AFLP linkage map of an intraspecific cross in Chlamys farreri.
KEY WORDS: Chlamys farreri, genetic linkage map, AFLP, intraspecific cross, segregation distortion
Genetic linkage maps have become powerful research tools in genetic studies of many species (Saliba-Colombani et al. 2000, Wu et al. 2000, Tan et al. 2001). A saturated linkage map can efficiently carry out molecular-based analyses such as molecular marker-assisted selection tRance et al. 2001, Kelly et al. 2003), quantitative trait loci (QTL) mapping (Linde et al. 2001, Chen et al. 2001, Mignouna et al. 2002), genetic basis of heterosis (Jones et al. 2003, Hua et al. 2003), and comprehensive investigations of genome evolution (Naruse et al. 2000, Doganlar et al. 2002, Slate et al. 2002).
Although there have been many techniques such as RFLP, RAPD, and SSR available for constructing genetic linkage maps, their applications are limited for various reasons. Amplified fragment length polymorphism (AFLP) is a recently developed marker system that combines the advantages of different marker systems and provides a new opportunity for mapping species with large, but less understood genomes. It can detect a large number of genetic loci per reaction and thus can obtain more genetic information than RFLP and RAPD analysis.
AFLP markers have been widely applied to construct the linkage maps in a variety of economically important species, such as rice (Virk et al. 1998, Zhu et al. 1999), rye (Saal & Wricke 2002), soybean (Keim et al. 1997), chicken (Herbergs et al. 1999, Knorr et al. 1999), silkworm (Tan et al. 2001), tea (Hackett et al. 2000), coffee (Ky et al. 2000), fish (Young et al. 1998, Coimbra et al. 2003), oyster (Yu & Guo 2003), and shrimp (Wilson et al. 2002, Li et al. 2003). AFLP markers have also been successfully applied to important genes mapping such an the scab resistance Vf gene in apple (Xu & Korban 2000), the Lr19 gene for resistance to leaf rust in wheat (Prins et al. 2001), the rhm gene for resistance to Southern Corn Leaf Blight in maize (Cai et al. 2003), the powdery mildew resistance gene Run1 in grapevine (Pauquet et al. 2001), and the fertility restorer gene rf4 in sorghum (Wen et al. 2002).
Chlamys farreri belongs to Mollusca, Lamellibranchia, Pterimorphia, Pterioodae, and Pectinidae and distributes mainly in the northern China, the western Korea, and Japan. It is a diploid with 38 chromosomes (Wang et al. 1990) and is one of the most important aquaculture species in China. It is important to understand its genome organization by means of molecular markers for genetic studies and breeding purposes. However, till now, a linkage map of C. farreri has not been reported. The purpose of this study in to build the first AFLP-based linkage map of C. farreri.
MATERIALS AND METHODS
The mapping population of 51 F1 progeny was derived from a pair mating of C. farreri. The parents were sampled from a wild population around Changdao Island, Shandong Province, China. The muscle tissues of the parents were scissored off and stored at -20[degrees]C. The F1 progenies were sampled at the swimming trochophore larvae stage (about twenty hours after fertilization) and stored in ethanol at 4[degrees]C.
The parental DNA was extracted from frozen muscle tissues with phenol/chloroform extraction as described by Sambrook et al. (1989). The F1 larvae were individually collected in PCR tube through micro-operation and then lysed in 10-[micro]L sterile water for about 5 hours to serve as the DNA solution.
AFLP analysis was carried out essentially as described by Vos et al. (1995) with some minor modifications, such as reducing the dosage of the reagents, for AFLP procedure in F1 individuals. The DNA samples were digested with EcoRI and MseI, then ligated to restriction site-specific adaptors. Preamplification was carried out using adaptor-specific primers with no selective base on each primer. The preamplification product was diluted (20-fold for parents and 10-fold for progeny) and then used in selective amplification. The selective amplification used the primers with three selective bases on each primer. Totally, 21 primer combinations were selected for AFLP analysis.
