Genetic linkage maps of the noble scallop Chlamys nobilis reeve based on AFLP and microsatellite markers.
KEY WORDS: Noble scallop, Chlamys nobilis, linkage map, AFLP, microsatellite marker
Distributed mainly along the south coasts of China, Japan, and Malaysia, the noble scallop Chlamys nobilis Reeve has been one of the important economic species for the shellfish aquaculture industry along the south coast of China for several decades. The culture regions are distributed principally along four coastal provinces (Fujian, Guangdong, Guangxi, and Hainan) of the South China Sea. In China, commercial farming of C. nobilis commenced in the Fujian Province in the 1970s. The Fujian stock, which was the central seed stock for cultured populations, has been preliminarily confirmed in our previous research (Yuan et al. 2009). In recent years, the C. nobilis aquaculture industry has been in chaos, with a higher mortality rate than ever before. One potential explanation for the problem is that every year the cultured stock has been used as broodstock to produce the next cultured generation. Such a closed cycle could have led to reproductive isolation from the wild populations and a reduction in genetic variability resulting from low effective population size and inbreeding (Gosling 1982, Hedgecock & Sly 1990). Stable strains with desired traits have not yet been produced for aquaculture. Thus, domestication and genetic improvement remain big challenges for the C. nobilis aquaculture industry.
Genetic linkage maps are powerful research tools for the study of many organisms (Dib et al. 1996). For many major and important aquaculture species, genetic linkage maps have been developed. Examples include scallop (Wang et al. 2004a, Li et al. 2005, Qin et al. 2007), mussel (Lallias et al. 2007), oyster (Li & Guo 2004, Yu & Guo 2006), abalone (Sekino & Hara 2007), sea urchin (Zhou et al. 2006), shrimp (Li et al. 2006, Staelens et al. 2008), and fish (Lee et al. 2005, Bouza et al. 2007, Ning et al. 2007, Reid et al. 2007, Shen et al. 2007). A complete linkage map is necessary to conduct molecular-based analyses efficiently, such as location of quantitative trait locus and map-based cloning (Lander & Botstein 1989). In addition, genetic mapping is useful for studying genome structure and evolution in molluscs.
Construction of a linkage map requires a large number of molecular markers. Several classes of molecular markers are needed in linkage mapping to cover a wide range of the genome, and anonymous DNA markers such as amplified fragment length polymorphisms (AFLPs) and random amplified polymorphic DNA (RAPD) can serve as efficient tools to achieve extensive genome coverage. AFLP markers are more reliable than RAPD, require no prior knowledge of DNA sequence, and generate abundant markers per detection. Given the codominant property, microsatellites have a wealth of segregation information and the transferability across populations. Therefore, the homologies of microsatellite markers and thereby linkage groups among populations can readily be established. Nevertheless, only 23 microsatellite loci have been reported in C. nobilis (Hui et al. 2006, Wang et al. 2008).
In this study, our goal was to construct preliminary genetic linkage maps for C. nobilis. Two parent-specific genetic linkage maps of C. nobilis were constructed by analyzing the segregation of AFLP and microsatellite DNA markers in 80 F1 progenies, which furnished a basic tool kit for C. nobilis genomic analysis. The C. nobilis maps reported here will provide a tool that can be used for research into molluscan genome evolution and as a foundation for C. nobilis broodstock enhancement programs using marker-assisted selection (MAS).
MATERIALS AND METHODS
Mapping families were produced at the Daya Bay Research Station of the Chinese Academy of Science in 2007. All scallops were selected from the Fujian cultured population. Mating scallops were divided into 2 types of shell colors (bright orange and brownish purple). The mapping population consisted of a single-pair mating family produced by mating 2 scallops with the same shell color (bright orange) with progeny that had shell colors that significantly deviated from the parents. After spawning, the parents were kept in 95% alcohol. The candidate mapping families were reared for 6 mo. In March 2008, 80 progeny (40 bright orange and 40 brownish purple) were randomly selected for linkage analysis.
