AFLP linkage map of sea urchin constructed using an interspecific cross between Strongylocentrotus nudus (♀) and S. intermedius (♂)

Aquaculture 259 (2006) 56 – 65 www.elsevier.com/locate/aqua-online

AFLP linkage map of sea urchin constructed using an interspecific cross between Strongylocentrotus nudus (♀) and S. intermedius (♂) Zunchun Zhou a,b , Zhenmin Bao a,⁎, Ying Dong b , Shi Wang a , Chongbo He b , Weidong Liu b , Limei Wang b , Feng Zhu a a

b

College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China Key Lab of Molecular biology, Open Lab of Applied Marine Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian 116023, China Received 9 January 2006; received in revised form 10 May 2006; accepted 10 May 2006

Abstract The interspecific cross between Strongylocentrotus nudus (♀) and S. intermedius (♂) holds significant heterosis in growth and disease resistance. For the purposes of genetic studies and breeding, AFLP linkage maps were constructed for both S. nudus (♀) and S. intermedius (♂) using a hybrid population with a two-way pseudotestcross strategy. Forty-two selective amplification primer combinations produced 897 polymorphic AFLP bands that were polymorphic in either of the parents and segregated in the progeny. Twenty four linkage groups of S. nudus contained 194 AFLP markers, covering a total length of 2988.3 cM, with an average marker spacing of 17.1 cM. In S. intermedius, 199 AFLP markers were mapped in 23 linkage groups, covering a total length of 2614.8 cM, with an average of 15.4 cM between markers. The expected map length and coverage were estimated to be 3827.5 cM and 78.1% for S. nudus and 3307.8 cM and 79.1% for S. intermedius respectively. © 2006 Elsevier B.V. All rights reserved. Keywords: Strongylocentrotus nudus; Strongylocentrotus intermedius; Genetic linkage map; AFLP; Interspecific cross

1. Introduction Since the 1990s, sea urchin cultivation has expanded rapidly due to the increased consumption of their gonads and played an important role in aquaculture. In China, two main species are used in culture; Strongylocentrotus nudus, a native species in China, can be cultured mainly in the northern areas, while S. intermedius, which was introduced into China from Japan in 1989, can be cultured more extensively along the northern coast of China. The repro⁎ Corresponding author. Tel./fax: +86 532 82031960. E-mail address: [email protected] (Z. Bao). 0044-8486/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2006.05.010

ductive seasons of these two species overlap in September, making artificial hybridization convenient. The earliest hybridization was tried 30 years ago (Osanai, 1974) and the characters of the hybrids at the larval stages were analyzed (Masayoshi and Osanai, 1994). Wang et al. (2003, 2004a) found that the first generation (F1) of S. intermedius (♀) ×S. nudus (♂) was highly vigorous in growth and disease resistance. Our studies showed that F1 of S. nudus (♀) ×S. intermedius (♂) was more vigorous than that of S. intermedius (♀) ×S. nudus (♂) in practice, which implies that the hybrid of S. nudus (♀) ×S. intermedius (♂) has the potential of being cultivated. The heterosis between other sea urchins has also been reported (Rahman et al., 2005).

