Extended Linkage Disequilibrium in Noncoding Regions in a Conifer, Cryptomeria japonica [Genetics]
(Genetics Via Acquire Media NewsEdge) ABSTRACT We measured linkage disequilibrium in mostly noncoding regions of Cryptomeria japonica, a conifer belonging to Cupressaceae. Linkage disequilibrium was extensive and did not decay even at a distance of 100 kb. The average estimate of the population recombination rate per base pair was 1.55 × 1025 and was ,1/70 of that in the coding regions. We discuss the impact of low recombination rates in a large part of the genome on association studies.
LINKAGE disequilibrium (LD) in the coding genes and their surrounding regions of conifers has been reported to extend to only several hundred to a few thousand base pairs [Brown et al. (2004); Heuertz et al. (2006); Pyhäjärvi et al. (2007), but see Pyhäjärvi et al. (2011) for LD extending to several kilobases]. This observation led to the conclusion that conifers are not suitable for finding associations with traits using a genomic scan (Neale and Savolainen 2004). However, if the genomewide extent of LD is not the same as that in the regions studied thus far, the strategy may change. Indeed, the extents of LD differ between genomic regions in maize possibly due to differences in the recombination rate (Gore et al. 2009). Furthermore, conifers generally have lower recombination rates than angiosperms (Jaramillo-Correa et al. 2010). Thus, it is important to know the extent of LD in regions other than the coding genes and their surrounding regions. However, the extent of LD in such regions has not been investigated in conifers to our knowledge possibly because of the lack of sequence information on those regions [but see Hamberger et al. (2009); Keeling et al. (2010); Kovach et al. (2010)].
We recently constructed a bacterial artificial chromosome (BAC) library for Cryptomeria japonica, a conifer belonging to Cupressaceae sensu lato (Gadek et al. 2000; Kusumi et al. 2000) and determined the sequences of eight random clones from the library. We used these sequences to design primers and resequenced fragments in those regions using the same tree samples used by Fujimoto et al. (2008) for the study of the coding regions. From these data, we investigated the extent of LD at a scale of up to 100 kb in the mostly noncoding regions covered by those BACs (see Supporting Information, File S1, for more details on materials and methods).
First, we give a summary of the sequence analysis of the BAC clones relevant to this study. Details of the analysis will be published elsewhere (M. Tamura, A. Watanabe, K. Uchiyama, N. Futamura, K. Shinohara, Y. Tsumura, H. Tachida, unpublished results). Eight random clones (BAC1-BAC8) from the BAC library developed by the Forestry and Forest Products Research Institute in Japan were sequenced. The total length of the sequences was ~800 kb. These sequences were mostly noncoding, and at least 51% of them consisted of known (the majority being LTR retrotransposons) or unknown repeats. Only part (four exons) of one putative protein-coding gene, homologous to genes coding for calcium-dependent protein kinases, was found in BAC6. The ratio of nonsynonymous-tosynonymous divergence between the exons and the corresponding regions in Taxodium distichum, a close relative of C. japonica, was 0.086, suggesting that the gene was active when the two species diverged. The size of the largest intron of this gene was 70 kb (see Figure 1).
In the resequencing experiment, in total, 19 fragments, including the coding regions in BAC6, could be sequenced for at least 40 samples, and their total length was 15,517 bp (Figure 1). We will call them the BAC regions hereafter. The fragment size ranged from 509 to 2016 bp, and the maximum lengths between pairs of sites in the sequenced regions in BAC3, BAC6, and BAC7 were 65,409, 110,613 and 103,427 bp, respectively.
We aligned those 40 or more sequences along with the corresponding sequence of the BACs. Thus, the total number of samples was at least 41. We computed various statistics of genetic diversity excluding sites involving indel variation (Table S2) using DNAsp 5.0 (Rozas et al. 2003). The average nucleotide diversity across all sites was 0.35%, which was similar to those at the silent sites in the mostly coding regions of the same species studied by Kado et al. (2003) and Fujimoto et al. (2008).
Figure 2 shows the squared allelic correlation coefficient, r2, against the number of base pairs separating the two sites. It decayed very little in all three BACs. Thus, LD extended to sites separated by up to 110 kb. We also estimated the population recombination rate, 4Ner, where Ne and r are the effective size of the population and the recombination rate, respectively, in the BAC regions using the compositelikelihood method (Hudson 2001; McVean et al. 2002) implemented in LDhat 2.1. The results for the BAC regions are shown in Table 1, along with those for the five coding genes previously studied by Fujimoto et al. (2008). The composite-likelihood curves for the data set are shown in Figure S1 (the BACs) and Figure S2 (the coding genes). The average r/u and 4Ner per base was 5.04 × 1023 and 1.55 × 1025, respectively, for the BAC regions and 4.46 × 1021 and 1.18 × 1023, respectively, for the five coding genes. Thus, the population recombination rate seems to be ,1/70 in the BAC regions compared to that in the coding genes studied by Fujimoto et al. (2008).
