genome regions of approximately 700 kb long in chromosomes 12 and 15, two duplicated chromosomes that probably originated during the recent genome duplication event that occurred in this species [28]. These two clusters contained 17 and 15 hits respectively while the third one placed in chromosome 16 had only one hit. In contrast, the number of clusters in Arabidopsis and Vitis were 9 and 11, respectively, and none of them contained more than 11 hits. Furthermore, cluster number (and clustering density) of the homologues of 39E7 putative deleted genes was also lower (and higher) in Populus than in Arabidopsis or Vitis, although the differences were smaller: 26, 30 and 30 clusters were obtained for poplar, Arabidopsis and grapevine respectively (Figure 3).


Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome.

Ríos G, Naranjo MA, Iglesias DJ, Ruiz-Rivero O, Geraud M, Usach A, Talón M - BMC Genomics (2008)

Chromosome mapping of poplar, Arabidopsis and grapevine homologues of the 39E7 deleted genes. The TBLASTX hits for the 78 39E7 candidates, identified as stated in figure 2, are represented on linkage groups (LG) from Populus trichocarpa, Arabidopsis thaliana and Vitis vinifera. The number of hits contained in each cluster is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533677&req=5

Figure 3: Chromosome mapping of poplar, Arabidopsis and grapevine homologues of the 39E7 deleted genes. The TBLASTX hits for the 78 39E7 candidates, identified as stated in figure 2, are represented on linkage groups (LG) from Populus trichocarpa, Arabidopsis thaliana and Vitis vinifera. The number of hits contained in each cluster is shown.
Mentions: Microsynteny comparisons with homologous stretches from the sequenced genomes of Arabidopsis thaliana, Populus trichocarpa and Vitis vinifera [27-29] were performed in order to elucidate hypothetical clustering of Citrus deleted genes in the genome. TBLASTX, which searches for translations of a crude genome similar to a translated query, was utilized with an E-value cut-off of 10-5. The homologous regions produced by the best TBLASTX hit of each of the Citrus candidate genes were located on the chromosome maps of Arabidopsis, poplar and grapevine. Homologues of Citrus genes were then grouped into clusters in each species when the distance between them was shorter than 250 kb. The second and third TBLASTX best alignments were similarly placed in the respective maps when they were included in an existing cluster. In this case, a binding line was drawn linking the second and third hits to the best hit of the same Citrus query. Thus, two chromosomal maps, one for each mutant, in the three species was obtained. Figure 2 represents in detail chromosome mappings of the 39B3 mutation, which was subjected to further analyses. The results indicate that the Populus mapping exhibited rather lower complexity than the Arabidopsis and grapevine ones since it included fewer chromosomes and only 3 clusters although the number of 39B3 candidate genes represented in the map was identical (21) for the three genomes. Note that the number of represented hits in these mappings is higher than 21 due to the inclusion of second or third homologues. In Populus, most of the candidate genes mapped to two different genome regions of approximately 700 kb long in chromosomes 12 and 15, two duplicated chromosomes that probably originated during the recent genome duplication event that occurred in this species [28]. These two clusters contained 17 and 15 hits respectively while the third one placed in chromosome 16 had only one hit. In contrast, the number of clusters in Arabidopsis and Vitis were 9 and 11, respectively, and none of them contained more than 11 hits. Furthermore, cluster number (and clustering density) of the homologues of 39E7 putative deleted genes was also lower (and higher) in Populus than in Arabidopsis or Vitis, although the differences were smaller: 26, 30 and 30 clusters were obtained for poplar, Arabidopsis and grapevine respectively (Figure 3).

Bottom Line: Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues.In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina.The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Genómica, Instituto Valenciano de Investigaciones Agrarias, Carretera Moncada-Náquera km 4,5, 46113 Moncada (Valencia), Spain. rios_gab@gva.es

ABSTRACT

Background: Many fruit-tree species, including relevant Citrus spp varieties exhibit a reproductive biology that impairs breeding and strongly constrains genetic improvements. In citrus, juvenility increases the generation time while sexual sterility, inbreeding depression and self-incompatibility prevent the production of homozygous cultivars. Genomic technology may provide citrus researchers with a new set of tools to address these various restrictions. In this work, we report a valuable genomics-based protocol for the structural analysis of deletion mutations on an heterozygous background.

Results: Two independent fast neutron mutants of self-incompatible clementine (Citrus clementina Hort. Ex Tan. cv. Clemenules) were the subject of the study. Both mutants, named 39B3 and 39E7, were expected to carry DNA deletions in hemizygous dosage. Array-based Comparative Genomic Hybridization (array-CGH) using a Citrus cDNA microarray allowed the identification of underrepresented genes in these two mutants. Subsequent comparison of citrus deleted genes with annotated plant genomes, especially poplar, made possible to predict the presence of a large deletion in 39B3 of about 700 kb and at least two deletions of approximately 100 and 500 kb in 39E7. The deletion in 39B3 was further characterized by PCR on available Citrus BACs, which helped us to build a partial physical map of the deletion. Among the deleted genes, ClpC-like gene coding for a putative subunit of a multifunctional chloroplastic protease involved in the regulation of chlorophyll b synthesis was directly related to the mutated phenotype since the mutant showed a reduced chlorophyll a/b ratio in green tissues.

Conclusion: In this work, we report the use of array-CGH for the successful identification of genes included in a hemizygous deletion induced by fast neutron irradiation on Citrus clementina. The study of gene content and order into the 39B3 deletion also led to the unexpected conclusion that microsynteny and local gene colinearity in this species were higher with Populus trichocarpa than with the phylogenetically closer Arabidopsis thaliana. This work corroborates the potential of Citrus genomic resources to assist mutagenesis-based approaches for functional genetics, structural studies and comparative genomics, and hence to facilitate citrus variety improvement.

Show MeSH
Related in: MedlinePlus