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Although sea urchin has been widely used as a model organism for developmental biological studies, researches concerning the molecular biological bases of these genetic phenomena are scarce in the literature. As a powerful molecular marker system (Vos et al., 1995; Maughan et al., 1996; Maheswaran et al., 1997), amplified fragment length polymorphisms (AFLP) has been widely used in the construction of linkage maps of many important aquaculture species such as fish (Young et al., 1998; Kocher et al., 1998; Agresti et al., 2000; Liu et al., 2003; Coimbra et al., 2003; Ezaz et al., 2004; Watanabe et al., 2004), shrimp (Wilson et al., 2002; Li et al., 2003; Pérez et al., 2004), oysters (Yu and Guo, 2003, 2004; Li and Guo, 2004), scallops (Wang et al., 2004b, 2005; Li et al., 2005). The goal of this study was to construct AFLP linkage maps of S. nudus and S. intermedius. Currently, sea urchin inbred lines are not available. The two-way pseudotestcross strategy, which is based on the fact that an F1 population produced by crossing between two heterozygous parents will cause segregation, has been successfully applied to construct linkage maps of many trees (Grattapaglia and Sederoff, 1994; Hemmat et al., 1994; Wu et al., 2000; Cervera et al., 2001). Based on such a strategy, we performed the linkage mappings of S. nudus and S. intermedius. This study was an initial step toward understanding the genetic basis of heterosis, and should lay the foundation for studies of quantitative traits and markerassisted selection. 2. Materials and methods 2.1. Animals F1 progenies were derived from a controlled hybridization between a female parent of S. nudus and a male one of S. intermedius. The parents were collected in Dalian Island, Liaoning Province, China. Sixty F1 progenies, six months old, were obtained with their gonads stored at −70 °C. 2.2. DNA extraction and AFLP analysis Total DNA of F1 progenies and their parents was extracted using DNA Extracting Kit (TaKaRa Inc. Dalian, China). AFLP analysis was carried out essentially as described by Vos et al. (1995). DNA was double digested with EcoRI and MseI and then ligated to adaptors. Pre-amplification was carried out using two adaptor corresponding primers each overhanging a single selective base. After being diluted 20-fold, the pre-amplification product was used as the template of selective amplification. The primers used in selective amplification each have two additional

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selective bases. Of 60 primer combinations screened using two parents and two progenies, 42 were selected for the construction of AFLP linkage maps. The product of selective amplification was separated through 4.5% denaturing polyacrylamide gel electrophoresis at 60 W constant power for 1.5 h. The bands were visualized using silver staining (Bassam et al., 1991). Clear and unambiguous bands in length ranging from 50 to 1500 bp were considered as usable. Bands presenting AA or Aa genotypes were scored as “1” and those presenting aa genotype (absent) as “0”. Unreliable bands were considered as missing and recorded as “–”. The names of AFLP markers contained primer information, where E and two numbers following it referred to EcoRI primer, M and two following numbers to MseI primer. Markers were numbered serially in descending order of fragment length; thus the last two or three numbers of AFLP marker name referred to the relative position of its corresponding AFLP band on the gel. 2.3. Segregation analysis Two types of loci were used for segregation analysis: one reflected that the genotypes of two parents were Aa and aa, and the F1 progenies segregated in 1:1 ratio; and the other reflected that the genotypes of two parents were Aa and Aa, and F1 progenies segregated in 3:1 ratio. All segregation loci were checked with χ2 test (P = 0.05) for goodness-of-fit of the observed with expected Mendelian ratios. Theoretically, two types of loci should have a binomial distribution (Van der Lee et al., 1997). The probability of band presence in progenies was 0.5 for the first type of loci, and 0.75 for the second. In this study only the first type of loci was used for linkage map construction. 2.4. Map construction Linkage map was constructed using the two-way pseudo-testcross strategy (Grattapaglia and Sederoff, 1994). Markers segregated according to Mendelian 1:1 ratios (P N 0.05) and some distorted (0.01 b P b 0.05) were used in linkage analysis. Linkage analysis was performed using software MAPMAKER/EXP3.0 (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 avoided map expansion (Cervera et al., 2001). A minimum logarithm of odds ratio (LOD) score of 3.0 and a maximum recombination fraction of 0.3 were set as the linkage threshold for grouping markers. Small groups (≤8 markers) were ordered with multipoint exhaustive analysis using the “compare” command, while large groups were processed with the “compare”, “build” and “try”

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Table 1 Numbers of the polymorphic AFLP bands and the total (before and after slash) amplified by each primer combination Primer

M47 M00CAA

E32 E00- 32/91 AAC E33 E00- 26/78 AAG E35 E00- 34/91 ACA E38 E00- 36/101 ACT E39 E00- 30/116 AGA E44 E00- 23/89 ATC Mean 30/94 Total 181/566