DNA Extraction and Marker Analysis
DNA of the 2 parents and 80 progeny were extracted from adductor muscle tissue using proteinase K digestion and DNA binding columns (QIAGEN QIAamp DNA Mini Kit, Hilden, Germany) according to the manufacturer's instructions. DNA concentration was measured using an ultraviolet spectrophotometer (Hitachi U-2001, Tokyo, Japan) and adjusted to 100 ng/[micro]L for molecular analysis. The quality of extracted DNA was assessed by 1.0% agarose gel electrophoresis.
AFLP markers were generated following protocols described by Vos et al. (1995) and Wang et al. (2004b). Polymerase chain reaction (PCR) products were separated using 6% denaturing polyacrylamide gels (Bio-Rad Sequi-Gen GT, Hercules, CA) and visualized by silver staining. Visual scoring was conducted on clearly visible and well-defined bands. Molecular size standard files were generated by the software Cross Checker 2.91 (Buntjer 1999). To ensure that the obtained markers were highly reproducible and specific, a primer combination was amplified at least twice for the parents and for 4 progeny under the 2-step AFLP analysis.
A collection of 23 C. nobilis microsatellite DNA markers published previously by Hui et al. (2006) and Wang et al. (2008) and 14 markers for the of Zhikong scallop Chlamys farreri (Zhan et al. 2006) were used in this study. Amplification of microsatellites by PCR was performed as reported by Yu and Guo (2003). PCR products were separated by denaturing gels containing a 6% acrylamide/bis acrylamide mixture sized with the low-molecular weight DNA Ladder (NEB, Ipswich, MA). DNA bands were visualized by silver staining.
AFLP fragments ranging from 70-700 bp were counted and scored. Segregating markers were judged for deviations from the expected 1:1 (of AFLP marker and microsatellite DNA marker polymorphic in one parent) or 1:1:1:1 and 1:2:1 (of microsatellite DNA marker polymorphic in both parents) phenotypic ratios with chi-square testing. Segregating markers were used to construct linkage maps at the expected ratios. The segregation type was scored following backcross population, with H representing the presence of a band and A representing the absence of a band. Two data sets were obtained for the maternal and paternal parents, respectively (Zhang et al. 2007). Segregation of marker loci was analyzed as an F2 backcross model, using the microcomputer program MapMaker/Exp v. 3.0 (Lander et al. 1987).
A framework map composed of segregating markers at the expected ratios was constructed first, then the distorted markers (0.01 < P < 0.05) were incorporated. Markers distorted at high significance levels (P < 0.01) were excluded from the linkage analysis to avoid false linkages (Bert et al. 1999).
Linkage Analysis and Map Construction
Under the GROUP command at the minimum LOD = 3 and the maximum distance of 25 cM between 2 loci, markers were organized into linkage groups. Successive COMPARE, ORDER, and MAP commands were used to order the markers preliminarily in each linkage group with fewer than 9 markers. Marker order in linkage groups with more than 9 markers was established using the THREE POINT, ORDER, and MAP commands, or sublinkage groups were constructed first under more stringent conditions and then subgroups were connected. The RIPPLE command was used to verify marker order. Additional markers that could not be mapped with the previous commands and the distorted markers were incorporated using NEAR and TRY commands under less stringent conditions (minimum LOD = 2.9 and maximum distance of 30 cM). Map distances (measured in centi-Morgans) were calculated using Kosambi's mapping function (Kosambi 1944). Linkage groups were assigned based on descending size. The graphic maps were generated using MapChart 2.1 (Voorips 2002). AFLP markers were named after the primer pairs used to generate them and their size. EeoRI- and MseI-selective primers were coded by numbers and letters, respectively, followed by the letter f (fragment) and 3 digits representing the size in base pairs. Table 1 shows the abbreviated primer combinations. The numbers of microsatellites were named following the author's description.
Distorted markers (Hui et al. 2006, Wang et al. 2008).