The products of the selective amplification were separated by polyacrylamide gel electrophoresis at 60 W for 1.5 hours and the bands were detected by silver staining. The electrophoretic images were scanned and then saved in a computer for further analysis.
Scoring of Data and Maker Nomenclature
In preliminary study we found that the bands ranged 50-1500 bp, had good reproducibility, and could unambiguously scored, so only these bands were scored in this study. Two persons scored all these bands independently. The consistent data were used for linkage analysis and scored as dominant markers. Bands present were scored as "1" and absent as "0". Unreliable bands were scored as missing "-".
The AFLP marker names were refereed to the primers used: E followed by two numbers refers to the EcoRI primer and M followed by two numbers to the MseI primer. Bands were numbered serially in descending order of fragment length; thus the last two numbers of the AFLP marker code refer to the relative position of the band on the gel.
The inheritance patterns of AFLP bands from parents to offspring in a diploid organism are shown in Table 1. Loci segregating in type 1, 3, 7, and 9 are not informative for mapping since all progeny are identical. Type 2 and 4 can only be scored on the basis of band intensities and were thus excluded due to their low reliability. Only type 5, 6, and 8 containing locus segregation information in the F1 progeny can be used for segregation analysis.
For each marker of all 3 types (type 5, type 6, and type 8), goodness-of fit of observed-to-expected allelic ratios was analyzed with [chi square] test ([alpha] = 0.05). Theoretically all three types should have a binomial distribution (Van der Lee et al. 1997). For type 6 and type 8 markers the probability of band presence in progeny is 0.5 and for type 5 markers, the probability is 0.75. The distribution of the segregation ratios in F1 progeny was used to compare with the expected. The goodness-of-fit of observed-to-expected distribution was also analyzed with [chi square] test ([alpha] = 0.05).
Type 6 and 8 markers were used to construct separate genetic linkage maps for female and male parents with the software program MAPMAKER/EXP (Whitehead Institute, F2 backcross model). The "error detection" feature was used to recognize the circumstance when an event was more probably the result of error than recombination. This feature avoids map expansion (Cervera et al. 2001). A LOD score of 3.0 and maximum recombination fraction [theta] = 0.30 were initially set as the linkage threshold for grouping markers. Groups were then analyzed with multipoint mapping functions to define the most likely map orders. Markers that did not significantly depart from Mendelian ratios at [alpha] = 0.05 level were used in the grouping analysis. Once the framework linkage groups were established, the relatively less stringent criteria (LOD = 2.5 and [theta] = 0.30) were applied to test whether there were any additional markers or distorted markers that could be mapped to the framework map. Map distance in CentiMorgans was calculated with Kosambi's mapping function. Linkage groups were drawn with the MAPCHART2.1 program.
Map Length and Coverage
Two methods were used to calculate the estimated genome length. First, we calculated the average marker spacing/interval(s) by dividing the total map length by the number of intervals (number of markers minus number of linkage groups). The estimated genome length ([G.sub.e1]) was determined by adding 2s to the length of each linkage group to account for chromosome ends (Fishman et al. 2001). Second, an estimated genuine length ([G.sub.e2]) was calculated by multiplying the length of each linkage group by (m+1)/ (m-1), where m is the number of framework markers in each group (Chakravarti et al. 1991). Lastly, the average of the two estimates was used as the estimated genome length (Ge) for C. farreri. Observed genuine lengths were calculated as the total length considering all markers (Goa). The observed genome coverage Coc was determined by Goa/Ge.
Distribution of AFLP Markers
The distribution of AFLP markers was analyzed from 3 aspects: First, under the hypothesis that AFLP markers are randomly distributed over a linkage group, a Poisson distribution function was assumed with the average marker number per 10 cM interval ([mu]) as the expected mean (Saal & Wricke 2002). Any interval on a specific chromosome, where the observed number of AFLP markers exceeded the 99% quantile of the cumulative distribution function, was considered an AFLP cluster.