One explanation for the difference of LD between the BACs and the coding regions is the difference in the recombination rate. In angiosperms, gene density and the recombination rate are positively correlated, possibly due to low gene density and low recombination rates in heterochromatic regions (Gaut et al. 2007). Therefore, if the genome of C. japonica is mostly heterochromatic, we expect to observe low recombination rates and low gene density in random BAC clones. Alternatively, the effective size, Ne, in the BAC regions might be smaller than that in the coding genes because of genetic draft (Gillespie 2000), background selection (Charlesworth et al. 1993), and/or the weak selection Hill-Robertson effect (McVean and Charlesworth 2000). However, because the levels of the silent nucleotide diversity in the two regions were comparable, this explanation seemed unlikely.
The slow decay of LD in the BAC regions is quite different from the rapid decay of LD within 500-2000 bp observed in Pinaceae and poplars (Brown et al. 2004; Ingvarsson 2005, 2008; Krutovsky and Neale 2005; González-Martínez et al. 2006; Heuertz et al. 2006; Pyhäjärvi et al. 2007; Ingvarsson et al. 2008; Li et al. 2010) although more extensive LD has been found in a few cases (Eckert et al. 2010; Pyhäjärvi et al. 2011). C. japonica is distantly related to these species (Chaw et al. 2000), and the difference may be ascribed to differences in the species studied; however, a more plausible explanation would be the differences in the regions studied. The other studies examined relatively narrow regions that included coding genes [except for the 80-kb gene-rich region surrounding the phytochrome B2 locus in aspen studied by Ingvarsson et al. (2008)], while our BAC regions were in mostly noncoding regions. Indeed, the extensive linkage disequilibrium found here might be consistent with the interlocus linkage disequilibrium found in coastal Douglas fir (Eckert et al. 2009) and in Pinus taeda (Eckert et al. 2010).
On the basis of the rapid decay of linkage disequilibrium in conifers, Neale and Savolainen (2004) concluded that genome-wide association studies would not be possible in conifers because of the enormous SNP marker density required (see also Neale and Kremer 2011). However, the number of necessary SNP markers may not be as large if most of the genome is segregating as blocks, as found in the BAC regions studied here. Thus, genome-wide association studies may be feasible in conifers whose genome sizes are generally large (Ohri and Khoshoo 1986), although it would be difficult to locate the causal polymorphism if LD is extensive (Atwell et al. 2010). Nevertheless, identifying causal polymorphisms is not necessary for genomic selection (Meuwissen et al. 2001). Of course, we need to choose the positions of SNP markers carefully so that they are spaced appropriately in terms of recombination rates by identifying recombination-poor and -rich regions (see Flint-Garcia et al. 2003). If heterochromatic regions are recombination-poor in conifers as they are in angiosperms (Gaut et al. 2007), then identifying heterochromatic regions is important for designing efficient markers.
In this article, we have shown that linkage disequilibrium in the BAC regions was much more extensive than that in the coding regions. However, our data are restricted to those from only three random BAC clones. We need to test the generality of these features in future studies.
We thank two anonymous referees for their helpful comments. This study was partially supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. 22370083).
Materials and Methods
Plant materials: In the present study, we used 48 seed samples of Cryptomeria japonica. Genetic characterizations of natural populations of C. japonica are described in Tsumura et al. (2007). The samples were the same as those used in the study by Kado et al. (2003) and Fujimoto et al. (2008) and were collected from three areas in Japan (Kantou-Toukai, Hokuriku, and Iwate). The average estimates of FST between populations based on the data of seven nuclear loci were 0.01-0.04 (Kado et al. 2003). Hence, we regard the present samples of C. japonica as homogeneous and pool the data of the three populations. Also, although the samples were collected from artificial forests, their genetic compositions were very similar to those of the natural populations of C. japonica (Kado et al. 2006). We also used two seed samples of Taxodium distichum, one of the closest relatives of C. japonica. These samples were used as outgroups for the analysis of a putative gene found in the studied region. All seed samples were provided from The Forestry and Forest Products Research Institute in Japan, and haploid genome DNA was extracted from the megagametophytes using a modified SDS method. Briefly, the megagametophytes from seeds were homogenized in extraction buffer (0.1 M Tris-HCl pH 8.0, 10 mM EDTA, 0.5% SDS, and 0.1 mg/ml Proteinase K), and haploid DNA was extracted from it using phenol/chloroform and ethanol.
Sequence analysis of BAC clones: Eight BAC clones (BAC1-BAC8) were randomly chosen from the BAC library developed by The Forestry and Forest Products Research Institute in Japan and sequenced using a Roche-454 Genome Sequencer FLX Titanium (Roche, Indianapolis, IN, USA) by Takara Bio Inc. (Ohtsu, Japan). The sequences of the fragments of each BAC clone were assembled using GS De Novo Assembler version 2.0, with its default setting. We chose the largest contig in the assembled sequences of each BAC clone and used it as the sequence of the BAC for designing primers. The eight BAC sequences were named BAC1-BAC8. The average number of reads per base pair for those contigs ranged from 12.9 to 86.3. The full analysis of the BAC sequences will be published elsewhere (M. Tamura, A. Watanabe, K. Uchiyama, N. Futamura, K. Shinohara, Y. Tsumura, H. Tachida, unpublished results).