M48 M00CAC

M49 M00CAG

M50 M00CAT

M59 M00CTA

M60 M00CTC

M61 M00CTG

Mean

Total

27/62

34/95

37/119

28/90

25/72

33/97

31/89

216/626

33/99

29/67

35/91

23/70

34/101

36/121

31/90

216/627

33/75

33/120

34/95

24/71

22/70

31/110

30/90

211/632

31/83

33/96

18/57

37/97

28/96

25/86

30/88

208/616

37/126

27/85

45/123

38/85

27/94

35/99

34/104

239/728

33/122

31/77

31/109

32/75

18/93

30/93

28/94

198/658

32/94 194/567

31/90 187/540

33/99 200/594

30/81 182/488

26/88 154/526

31/101 190/606

31/93 1288/3887

command. The resulting linkage order was checked using the “ripple” command. Once the framework linkage groups were established, the relatively less stringent criteria (LOD ≥ 2.0) 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 (Ott, 1999). Linkage groups were assigned in descending size. The map was drawn with the MAPCHART2.1 program (Voorrips, 2001).

ter (Saal and Wricke, 2002). 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 and Guo, 2003). The t-testing was applied to check the significance of correlation coefficient at P = 0.01 level.

2.5. Map length and coverage

Among the 3887 bands obtained, 1288 were polymorphic, accounting for 33.3% of the total (Table 1). Each primer combination yielded 57–126 bands in length of 50 to 1500 bp. Polymorphism level detected by different primer combination ranged from 25.8% (E44M47) to 44.7% (E39M59). On average, each primer combination produced 30 bands appropriate for serving as markers.

Estimated genome length was calculated by multiplying the length of each linkage group by (m + 1) / (m − 1), where m was the number of framework markers in each group (Ge1), or by adding 2 s to the length of each linkage group to account for chromosome ends (Ge2) (Chakravarti et al., 1991; Fishman et al., 2001). The average of the two estimated genome lengths (Ge) was used to describe the genome length. Observed genome length was calculated as the total length considering all markers (Goa). The observed genome coverage Coa was obtained by dividing Goa with Ge. 2.6. Distribution of AFLP markers Under the hypothesis that AFLP markers are randomly distributed over a linkage group, Poisson distribution function (P(x) = e−μμx / x!) is assumed with the average marker number per 10 cM interval (μ) as the expected mean (Young et al., 1999). 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 clus-

3. Results 3.1. Polymorphism level

3.2. Segregation analysis Of 1288 polymorphic bands or markers accordingly, 391 segregated in both parents, of which 169 (43.2%) Table 2 Segregating AFLP markers and distorted (in parenthesis) in a F1 hybrid cross between S. nudus (♀) and S. intermedius (♂) Female Segregating markers Number of markers in linkage analysis Mapped markers Eliminated markers Proportion of mapping markers

Male a

451 (189 ) 324 (62) 194 (44) 17 43.0%

446 (163) 339 (56) 199 (39) 8 44.6%

Significantly distorted markers (P b 0.01) were excluded from linkage analysis. a

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Fig. 1. Genetic linkage map of the female, S. nudus. Distorted markers were marked by asterisks. Three doublets were excluded.

deviated from the expected Mendelian 3:1 ratios (P b 0.05); the remaining 897 segregated in either the female or the male parent. Of the 897 polymorphic markers, 451 segregated through the female parent, of which

189 (41.9%) deviated from the expected Mendelian 1:1 ratios (P b 0.05); and 446 segregated through the male parent, of which 163 (36.6%) showed segregation distortion (P b 0.05).

