High-throughput discovery of chloroplast and mitochondrial DNA polymorphisms in Brassicaceae species by ORG-EcoTILLING

Abstract

Background: Information on polymorphic DNA in organelle genomes is essential for evolutionary and ecological studies. However, it is challenging to perform high-throughput investigations of chloroplast and mitochondrial DNA polymorphisms. In recent years, EcoTILLING stands out as one of the most universal, low-cost, and high-throughput reverse genetic methods, and the identification of natural genetic variants can provide much information about gene function, association mapping and linkage disequilibrium analysis and species evolution. Until now, no report exists on whether this method is applicable to organelle genomes and to what extent it can be used.

Methodology/principal findings: To address this problem, we adapted the CEL I-based heteroduplex cleavage strategy used in Targeting Induced Local Lesions in Genomes (TILLING) for the discovery of nucleotide polymorphisms in organelle genomes. To assess the applicability and accuracy of this technology, designated ORG-EcoTILLING, at different taxonomic levels, we sampled two sets of taxa representing accessions from the Brassicaceae with three chloroplast genes (accD, matK and rbcL) and one mitochondrial gene (atp6). The method successfully detected nine, six and one mutation sites in the accD, matK and rbcL genes, respectively, in 96 Brassica accessions. These mutations were confirmed by DNA sequencing, with 100% accuracy at both inter- and intraspecific levels. We also detected 44 putative mutations in accD in 91 accessions from 45 species and 29 genera of seven tribes. Compared with DNA sequencing results, the false negative rate was 36%. However, 17 SNPs detected in atp6 were completely identical to the sequencing results.

Conclusions/significance: These results suggest that ORG-EcoTILLING is a powerful and cost-effective alternative method for high-throughput genome-wide assessment of inter- and intraspecific chloroplast and mitochondrial DNA polymorphisms. It will play an important role in evolutionary and ecological biology studies, in identification of related genes associated with agronomic importance such as high yield and improved cytoplasmic quality, and for identifying mitochondrial point mutations responsible for diseases in humans and other animals.

Figure 1. Overview of the ORG-EcoTILLING procedure for detecting DNA polymerphisms in Chloroplast and Mitochondrial genes, modified from Haughn and Gilchrist (2006).

A, Total DNA is extracted from plants. B, primers in which a sequence from the universal primer M13F (CACGACGTTGTAAAACGAC) and M13R (GGA TA A CAAT TTCACACAGG) are added to the 5′ end of a target organelle gene primers as adaptors, and query DNA and the reference DNA are amplified, respectively. Then the PCR products of query DNA and reference DNA with the ratio of one to one are pooled. C, the mixture is amplified using the universal primers tagged fluorescently. The forward and reverse primers are labeled with different fluorophors (IRD700 and IRD800) that each label a different end of the PCR fragments. D, the amplified products are denatured by heating and then allowed to cool slowly so that they randomly re-anneal. Heteroduplex molecules form when single DNA strands of mutant and wild-type PCR products anneal together. E, these heteroduplexes become targets for a single-strand-specific nuclease as found in celery juice extract. The nuclease cleaves these heteroduplex fragments at one of the two strands, 3′ to the site of the mismatch in the DNA. F, the PCR products that retain one of the labeled primers can be detected on polyacrylamide denaturing LI-COR gels. G, because the nuclease cleaves either of the two strands randomly, cleavage products can be detected in both the IRD700 and IRD800 channels of the gel image. The position of the mutation within the PCR amplicon can be calculated from the size of the fragments carrying the IRD700-labeled forward primer and the IRD800-labeled reverse primer. The additive size of the cleaved fragments should equal the total length of the entire product.

Figure 2. Detection of amplified fragments of selectcd chloroplast genes by agarose gel electrophoresis.

a. accD gene, the size of PCR products in accD-1 and accD-2 amplified by two pairs of primers were 974 and 789 bp, respectively. b. matK gene, the size of PCR products in matK-1 and matK-2 amplified by two pairs of primers were 959 and 948 bp, respectively. c. rbcL gene, the size of PCR products in rbcL-1 and rbcL-2 amplified by two pairs of primers were 974 and 1081 bp, respectively. The sizes of the DNA fragments produced for the three genes corresponded with what we expected, and the abundance of DNA was fit for ORG-EcoTILLING analysis. The samples from 1 to 5 were B12, B20, B26, B85 and B94 in accD-1 gene fragment, and the same in other gene fragments. The molecular size of DNA is shown at the left with arrowheads. M represented the DNA ladder DL 2000 (Promega. Inc).

Figure 3. Overview of mutation detections in matK gene of matK-2 fragment by ORG- EcoTILLING.