On the basis of the synthesized maps, expected genome length was obtained using the following 2 methods. First, the average spacing s between markers, which is calculated by dividing the total observed map length by the number of marker intervals, was estimated, followed by adding twice the s value to the observed map length of each linkage group (Gel, Fishman et al. 2001). Second, the observed map length of each linkage group was multiplied by (m + 1)/(m - 1), where m is the number of markers placed at different positions on the linkage group ([G.sub.e2], method 4 in Chakravarti et al. 1991). The average of the 2 estimates was used as the estimated genome length ([G.sub.e]). The observed map length was calculated as the length of the map ([G.sub.of]) and the total length ([G.sub.oa]) considering all the markers on the map, the triplets, and doublets. The observed genome coverages, [C.sub.of] and [C.sub.oa], were calculated as [G.sub.of]/[G.sub.e] and [G.sub.oa]/[G.sub.e], respectively.
Marker Polymorphism and Segregation
Sixty-four AFLP selective primer combinations were tested in this study. Following the preselective amplification step, 26 primer combinations were selected for genotyping the parents and 80 progeny. In total, 1,878 markers were obtained and 475 were polymorphic between the 2 parents. On average, each primer combination produced 45-100 markers. Of the 475 polymorphic markers, 404 segregated in either of the parents (Table 1). In addition, the chi-square analysis revealed that 350 (179 in female and 171 in male) segregated with a 1:1 ratio and 54 (30 in female and 24 in male) deviated significantly from the Mendelian ratio at P < 0.05.
Of the 80 progeny, 40 were bright orange and 40 were brownish purple. Marker Blf176 was present in both parents (bright orange) and all the bright-orange progeny, but was absent in all the brownish purple progeny. Consequently, the Blf176 marker was excluded from the framework map, which was polymorphic in both parents.
Thirty-seven pairs of microsatellite primers were screened for segregation in the mapping population. In total, 26 microsatellite DNA markers amplified robust bands, and 15 of them were polymorphic and therefore informative in one or both parents. Among these, CN005, CN010, CN021, CN026, CN028, CN035, CN070, and CF019 in the female and CN003, CH007, CN013, CN064, and CN067 in the male segregated at a 1:1 ratio; CN036 segregated at a 1:1:1 : 1 ratio and CN048 segregated at 1:2:1 ratio. Chi-square analysis indicated that CN005, CN026, CN028, CN003, CN013, and CN067 were not in agreement with Mendelian ratios (P < 0.05). Distorted markers were excluded from the linkage analysis.
The majority of AFLP and microsatellite loci of C. nobilis were mapped successfully. A total of 117 (115 AFLPs and 2 microsatellites) segregating markers were mapped in the female data set and the male data set was composed of 108 (106 AFLPs and 2 microsatellites) segregating markers. According to the linkage analysis, markers could be classified into 3 types: framework markers; unplaced markers, which were linked to the framework markers but had irresolvable conflict and their addition would significantly expand the map length; and unlinked markers, which had no evidence of linkage to the framework markers but could form triplets or doublets or remain single. The doublet composed of CN036 and CN048 segregated in both the male and female parent maps. Therefore, CN036 and CN048 were considered as one group in the framework maps.
In the female linkage map, 16 linkage groups with an average number of 7.3 loci per linkage group were found and mapped (Table 2). The marker number per linkage group ranged from 2 (group 16) to 14 (group 1). Including all loci, the average distance between loci was 16.8 cM and the overall map length was 1,698.6 cM ([G.sub.of]). The markers not included in the framework were 3 unlinked triplets, 8 doublets, 42 unlinked singles, and 15 unmapped markers. In addition, 3 distorted markers were mapped--1 to group 1 and 2 to group 9. Including the triplets and doublets, the total length of the map ([G.sub.oa]) was 2,002.1 cM. The identified linkage groups corresponded to the expected haploid number of 16(n) chromosomes (Fig. 1).
In the male linkage map, a total of 61 loci remained unassigned, with 2 triplets, 12 doublets, 39 singles, and 8 unmapped. Sixteen male linkage groups were mapped with an average number of 6.8 loci per linkage group, and each linkage group had 2-15 markers (Table 2). The map length was 1,519.0 cM ([G.sub.of]) with an average distance of 16.5 cM between loci. Two distorted markers were mapped to the framework map--one to group 10 and the other to group 15. The total length of the map ([G.sub.oa]) including triplets and doublets was 1,864.2 cM. The identified linkage groups corresponded to the expected haploid number of 16(n) chromosomes (Fig. 2).