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Table 3 Length, number of markers and marker intervals of linkage groups of the female S nudus linkage map Group

Length (cM)

No. of markers

Average interval (cM)

Maximum interval (cM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Total

311.4 290.1 274.4 233.8 213.1 195.2 177.2 170.0 164.5 122.5 94.8 119.5 140.6 126.1 93.6 48.6 48.0 46.6 31.4 27.6 25.4 15.7 12.8 5.4 2988.3

21 22 15 11 15 9 8 12 8 6 8 6 10 9 6 3 4 4 3 4 4 2 2 2 194

15.6 13.8 19.6 23.4 15.2 24.4 25.3 15.5 23.5 24.5 13.5 23.9 15.6 15.8 18.7 24.3 16.0 15.5 15.7 9.2 8.5 15.7 12.8 5.4 17.1a

31.3 27.2 32.2 29.7 24.3 33.6 32.7 32.2 33.4 28.3 22.2 35.5 31.1 27.8 34.2 33.4 35.3 29.3 22.7 16.7 11.4 15.7 12.8 5.4

a

Average of all markers.

Among all the distorted markers that were segregating in either of the parents, 133 (37.8%) showed heterozygote (Aa) deficiency, and 219 (62.2%) showed homozygote (aa) deficiency. For the distorted markers that were segregating in both parents, 108 (63.9%) showed homozygote (aa) deficiency. The observed distribution of segregation ratios (1:1 or 3:1) did not resemble the expected at P = 0.05 level, and for all the distorted markers, significant homozygote (aa) deficiency was found compared with the expected distribution. 3.3. Linkage map construction Based on female and male segregating data, two linkage maps were constructed: one for the female S. nudus parent, and the other for the male S. intermedius parent. Among 451 markers segregating through the female, 262 segregated in 1:1 ratios (P N 0.05) and 62 distorted (0.01 b P b 0.05) were used in linkage analysis (Table 2). In total, 194 markers were mapped in the female linkage map. Twenty four linkage groups covered 2988.3 cM in length, with a maximum interval of 35.5 cM and an average interval of 17.1 cM (Fig. 1; Table 3). The length of the

linkage groups ranged from 5.4 to 311.4 cM and the number of markers varied from 2 to 22 per group. The markers used in female linkage map construction included also 113 unlinked and 17 eliminated because they spanned very long map distances. Among 446 markers segregating through the male parent, 283 segregated in 1:1 ratios (P N 0.05) and 56 distorted (0.01 b P b 0.05) were used in linkage analysis. The male map consisted of 199 markers clustered into 23 linkage groups that covered 2614.8 cM in length. The maximum interval found was 34.8 cM. The average interval was 15.4 cM (Fig. 2; Table 4). The lengths of linkage groups ranged from 18.7 to 268.8 cM, and the number of markers varied from 3 to 15 per group. The markers used contained also 132 unlinked and 8 eliminated markers. 3.4. Map length and coverage The genome length estimated using two methods were 3811.1 cM and 3843.9 cM respectively for the female, and 3324.1 cM and 3291.5 cM respectively for the male (Table 5). Two estimates were similar each other. The average of the two estimates, 3827.5 cM for the female and 3307.8 cM for the male, was used as the expected genome length. Accordingly, the genome coverage of the female and male framework maps was 78.1% and 79.1% respectively. 3.5. Distribution of AFLP markers Some marker clusters were found in the linkage maps of both parents. Four clusters located on linkage group 2, group 3, group 8 and group14 of the female map, whereas ten clusters located on linkage group 2, group 3, group 7, group 8, group 11, group 14, group 15, group 19, group 21 and group22 of the male map. AFLP marker distribution in these regions deviated significantly from Poisson distribution (P = 0.01). The Pearson correlation coefficient analysis (female: r = 0.94, t = 5.95 N t0.01; male: r = 0.85, t = 9.39 N t0.01) exhibited highly positive correlation between the size of the linkage group and the number of AFLP markers in linkage group. On average, the proportions of markers employed for mapping were 43.0% (female) and 44.6% (male) respectively. Most primer combinations generated 4–14 usable markers. In comparison, E35M48, E35M59 and E44M59 generated few informative markers for mapping. The numbers of mapped markers of different primer combinations ranged from 1 to 18. Markers generated by any primer combination that were clustered on only one linkage group or in only one region were not found.

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Fig. 2. Genetic linkage map of the male, S. intermedius. Distorted markers were marked by asterisks.