Second, the AFLP marker distribution was also analyzed by calculating the Pearson correlation coefficient between the number of AFLP markers in the linkage groups and the size of the linkage groups (Yu & Guo 2003). Then t-test was applied to test the significance of correlation coefficient at [alpha] = 0.01 level.
Lastly, the mapped AFLP markers were classified according to primer combination for analyzing its distribution among the 21 primer combinations used.
Polymorphism Level of AFLP Markers in C. farreri
In total, 1244 bands have been detected, of which 783 were polymorphic, that is, 62.94% polymorphic markers. The polymorphism level of each primer combination is listed in Table 2. Depending on the different primer combinations, the number of bands was counted from 42 to 84 in the range of 50 to 1500 bp. Levels of polymorphism individually ranged from 27.69% for E33M61 to 78.85% for E42M58. A typical amplification profile by E33M58 is shown in Figure 1. For all primer combinations, significant correlation between the total number of bands and the number of polymorphic bands could be observed (r = 0.68, t = 4.04 > [t.sub.0.01]).
[FIGURE 1 OMITTED]
In the 783 polymorphic markers detected, 257 belonged to type 5, of which 190 segregated in 3:1 ratio and 67 showed segregation distortion ([alpha] = 0.05). The other 526 belonged to type 6 or 8, that is, were segregated in either the male or the female parent. For the male parent, 165 segregated in a 1:1 ratio showing 37.5% segregation distortion, whereas 152 segregated in a 1:1 ratio showing 41.98% segregation distortion for the female parent.
The distribution of the segregation ratios observed in F1 progeny did not resemble the expected distribution ([alpha] = 0.05) (Fig. 2). The tendency of 1:1 segregation markers' distribution is similar to the expected distribution. However, compared with the expected distribution, the distribution of 1:1 segregation markers showed homozygote excess in the left and heterozygote excess in the right. For the distribution of 3:1 segregation markers, it shows significant allozygote deficiency compared with the expected distribution.
[FIGURE 2 OMITTED]
Linkage Map Construction
Two linkage maps were constructed: one for the male parent, the other for the female parent. The male map consisted of 94 markers in 19 linkage groups that covered 1511.4 cM in length, with a maximum interval of 36.9 cM and an average interval of 20.15 cM (Fig. 3; Table 3). The length of the linkage groups ranged from 3.9 to 265.4 cM and the number of markers varied from 2 to 15 per group.
[FIGURE 3 OMITTED]
The female map consisted of 97 markers in 20 linkage groups that covered 1610.2 cM in length, with a maximum interval of 36.9 cM and an average interval of 20.91 cM (Fig. 4; Table 4). The length of the linkage groups ranged from 12.0 to 316.0 cM, and the number of markers varied from 2 to 14 per group.
[FIGURE 4 OMITTED]
Map Length and Coverage
As shown in Table 5, the genome lengths estimated by two methods were similar, being 2264.21 cM and 2277.1 cM respectively in male, whereas 2428.81 cM and 2446.6 cM respectively in female. The average of the 2 estimates, 2270.66 cM for male and 2437.7 cM for female, was used as the expected genome length. On the basis of the expected genome lengths, genuine coverages of the male and female framework maps were 66.56% and 66.05%, respectively.
Distribution of AFLP Markers
Three clusters were found: two being located on linkage group 2 in male map and one on linkage group 6 in female map. AFLP marker distribution in these regions deviated significantly from the Poisson distribution (P = 0.01). The Pearson correlation coefficient analysis (Male: r = 0.94, t = 11.26 > [t.sub.0.01], Female: r = 0.99, t - 27.13 > [t.sub.0.01]) exhibited highly positive correlation between the size of the linkage group and the number of AFLP markers in the linkage group.