Genome Estimation and Map Coverage
The estimated genome lengths were 2,236.2 ([G.sub.el]) and 2,233.9 cM ([G.sub.e2]) with an average of 2,235.1 cM for the female, and 2,047.0 (Gel) and 2,060.0 cM ([G.sub.e2]) with an average of 2,053.5 cM for the male. According to the observed length and the estimated genome length, genome coverage of female and male maps was 76.0% and 74.0%, respectively. When all the triplets and doublets were included, the map coverage increased to 89.6% for the female and 90.8% for the male maps.
A high-resolution linkage map requires a large number of molecular markers. AFLP markers have the potential to construct high-resolution maps efficiently and rapidly and to identify and isolate those closely linked with desirable traits. The assay efficiency index of AFLP is more than 10-fold that of microsatellites and RAPD (Pejic et al. 1998). In this study, polymorphic markers between the parents were 18.3 loci per primer combination, which is less than the 22.6 reported for the Pacific oyster (Li & Guo 2004), 23.3 for the eastern oyster (Yu & Guo 2003), 20.3 for C. farreri (Li et al. 2005), and 22.0 for the blue mussel (Lallias et al. 2007), but much higher than the 5.8 reported for the bay scallop (Qin et al. 2007). The high levels of polymorphism make it efficient to use the F1 progeny of a single cross for linkage analysis.
However, the application of AFLP mapping is restricted by the difficulties of transformation among laboratories and populations (Wang et al. 2004a). Microsatellite DNA markers have prepotency of linkage mapping than AFLP (Rafalski et al. 1996), which benefit from their high polymorphism, heterozygosity, codominance, and wide transportability across different mapping populations. Microsatellite linkage maps have been constructed for zebrafish (Knapik et al. 1998), cichlid fish (Kocher et al. 1998), rainbow trout (Sakamoto et al. 2000), Pacific oyster (Hubert & Hedgecock 2004), Pacific abalone (Sekino & Hara 2007), and turbot (Bouza et al. 2007). Nevertheless, the number ofmicrosatellite DNA markers known for C. nobilisis still very limited. Consequently, microsatellite-based genetic maps of C. nobilis are unavailable. A total of 23 microsatellites have been published to date, and only 13 of them were polymorphic between the 2 parents and their progenies in this study. Only one linkage group was matched between male and female maps. To optimize the framework maps, more microsatellite markers or other kinds of codominant markers (e.g., single-nucleotide polymorphisms) should be developed and added for C. nobilis. Thus, future development of microsatellite DNA markers will facilitate the exploration of the genetic basis of diverse biological phenomena and will promote the breeding of C. nobilis and the management of its genetic resources.
Cross-species amplifications of microsatellite DNA markers have been applied recently to construct genetic maps of some aquacultural species (Reid et al. 2007, Sekino & Hara 2007, Shen et al. 2007). In our research, 14 microsatellite DNA markers of C. farreri (Hui et al. 2006) have been tested. Five microsatellite markers amplified robust bands and 2 of them were informative in either of the parents. Therefore, in C. nobilis, cross-species amplifications of microsatellites were able to compensate for the lack of marker needed for genetic map construction.
Based on female and male segregating data, 2 separate linkage maps were constructed. Theoretically, the 2 different maps could be merged into a composite map by using markers that are heterozygous in both the parents (Barreneche et al. 1998), but such a merge can be poorly constructed with dominant markers like AFLPs (Malipaard et al. 1997). Currently, only 2 microsatellite markers were common to both maps, hence it was impossible to merge them.
Except for the chromosome number (2n = 32), there have been no reports about the genome of C. nobilis. The 2 framework maps constructed in this study had 16 major linkage groups, and they might correspond to the 16 chromosomes in the haploid C. nobilis. Estimation of the genome length and map coverage suggested that the primary linkage maps had acceptable coverage and completeness. Compared with the average intervals of 20.91 cM for females and 20.15 cM for males on the map of C. farreri reported by Wang et al. (2004a), the maps presented here are more saturated (16.8 cM for the female and 16.5 cM for the male), but they are incomparable with those of the bay scallop (7.0 cM for the female and 7.2 cM for the male) reported by Qin et al. (2007). These differences are an indication of large gaps and low marker density. Without common markers, it is impossible to detect differences in recombination between the male and female. The linkage groups varied substantially in length, with 5.0-174.3 cM for the female and 5.0-167.7 cM for the male. These values might have been influenced by unequal recombination rates between different chromosomes; however, they might also reflect the incomplete nature of the relevant small linkage groups.