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4. Discussion

Table 5 Length and genome coverage of female and male linkage maps

4.1. Polymorphism level of AFLP markers

Map length (cM)

The efficiency of constructing a genetic linkage map depends on the heterozygosity level of markers in a species. Relatively high polymorphism level of AFLP markers were detected in F1 progenies (33.3%) as might be expected from an interspecies cross, and from the heterozygosity level of the parent sea urchin genomes. High heterozygosity level has been demonstrated by different studies on sea urchin. The genome of S. purpuratus owns very high sequence polymorphism, which exceeds those reported for any other metazoan so far (Cameron et al., 1999). High heterozygosity level was also revealed in sea urchin based on different molecular markers (Cameron et al., 1999; Addison and Hart, 2004; Miller et al., 2004). Most of the 42 primer combinations not only detected high polymorphism, but also yielded a large number of polymorphic bands. Significant correlation (r= 0.63, t= 27.17 N t0.01) was found between the total number of bands and the number of polymorphic bands in all primer combinations used, which made it easier to select primer combinations for obtaining highly informative markers. Table 4 Length, number of markers and marker intervals of linkage groups of the male S. intermedius linkage map Group

Length (cM)

No. of markers

Average interval (cM)

Maximum interval (cM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Total

268.8 199.3 177.6 177.2 169.6 159.1 144.9 123.9 123.5 116.1 111.7 107.3 92.1 82 77.9 76.7 74.5 70.8 68 62.8 60.5 51.8 18.7 2614.8

13 13 15 14 10 12 11 11 10 7 12 8 5 10 8 4 4 6 5 5 6 7 3 199

22.4 16.6 12.7 13.6 18.8 14.5 14.5 12.4 13.7 19.4 10.2 15.3 23.0 9.1 11.1 25.6 24.8 14.2 17.0 15.7 12.1 8.6 9.4 15.4a

33.1 31.4 32.1 33.8 34.1 32.8 33.4 28.7 32.3 28.6 20.7 33.3 32.6 34.8 27.2 27.3 27.1 23.9 23.2 30.8 19.1 19.5 10.3

a

Average of all markers.

Observed Estimated length length Goa Ge1 Ge2

S. nudus (female) 2988.3 S. intermedius 2614.8 (male)

Average Genome Ge coverage (%) Coa

3811.1 3843.9 3827.5 3324.1 3291.5 3307.8

78.07 79.05

Goa, observed length (cM); Ge1 and Ge2, estimated lengths using 2 methods; Ge, average; Coa, genome coverage (%).

4.2. Segregation distortion Segregation distortion was found in about 40.5% of polymorphic bands in the F1 progenies (41.9% in female and 36.6% in male respectively when considering 1:1 segregated bands only). Deviations from Mendelian segregation have been reported in constructing AFLP linkage maps of other species, for example, channel catfish (16%) (Liu et al., 2003), oyster (8.2%) (Yu and Guo, 2003), Chlamys farreri (35.24% and 17.8%) (Wang et al., 2004b; Li et al., 2005), Brassica oleracea (64%) (Voorrips et al., 1997). In order to test if the small sample size (60 individuals) resulted in high segregation distortion, 4 primer combinations (E32M47, E33M49, E38M50 and E44M59) were used to analyze 40 additional individuals. The markers from 100 individuals also showed homozygote (aa) deficiency, illustrating that the high segregation distortion may not result from the limited number of mapping individuals. In oysters, a high level of segregating distortion was well explained by high genetic load and selection against the gametes with homozygous deleterious recessive mutations and the deleterious recessive lethal genes resulted in the homozygote deficiency (Launey and Hedgecock, 2001; Yu and Guo, 2003; Li and Guo, 2004). Similar results were also reported in scallop (Wang et al., 2004b; Li et al., 2005) and tilapia (Palti et al., 2002). In this study, the distribution of 1:1 and 3:1 segregating markers observed did not resemble the expected distribution. Significant homozygote (aa) deficiency suggested that high distortion may be caused by deleterious recessive lethal genes. Distorted markers tended to form clusters on linkage maps in many organisms like Pacific oyster (Li and Guo, 2004), scallop (Wang et al., 2004b; Li et al., 2005) and oak (Barreneche et al., 1998). In this study, some clusters formed by distorted markers were also found; one was on group 8 of female map and three on group 3, group 7 and group 14 of male map respectively. Some studies indicated that clustered distorted markers may link to deleterious genes. Mapping skewed