Samples were resolved by denaturing polyacrylamide gel electrophoresis and visualized with the LI-COR DNA analyzer. The sample in each lane from left to right was B49–B96 samples. The IRD 700 and IRD 800 channels were shown respectively. CEL I-cleaved heteroduplexes appeared as intense bands (circled in different color rectangles of IRD 700 channel and corresponding same color ones of IRD 800 channel). The size of the specific cleavage products in the IRD700 labeled plus the size of the IRD800 labeled (indicated by arrowheads in the same color geometries, respectively) was equal to that of uncleaved fragment (948 bp). The fragment sizes also indicated the position of the mutation present in the locus matK gene of the representative samples. The reference sample was B26.

Figure 4. Examples of DNA amplified regions detection of plastidic genes separated by agarose gel electrophoresis.

The picture shows the results from PCR analyses of plastidic gene accD (primer pair: M13-CAF and M13-CAR), and atp6 (primer pair: M13-CP F and M13-CP R), respectively. The samples of accD gene and atp6 came from 91 various species in Cruciferae. The sizes of the DNA fragments in the two genes detected by agarose gel electrophoresis were consistent with the result of primers design, and the abundance of DNA was fit for ORG-EcoTILLING analysis. The molecular size of DNA is shown at the left with arrowheads. M represented the DNA ladder DL 2000 (Promega. Inc).

Figure 5. Overview of mutation detections in Brassicaceae samples by ORG- EcoTILLING Electrophoresis on a 6.5% KBplus Gel of CEL I-digested products of APCR fragment of accD gene, 827 bp in ALI-COR 4300 DNA Analyzer were displayed here.

The sample in each lane from one to six was A64, A65, A61, A67, A68 and A69, respectively. CEL I-cleaved heteroduplexes appeared as intense bands (circled in different color geometric figures of IRD 700 channel and corresponding same color ones of IRD 800 channel). The fragment sizes indicated the position of the mutation present in the locus accD gene of the collected samples. The reference sample in this gene was A64.

Figure 6. Images of ORG-EcoTILLING gels of CEL I-digested products of a PCR fragment of atp6 gene obtained from each of the two fluorescent channels of the LI-COR 4300 DNA Analyzer.

The intense bands (circled in different color geometric figures of IRD 700 channel and corresponding same color ones of IRD 800 channel) came from the product of CEL I-cleaved heteroduplexes. The sample in each lane from left to right was A24, A18, A32, A67, A76, A83 and A90, respectively, and sample A24 acted as the reference sample of atp6 gene for ORG-EcoTILLING. Therefore, it was obvious that 13 polymorphism sites were detected in atp6 gene among these seven samples.

Figure 7. Gel image showing the result of validation in the difference of the same polymorphic position discovered by ORG-EcoTILLING among Brassica species or Cruciferae.

(a) Based on the results of ORG-EcoTILLING in matK-2 fragment of Brassica intraspecies in Figure 3, 19 mutated samples and no mutation sample B26 (as the reference in Figure 3) were selected to mix with B76 (as the reference), respectively. The sample in each lane from left to right in the channel of IRD700 or IRD800 was B26, B51, B60, B64, B65, B66, B68, B69, B73, B74, B76, B79, B81, B90, B91, B92, B93, B94, B95 and B96. CEL I-cleaved heteroduplexes appeared as intense bands (circled in different color geometric figures of IRD 700 channel and corresponding same color ones of IRD 800 channel). (b) ORG-EcoTILLING gel image of validation in polymorphism site 474 of accD gene (Figure S1, Table 7) by using one of the three samples of A64, A65 and A67 as reference with the others. In the channel of IRD700 or IRD800, the lane of 1, 2, and 3; 4, 5 and 6; 7, 8 and 9 were sample A64, A65 and A67, respectively, but the reference of 1, 2, and 3 was A64; the reference of 4, 5 and 6 was A65, and the reference of 7, 8 and 9 was A67. It was obvious that there was different polymorphism in the same position 474 because polymorphism appeared when 65 mixed with A67 (blue arrow showed polymorphism band).

Figure 8. Phylogenetic tree was constructed based on the integrating data of the three chloroplast genes (accD, matK and rbcL) in Brassica species.

A total of accD, matK and rbcL sequences of 90 accessions of B. napus and each of 3 accessions of B. rapa and B. oleracea were collected for analysis. The tree was constructed by the neighbour-joining method and bootstrap values above 750 from 1000 resamplings were shown for each node. The code in the tree corresponded to the locality code of Table 2. M represented 69 no mutation samples in B. napus. The bar corresponded with 100 substitutions per site.