AFLP clustering is a pervasive phenomenon of AFLP maps in many organisms, such as rainbow trout (Young et al. 1998), tilapia (Agresti et al. 2000), channel catfish (Liu et al. 2003), and the bay scallop (Qin et al. 2007). Nevertheless, as in the Pacific oyster (Li & Guo 2004) and C. farreri (Li et al. 2005), no obvious clusters were found in this study. The relatively even distribution of the markers increases the effectiveness of AFLPs in linkage map construction.
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In most species, including humans (Gyapay et al. 1994), dogs (Neff et al. 1999), rainbow trout (Sakamoto et al. 2000), zebrafish (Singer et al. 2002), shrimp (Staelens et al. 2008), and oysters (Yu & Guo 2003, Li & Guo 2004), female maps are longer than male maps; birds can show the reverse pattern (Groenen et al. 1998). By reason of discrepancy, the heterogametic sex shows slightly lower recombination, which may be explained by Haldane's prediction (Haldane 1922). The linkage map of the female C. nobilis in this study showed greater distances (1698.6 cM) than that of the male (1,519.0 cM), but the opposite was found in both C. farreri (Li et al. 2005) and the bay scallop (Qin et al. 2007). Sex-linked markers have been mapped in several marine molluscs. In female C. farreri (Li et al. 2005) and in male Haliotis discus hannai (Liu et al. 2006), the heterogametic sex shows a lower recombination rate. The unusually large recombination differences between males and females are postulated to arise from the differential chromosome pairing affinities observed between the sexes (Sakamoto et al. 2000). However, the molecular mechanism responsible for differences in recombination rates between the 2 sexes is not well understood in most species.
According to the observed data, the proportion of distorted AFLP markers was 11.4%, which was lower than the 14.5% reported for the bay scallop (Qin et al. 2007) and the 17.8% for C. farreri (Li et al. 2005), but higher than the 8.2% of the eastern oyster (Yu & Guo 2003). Widespread distortions of segregation ratios have been reported in many previous studies of inheritance and linkage in bivalve molluscs (reviewed by Mcgoldrick et al. 2000). Segregation distortion can be explained by high genetic load and selection against zygotes with homozygous deleterious recessive mutations (Launey & Hedgecock 2001). High genetic load in the Pacific oyster was confirmed by Hubert and Hedgecock (2004), who made a linkage map by genotyping 11-day-old larvae. They successfully reduced segregation distortion caused by genotyping 11-day-old outbred larvae of the Pacific oyster, which successfully reduced segregation distortion caused by selection acting in the late larval or early juvenile stage. Although most of the distorted markers were excluded in our study, one similar clustering (on linkage group 9) in the female map was observed. The clustered distorted markers in the female map might also be linked to deleterious genes. In addition, distorted segregation may result from amplifcation of the same size fragment from different regions (Faris et al. 1998, Negi et al. 2000) and from a technical artifact in genotyping. The parents used in this study were from the same population with low genetic distance, and the low ratio of segregation distortion might also have been more or less caused by the genome divergence of the parents as in interspecific crosses (Whitkus 1998, Fishman et al. 2001).
In this study, the mating scallops were divided into 2 groups based on shell color: O (bright orange) and P (brownish purple). The analysis of the shell color segregation will be discussed in a separate article. The marker Blf176 was linked to shell color, as we described earlier. Hence, the marker Blf176 can be isolated, sequenced, and converted into sequence characterized amplified regions in the additional study. This would provide a good opportunity for studying shell color genetics and marker-assisted selection.
The scallops used in the study were reared at the Marine Biology Research Station at Daya Bay, Chinese Academy of Sciences, Shenzhen, China.