The distributions of the AFLP mapping markers among 21 primer combinations were shown in Table 6. The average proportions of markers used fur mapping were 35.61% (male) and 37.02% (female), respectively. Most primer combinations generated 5-12 markers that were useful for mapping; but E32M54 and E33M61 generated poorly informative markers for mapping. The numbers of mapped markers among all primer combinations ranged from 2 to 20. Markers generated by any primer combination that were clustered on only one linkage group or in only one region were not found.
Polymorphism Level of AFLP Markers in C. farreri
The efficiency of constructing a genetic linkage map depends on the heterozygosity level of markers in a species. For C. farreri, its genome should have relatively high heterozygosity because of its mix-mating reproduction system in the nature. In this study, the high polymorphism level (62.94%) as revealed by AFLP markers in the F1 progeny of C. farreri reflected high heterozygosity level of the parent genome. The polymorphism level in C. farreri based on RAPD markers and isozyme markers is 75.82% and 41.18%, respectively (Song et al. 2002, Li et al. 2001). These results indicate that the heterozygosity level of C. farreri is relatively high.
Most of the 21 primer combinations could not only give high polymorphism ratios, but also produce a large number of polymorphic bands. Significant correlation (r = 0.68, t = 4.04 > [t.sub.0.01]) was found between the total number of bands and the number of polymorphic bands for all primer combinations in the present study, which made it easier to select primer combinations for obtaining highly informative markers.
In this study, segregation distortion ratio is about 35.24% when considering all polymorphic bands in F1 progeny. Segregation distortion ratio is 37.50% (male) and 41.98% (female) when considering 1:1 segregation pattern in either male parent or female parent. Deviations from Mendelian segregation have been reported in constructing genetic linkage maps with molecular markers: 25% of RFLP markers in the soybean (Deshui et al. 1997), and 40% of RAPD markers in the Medicago (Jenczewski et al. 1997). Distorted segregation ratios of AFLP markers were also observed: 8.2% in the oyster (Yu & Guo 2003), 14% in the melon (Wang et al. 1997), 32% in the coffee (Ky et al. 2000), 56% in the silkworm (Tan et al. 2001), and 65% in the clubroot (Voorrips et al. 1997). Distorted segregation of molecular markers may result from sampling in finite mapping populations, preferential fertilization, breaking of DNA chains during extraction of DNA samples from tissues, or amplification of a single-sized fragment derived from several different regions (Tan et al. 2001). The above explanations cannot account for the relatively high-distorted segregation in this study. Deviation from Mendelian segregation ratios in bivalves grow more severe as progeny age (Gaffney & Stott 1984), which may result from natural selection against recessive deleterious genes (Launey & Hedgecock 2001). The distribution of 1:1 and 3:1 segregation markers observed in C. farreri in this study did not resemble the expected distribution (see Fig. 2) and the tact of significant allozygote deficiency support the hypothesis. Genetic map also provides an efficient approach to investigate the cause of segregation distortion. In this study, the skewed markers assembled together in specific regions of only a few linkage groups, which nut only corresponds with the hypothesis but also implies that the specific regions may contain genes influencing the development of the early larvae. Mapping the skewed markers may have little effect on estimating the recombination frequency (Hackett & Broadfoot 2003) but can provide useful information for genetic research and breeding in C. farreri, so our final genetic maps include these markers.
Genetic Mapping of AFLPs in C. farreri
The haploid genome of C. farreri contains 19 chromosomes (Wang et al. 1990). Our male genetic map is composed of 19 linkage groups and female genetic map is composed of 20 linkage groups. Nonequivalence between the number of linkage groups and the number of chromosomes has also been reported in other studies (Young et al 1998, Vivek & Simon 1999). It is difficult to construct maps without missing some chromosomes for species with large number of chromosomes in haploid genomes (Yasuko chi 1998), especially when the total number of markers and the coverage are not large enough.