Loci and primers: The PCR primers were based on the BAC sequences of C. japonica. The BAC sequences contained putative transposable elements and many other repetitive sequences (M. Tamura, A. Watanabe, K. Uchiyama, N. Futamura, K. Shinohara, Y. Tsumura, H. Tachida, unpublished results). In the design of primers, we avoided the repetitive regions. The accuracy of the distances on the BAC sequences for which genetic diversity was investigated (BAC3, BAC6 and BAC7) was confirmed using long PCR and electrophoresis. To increase the specificity of the amplification, we carried out nested PCR at all loci. First, PCR primers were designed to generate fragments of approximately 3 to 15 kbp in size. Then, a second set of PCR primers was designed to amplify one or more regions within the first PCR product so that the lengths of the fragments for which sequences were determined would be approximately 700 to 2000 bp. BAC6 contained a partial coding gene that was homologous to the calcium-dependent protein kinase of Populus trichocarpa (XP_002322129.1). The size of one of the introns was greater than 70 kb. We designed primer pairs so that all exons contained in BAC6 could be sequenced. The positions of the regions used for the diversity analysis and structure of the coding region are shown in Figure 1. If necessary, we designed internal primers for sequencing. All primers were designed using Primer3 software (http://primer3.sourceforge.net/). The primer sequences used are listed in Table S1 (supporting information).
Amplification and sequencing: We carried out long PCR using TaKaRa LA Taq (Takara, Ohtsu, Japan) for long fragments (> 10 kb), but for those with smaller sizes, we used TaKaRa ExTaq (Takara, Ohtsu, Japan). The first PCR conditions were 3 min at 98°, followed by 35 cycles of 10 sec at 98° and 15 min at 68°, and finally, 15 min at 68°. The products were directly used as templates for the second PCR. The second PCR conditions were 3 min at 95°; followed by 25 cycles of 30 sec at 95°, 30 sec at 50°, and 1-2 min at 72°; and a final extension of 7 min at 72°. The products were purified by PEG precipitation treatment to remove surplus primers and dNTP. The DNA fragments were directly sequenced on an ABI Prism 3100 automatic sequencer or Applied Biosystems 3730 DNA Analyzer using a Big Dye Terminator v3.1 Cyclesequence Kit (Applied Biosystems, Foster City, CA, USA). All sequences described in this article have been deposited in the GenBank/EMBL/DDBJ databases (accession nos. AB686666-AB687489).
Analyses of data: All sequences were assembled using the SeqMan package in Lasersene 7.1 (DNASTAR, Inc., Madison, WI, USA) and aligned manually using MEGA3 (Kumar et al. 2004). By comparing these sequences with those of the cDNA clones, we could determine the coding regions. Estimates of standard population genetics statistics, such as p, Tajima's D (Tajima 1989), Fu and Li's F* and D* (Fu and Li 1993) and the squared allelic correlation coefficient (r2), between sites were calculated using DnaSP 5.0 (Rozas et al. 2003).
We also estimated the population recombination rate, 4Ner, where Ne and r are the effective size and the recombination rate, respectively, using the composite likelihood method (Hudson 2001; McVean et al. 2002) implemented in LDhat 2.1 (www.stats.ox.ac.uk/~mcvean/LDhat.html). First, we made FASTA-like files in which undetermined nucleotides were represented by "?" and the program convert was run. We used all polymorphic sites as data. Next, the "locs" and "sites" files in the outputs of convert were used as inputs for the program pairwise. The program pairwise assumes a constant recombination rate across the region. We used the crossing-over model to estimate 4Ner, and the parameter region searched was 4Ner = 0.0-100.0. The minimum number of recombination events (Hudson and Kaplan 1985) was also obtained by the program.
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Communicating editor: O. Savolainen
Etsuko Moritsuka,*,1 Yosuke Hisataka,[dagger],1 Miho Tamura,[dagger] Kentaro Uchiyama,[double dagger] Atsushi Watanabe,§
Yoshihiko Tsumura,[double dagger] and Hidenori Tachida*,2
*Department of Biology, Faculty of Sciences, Kyushu University, Fukuoka 812-8581, Japan, [dagger]Graduate School of Systems Life
Sciences, Kyushu University, Fukuoka 812-8581, Japan, [double dagger]Department of Forest Genetics, Forestry and Forest Products Research
Institute, Tsukuba, Ibaraki 305-8687, Japan, and §Tree Breeding Center, Forestry and Forest Products Research Institute,
Hitachi, Ibaraki 319-1301, Japan
Copyright © 2012 by the Genetics Society of America
Manuscript received November 9, 2011; accepted for publication December 12, 2011
Supporting information is available online at http://www.genetics.org/content/ suppl/2011/12/30/genetics.111.136697.DC1
1These authors contributed equally to this study.
2Corresponding author: Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581, Japan. E-mail: firstname.lastname@example.org
(c) 2012 Genetics Society of America
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