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TAG YUAN, (1,2) MAOXIAN HE, (1) *. LIANGMIN HUANG (1) AND JIANXING HU (1)
(1) LAMB, LMB, South China Sea Institute of Oceanology, Chinese Academy of Science, 164 West Xingang Road, Guangzhou 510301, Guangdong Province, China; (2) Graduate University of Chinese Academy of Sciences, Beijing 100049, China
* Corresponding author: E-mail: email@example.com
TABLE 1. The mapped AFLP markers (before slashes), the total segre gating markers (after slashes), and the distorted segregating makers (in the parentheses) in female and male Chlamys nobilis. Primer Combination EcoRI MseI Maternal Paternal AAC(A) CAA(l) 1/8 (0) 1/10 (1) AAC(A) CAC(2) 3/8 (0) 8/9 (0) AAC(A) CAG(3) 0/10 (3) 0/6 (1) AAC(A) CAT(4) 7/9 (1) 5/7 (1) AAC(A) CTC(6) 2/7 (0) 2/8 (0) AAC(A) CTG(7) 3/6 (1) 1/4 (1) AAG(B) CAA(1) 0/3 (0) 0/2 (0) ACA(C) CAG(3) 1/6 (2) 1/3 (0) ACA(C) CAT(4) 0/7 (2) 4/10 (3) ACA(C) CTA(5) 3/5 (1) 5/6 (0) ACA(C) CTC(6) 6/7 (1) 6/7 (1) ACC(D) CAG(3) 1/8 (4) 3/6 (0) ACC(D) CTC(6) 9/12 (1) 5/13 (3) ACT(F) CAA(1) 9/15 (3) 8/13 (1) ACT(F) CAC(2) 6/10 (1) 9/15 (3) ACT(F) CAG(3) 10/15 (1) 2/7 (0) ACT(F) CAT(4) 6/8 (1) 3/9 (1) ACT(F) CTC(6) 7/7 (2) 5/5 (0) ACT(F) CTG(7) 3/6 (1) 5/8 (0) ACT(F) CTT(8) 2/2 (0) 1/1 (0) AGC(G) CAA(1) 7/12 (2) 10/11 (0) AGC(G) CAC(2) 5/8 (0) 10/11 (0) AGC(G) CTA(5) 1/1 (0) 4/7 (2) AGC(G) CTC(6) 7/9 (0) 1/3 (1) AGC(G) CTG(7) 1/1 (0) 1/2 (0) AGG(H) CAT(4) 15/19 (3) 6/12 (5) Total 115/209 (30) 106/195 (24) TABLE 2. Length, number of markers, average spacing, and largest interval of linkage groups in the female and male parent maps of Chlamys nobilis. Female Linkage Map Length Average Interval Linkage Group (CM) No. of Markers (CM) 1 174.3 14 13.4 2 160.9 10 17.9 3 155.0 11 15.5 4 138.3 9 17.3 5 130.8 8 18.7 6 128.3 9 16.0 7 117.4 7 19.6 8 112.0 6 22.4 9 108.0 9 13.5 10 106.6 6 21.3 11 96.1 7 16.0 12 84.8 5 21.2 13 78.8 6 15.8 14 62.1 4 20.7 15 40.2 4 13.4 16 5.0 2 5.0 Total 1,698.6 117 16.8 Male Linkage Map Length Average Interval Linkage Group (CM) No. of Markers (CM) 1 167.7 15 11.9 2 151.9 10 15.2 3 140.3 11 14.0 4 126.6 8 18.1 5 124.4 7 20.8 6 105.8 7 17.6 7 101.3 7 16.9 8 89.3 7 14.9 9 83.9 5 21.0 10 83.3 5 20.8 11 82.6 7 13.8 12 81.9 5 20.5 13 69.7 4 23.2 14 54.9 4 18.3 15 50.4 4 12.6 16 5.0 2 5.0 Total 1,519.0 108 16.5
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|Title Annotation:||amplified fragment length polymorphisms|
|Author:||Yuan, Tao; He, Maoxian; Huang, Liangmin; Hu, Jianxing|
|Publication:||Journal of Shellfish Research|
|Date:||Apr 1, 2010|
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