Our AFLP map with an average of 20.15 cM (male) and 20.91 cM (female) per interval was not particularly dense. About 64.39% markers in male and 62.98% in female that show Mendelian segregation were not linked in our maps. Higher proportions of unlinked markers were observed in mapping studies in other species (Grattapaglia & Sederoff 1994). However, these markers may be linked when more markers obtained from other AFLP, RFLP, RAPD, and microsatellite analyses were used to construct denser maps.
Map Length and Coverage
The study provides estimated genetic map length for male and female of C. farreri for the first. The male map length is 98.8 cM shorter than the female map length, which may reflect sex-specific recombination rates in C. farreri. Similar differences have been noted in aquatic animals including rainbow trout (Sakamoto et al. 2000), zebrafish (Knapik et al. 1998), and Pacific oyster (Yu & Guo 2003). Several possible explanations, such as time spent in meiotic prophase in one sex, transcriptional activity of certain genes during meiosis in one sex, and presence of sequences that are recognized by sex-specific enzymes (Coimbra et al 2003), have been applied to explaining the phenomenon. So far, the genetic mechanism of suppression in recombination is not yet clear, and there is no adequate explanation for the difference in recombination rate between the two sexes in C. farreri.
The map coverage in the C. farreri was 66.56% in male and 66.05% in female. The current map coverage is not high compared with other species. To obtain a saturated genetic map in C. farreri, effective resolution is not only to integrate the male and female maps but also to add genetic markers obtained from RFLP, RAPD, EST, and microsatellite analyses as well as isozyme loci into current genetic map.
Distribution of AFLP Markers
The distribution of AFLP markers is relatively even in chromosomes of male and female map, because only few of clusters were observed. The number of AFLP markers in the linkage groups that had high positive correlation with the size of the link age groups also supports this conclusion. In published literature, AFLP markers distribute randomly in some species (Castiglioni et al. 1999, Remington et al. 1999, Cervera et al. 2001), but they form cluster in others (Young et al. 1998, Waldbieser et al. 2001, Sakamoto et al. 2000). Some studies suggest the presence of AT rich sequence blocks, which are more likely to be recognized by EcoRI and MseI in connection with suppressed recombination even though such distinct AT-rich regions are not in the centromeric and pericentromeric parts.
Analysis of the distributions of informative markers among primer combinations indicated that neither all primers nor all primer combinations could produce informative polymorphic markers for mapping (see Table 6). This suggests that, similar to the RAPD technique, AFLP analysis of the whole genome such as performed here need to search for primer combinations with highly informative markers.
Applicability of AFLPs in C. farreri Genetics and Breeding
Genomic mapping provides a new approach to the identification of genes that control commercially important traits (Dudley 1993). AFLPs have the potential to efficiently and rapidly construct high-resolution maps, which will make gene isolation more convenient. The identification and isolation of specific markers closely linked with desirable traits is necessary for marker-assisted breeding. Also, the comparison of genetic maps from different species or genera can provide insights into animal evolution and genome structure (Hohmann et al. 1995).
However, AFLP markers, especially the gel-based, are difficult to transfer among laboratories and populations, which limits the extension of the AFLP maps' application. In some cases, poor transferability can be compensated for by the ease of developing a large set of AFLP markers in any population of interest, thus limiting the need for transfer (Yu & Guo 2003). Microsatellite markers are better markers for linkage mapping than AFLP markers, because of high levels of polymorphism, codominance, and good transferability. At present, our laboratory is developing a set of microsatellite markers in C. farreri. In the near future, these microsatellite markers will be contained in the linkage maps, which will enhance the transferability of the current maps.