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markers may have little effect on estimating recombination frequency (Hackett and Broadfoot, 2003). However, it can provide useful information for genetic research and breeding of S. nudus, S. intermedius and their hybrid. Therefore, these markers were included in our final genetic maps. 4.3. Genetic mapping of AFLP in S. nudus and S. intermedius The haploid genome of S. nudus and S. intermedius contains 21 chromosomes (Saotome, 1987, 1989). Our female genetic map was composed of 24 linkage groups. If three doublets were excluded, the number (21) of female linkage groups would be the same as the number of haploid chromosomes. The male genetic map was composed of 23 linkage groups. Nonequivalence between the number of linkage groups and the number of chromosomes were also reported in other studies (Young et al., 1998; Vivek and Simon, 1999). It is difficult to construct linkage maps in which the number of linkage groups is consistent with that of haploid chromosomes (Yasukochi, 1998), especially when the total number of markers and the coverage are not large enough. Our AFLP maps were not particularly dense; the average intervals were 17.1 cM (female) and 15.4 cM (male). Only about 43.0% markers in female and 44.6% in male segregated in Mendelian patterns were assigned to our maps. High proportions of unmapped markers were also reported in the construction of linkage maps of many species (Grattapaglia and Sederoff, 1994; Roussot et al., 2003; Wang et al., 2004b). We expect that the small linkage groups and unmapped markers will be merged into larger linkage groups when more markers are included.

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AFLP markers was relatively even in both female and male maps. The distribution of AFLP markers was more even in the female map than that in the male map; only four clusters were observed in the female map, while 10 were found in the male map. AFLP marker clustering is common and has been observed in many organisms, such as rainbow trout (Young et al., 1998), tilapia (Agresti et al., 2000) and channel catfish (Liu et al., 2003). Analysis of informative marker distribution among primer combinations indicated that not all primer combinations produced informative polymorphic markers for mapping. This suggested that primer combinations should be screened ahead of AFLP map construction so that highly informative markers were found and used. 4.6. Applicability of AFLPs in sea urchin AFLP markers have the potential of efficiently and rapidly constructing high-resolution maps, which can be used in the identification of markers closely linked with QTL of economically important traits (such as growth rate and disease resistance). It was expected that greater successes will be achieved in the near future in the QTL mapping of economically important species, which in turn will promote marker-assisted selection (Liu and Cordes, 2004). In addition, comparison of genetic maps of different species can provide insights into genome structure evolution (Hohmann et al., 1995). However, AFLP markers, especially the gel-based, are difficult to transfer among laboratories and populations, and this limits the extension of the AFLP maps' application. Currently, we are developing microsatellite markers of S. nudus and S. intermedius which will be integrated to present linkage maps in the near future.

4.4. Map length and coverage

Acknowledgments

This study provided estimated genetic map length for sea urchin S. nudus (female) and S. intermedius (male) for the first time. The male map length was 373.5 cM shorter than the female. Because two parents used came from different species, the difference in map lengths may reflect the difference in recombination rate between the two species and two sexes. The map coverage of 78.1% (S. nudus) and 79.1% (S. intermedius) could be improved by the integration of female and male maps and the addition of additional genetic markers.

We would like to thank Prof. Guanpin Yang for reviewing this manuscript. This work is mainly supported by Natural Science Foundation of Liaoning Province (No. 20042127) and National Nature Science Foundation of China (No. 30571410).

4.5. Distribution of AFLP markers Based on the correlation between the number of AFLP markers and the size of linkage groups, the distribution of

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