TABLE 1. Inheritance of AFLP fragments in the F1 progeny of diploid organisms. AFLP Phenotypes Expected Type Genetic Model Ratio Parents 1 AAxAA[right arrow]AA 1:0 -- -- -- -- -- 2 AAxAa[right arrow]AA,Aa 1:0 -- -- -- -- -- 3 AAxaa[right arrow]Aa 1:0 -- -- -- -- 4 AaxAA[right arrow]AA,Aa 1:0 -- -- -- -- -- 5 AaxAa[right arrow]AA,Aa,aa 3:1 -- -- -- -- -- 6 Aaxaa[right arrow]Aa,aa 1:1 -- -- -- -- 7 aaxAA[right arrow]Aa 1:0 -- -- -- -- 8 aaxAa[right arrow]Aa,aa 1:1 -- -- -- -- 9 aaxaa[right arrow]aa 0:1 AFLP Phenotypes Type Genetic Model Offspring 1 AAxAA[right arrow]AA -- -- -- -- -- 2 AAxAa[right arrow]AA,Aa -- -- -- -- -- 3 AAxaa[right arrow]Aa -- -- -- -- -- 4 AaxAA[right arrow]AA,Aa -- -- -- -- -- 5 AaxAa[right arrow]AA,Aa,aa -- -- -- 6 Aaxaa[right arrow]Aa,aa -- 7 aaxAA[right arrow]Aa -- -- -- -- -- 8 aaxAa[right arrow]Aa,aa -- 9 aaxaa[right arrow]aa TABLE 2. AFLP polymorphism of an intraspecific cross in C. farreri. EcoRI MseI Approximate Primer Primer Primer Number of Combination (5'-3') (5'-3') Bands (n) E32M49 E00 (1) +AAC M00 (2) +CAG 83 E32M54 E00+AAC M00+CCT 50 E32M55 E00+AAC M00+CGA 54 E32M58 E00+AAC M00+CGT 70 E32M61 E00+AAC M00+CTG 45 E32M48 E00+AAG M00+CAC 55 E32M58 E00+AAG M00+CGT 52 E32M61 E00+AAG M00+CTG 65 E35M55 E00+AAC M00+CGA 46 E35M61 E00+AAC M00+CTG 70 E38M48 E00+ACT M00+CAC 84 E38M58 E00+ACT M00+CGT 64 E38M61 E00+ACT M00+CTG 82 E39M48 E00+AGA M00+CAC 58 E42M54 E00+AGT M00+CCT 43 E42M58 E00+AGT M00+CGT 52 E42M61 E00+AGT M00+CTG 55 E44M61 E00+ATC M00+CTG 42 E45M55 E00+ATG M00+CGA 54 E45M58 E00+ATG M00+CGT 63 E45M61 E00+ATG M00+CTG 57 Total -- -- 1244 Average -- -- 59 Primer Polymorphic Polymorphic Combination Bands (n) Bands (%) E32M49 58 69.88 E32M54 31 62.00 E32M55 34 62.96 E32M58 47 67.14 E32M61 33 73.33 E32M48 44 80.00 E32M58 27 51.92 E32M61 18 27.69 E35M55 21 45.65 E35M61 25 35.71 E38M48 60 71.43 E38M58 45 70.31 E38M61 51 62.20 E39M48 44 75.86 E42M54 31 72.09 E42M58 41 78.85 E42M61 29 52.73 E44M61 25 59.52 E45M55 36 66.67 E45M58 41 65.08 E45M61 42 73.68 Total 783 -- Average 37 63.08 (1) E00, 5'-GACTGCGTACCAATTC-3' (2) M00, 5'-GATGAGTCCTGAGTAA-3' TABLE 3. Length, number of markers, average spacing. and largest intervals of linkage groups in the male of C. farreri. Linkage Length Number of Average Largest Group (cM) Markers Interval (cM) Interval (cM) 1 265.4 15 18.96 36.9 2 93.2 9 11.65 25.5 3 176.8 8 25.26 33.7 4 137.2 7 22.87 30.8 5 93.0 6 18.6 23.1 6 103.9 5 25.98 30.8 7 78.9 5 19.73 30.8 8 86.5 5 21.63 30.8 9 40.3 4 13.43 16.2 10 67.6 4 22.53 29.7 11 61.8 4 20.6 25.5 12 78.3 4 26.1 28.1 13 47.0 3 23.5 28.1 14 49.2 3 24.6 26.1 15 44.9 3 22.45 30.8 16 56.4 3 28.2 30.8 17 14.1 2 14.1 14.1 18 3.9 2 3.9 3.9 19 12.0 2 12.0 12.0 Total 1511.4 94 20.15 (a) (a) Average of all linked markers. TABLE 4. Length, number of markers, average spacing, and largest intervals of linkage groups in the female of C. farreri. Linkage Length Number of Average Largest Group (cM) Markers Interval (cM) Interval (cM) 1 316.0 14 24.31 30.8 2 241.5 12 21.95 36.9 3 184.1 10 20.46 25.5 4 133.3 8 19.04 30.8 5 133.2 7 22.2 28.1 6 79.0 6 15.8 25.7 7 73.1 5 18.28 30.8 8 49.2 4 16.4 23.1 9 60.2 4 20.07 24.6 10 51.6 3 25.8 30.8 11 32.7 3 16.35 20.7 12 39.2 3 19.6 20.7 13 44.4 3 22.2 28.1 14 51.7 3 25.85 26.1 15 25.5 2 25.5 25.5 16 12.0 2 12.0 12.0 17 18.4 2 18.4 18.4 18 14.1 2 14.1 14.1 19 25.5 2 25.5 25.5 20 25.5 2 25.5 25.5 Total 1610.2 97 20.91 (a) (a) The symbols are the same as those in Table 3. TABLE 5. Map length and genome coverage for C. farreri. Map Length (cM) Male Female Observed length [G.sub.oa] 1511.4 1610.2 Estimate length [G.sub.e1] 2277.1 2446.6 [G.sub.e2] 2264.21 2428.81 Average [G.sub.e] 2270.66 2437.7 Genome coverage (%) [C.sub.oa] 66.56 66.05 TABLE 6. The distribution of the AFLP mapping markers among primer combinations. Male Female Primer Combinations A (a) B (b) A/B (%) A (a) B (b) A/B (%) E32M49 7 22 31.82 5 21 23.81 E32M54 2 10 20.00 0 12 0.00 E32M55 4 12 33.33 7 14 50.00 E32M58 5 17 29.41 3 11 27.27 E32M61 3 12 25.00 3 5 60.00 E33M48 3 16 18.75 9 18 50.00 E33M58 4 6 66.67 4 10 40.00 E33M61 0 2 0.00 2 6 33.33 E35M55 5 8 62.50 0 6 0.00 E35M61 3 10 30.00 4 9 44.44 E38M48 12 23 52.17 8 21 38.10 E38M58 4 12 33.33 6 15 40.00 E38M61 5 21 23.81 2 12 16.67 E39M48 3 11 27.27 4 13 30.77 E42M54 9 11 81.82 5 11 45.45 E42M58 3 11 27.27 7 15 46.67 E42M61 4 13 30.77 1 8 12.50 E44M61 3 11 27.27 1 6 16.67 F45M55 3 9 33.33 7 15 46.67 E45M58 8 17 47.06 10 18 55.56 E45M61 4 10 40.00 9 15 60.00 (a) A is the number of mapping markers. (b) B is the number of markers segregating in male or female parent.
The authors thank Prof. Guanpin Yang and Dr. Jingjie Hu for reviewing this manuscript. This work is mainly supported by grants 2002AA628l10 and 2003AA603022 from Hi-Tech Research and Development Program of China; G1999012009 from "973" Program of China; 30300268 from National Natural Science Foundation of China; 01BS10 from Outstanding Scientists Research Fundation of Shandong Province.
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SHI WANG, ZHENMIN BAO, * JIE PAN, LINGLING ZHANG, BING YAO, AIBIN ZHAN, KE BI AND QUANQI ZHANG
Laboratory of Marine Genetics and Breeding (MGB), Division of Life Science and Technology, Ocean University of China, Qingdao 266003, People's Republic of China
* Corresponding author. E-mail: email@example.com