Genetic Mapping of Laminaria japonica and L. longissima Using Amplified Fragment Length Polymorphism Markers in a “Two-Way Pseudo-Testcross” Strategy

Journal of Integrative Plant Biology 2007, 49 (3): 392−400

Received 15 Mar. 2006 Accepted 16 Sept. 2006

Supported by the NSFC projects (40376049, 30500383), the Knowledge

Innovation Program of the Chinese Academy of Sciences (D. L. Duan) and

the Shandong Agricultural Seedstocks Project.

*Author for correspondence.

Tel: +86 (0)532 8289 8556;

Fax: +86 (0)532 8289 8556;

E-mail: <[email protected]>.

© 2007 Institute of Botany, the Chinese Academy of Sciences

doi: 10.1111/j.1672-9072.2006.00397.x

Genetic Mapping of Laminaria japonica and L. longissimaUsing Amplified Fragment Length Polymorphism

Markers in a “Two-Way Pseudo-Testcross” Strategy

Yuhui Li1, 2, Yingxia Yang3, Jidong Liu2, Xiuliang Wang2, Tianxiang Gao1 and Delin Duan2*

(1Ocean University of China, Qingdao 266003, China;2Institute of Oceanology, the Chinese Academy of Sciences, Qingdao 266071, China;

3Ludong University, Yantai 264025, China)

Abstract

With a ‘’two-way pseudo-testcross’’ mapping strategy, we applied the amplified fragment length polymorphism(AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps weregenerated from the 60 progenies of the F1 cross family (Laminaria longissima Aresch. × L. japonica Miyabe) withtwenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in bothparents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were orderedin 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of thematernal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies betweentwo parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkagegroup. The linkage data developed in these maps will be useful for detecting loci-controlling commercially impor-tant traits for Laminaria.

Key words: amplified fragment length polymorphism makers; genetic linkage map; Laminaria japonica; Laminaria longissima; segre-gation ratio; two-way pseudo-testcross strategy.

Li Y, Yang Y, Liu J, Wang X, Gao T, Duan D (2007). Genetic mapping of Laminaria japonica and L. longissima using amplified fragmentlength polymorphism markers in a “two-way pseudo-testcross” strategy. J. Integr. Plant Biol. 49(3), 392−400.

Available online at www.blackwell-synergy.com/links/toc/jipb, www.jipb.net

Laminaria is one of the most important seaweeds cultivatedin China. Nowadays its production has reached annually about900 000 000 kg of dry and 13 000 000 kg of algins, rankingChina first in the world (Tseng 2001). The life history of

Laminaria is divided into two heterogeneous phases, the hap-loid gametophytes generation and the diploid sporophytesgeneration. The gametophyte generation goes though threestages of growth and development from the liberation of thezoospores to the formation of zygotes. The sporophyte gen-eration is subdivided into four different morphological stages:the sporeling stage, the young sporophyte stage, the robustsporophyte stage and the mature sporophyte stage (Tseng1958). Since the 1970s, male and female Laminaria gameto-phytes were isolated and cultured separately, and variousLaminaria varieties with specific phenotype characteristicswere obtained by crossbreeding with different male and fe-male gametophytes, for example, Haiqing No.1 and Laminaria“901” strain (Fang et al. 1962b; Zhang et al. 2001).

Previously, based on the statistical data for Laminariahybridization, many genetic characters, such as frond length,

Genetic Mapping of Laminaria species 393

stipe length and frond thickness, were believed to be con-trolled and accumulated by multi-loci (Fang et al. 1962a, 1965;Fang 1983; Zhang and Fang 1980). Wang et al. (2004, 2005b)conducted assessments of genetic diversities for Laminariagametophytes with random amplified polymorphic DNA (RAPD),inter simple sequence repeats (ISSR) markers. Xia et al. (2005)conducted the genetic study of the Laminaria “901” strain withRAPD markers. However, so far no genetic linkage map ofLaminaria has been constructed, let alone genetic loci corre-lated to specific characters of Laminaria. In order to under-stand the genetic behavior during hybridization, and to locatethe dominant loci related with important traits, which providepotential for Laminaria genetic improvement, it is needed toconstruct genetic linkage maps of Laminaria.

Genetic linkage mapping is a key field in genome research,and plays an important role in gene position, gene clone, fur-ther genome organization and function analysis. It gives waysto resolve the questions associated with cellular, developmen-tal and evolutionary processes, and has been successfullyapplied to genetic improvement in plant breeding (Danin-Poleget al. 2002; Hurtado et al. 2002). Most linkage maps in plantshave been obtained from segregating populations of F2 andBC1 (back-cross) progenies, but such populations are gener-ally unavailable in highly heterozygous organisms, for example,trees. The “two-way pseudo-testcross” strategy makes it pos-sible to generate single individual linkage maps quickly with thepopulations of F1 progenies for highly heterozygous organisms(Grattapaglia and Sederoff 1994). In a cross between het-erozygous parents, many single-dose polymorphic loci will beheterozygous in one parent and null in the other, so they seg-regate 1:1 in the progenies as in a testcross. Based on thisassumption, F1 progeny populations can be regarded as test-cross populations for constructing genetic linkage maps withthe loci segregated at 1:1 in their progenies. So far, geneticmaps of various plants have been constructed with this strategy,for example, eucalypts (Eucalyptus spp.) (Grattapaglia andSederoff 1994), apple (Malus spp.) (Hemmat et al. 1994), oilpalm (Elaeis guineensis Jacq.) (Moretzsohn et al. 2000), kiwi-fruit (Actinidia spp.) (Testolin et al. 2001), pineapple (Ananasspp.) (Carlier et al. 2004) and rhodesgrass (Chloris spp.) (Ubiet al. 2004).

The amplified fragment length polymorphism (AFLP) assay, avalid method to identify a large number of polymorphic loci with-out any prior knowledge of DNA sequences in an organism,has been widely used for genetic fingerprinting, genome map-ping and genetic variability studies (Castiglioni et al. 1999; Shimand Jorgensen 2000; Sun et al. 2005). Since a large number ofloci are amplified and analyzed in high-resolution sequencinggels, this method has been available to construct high-densitymaps for individual families with a small number of oligonucle-otide primers and minimal amounts of DNA.

The goals of this study were to probe the genetic mechanism

in sexual reproduction of Laminaria with AFLP makers, and togenerate their genetic linkage maps with the “two-way pseudo-testcross” strategy. It will be used to identify loci correlatedwith important commercial traits of Laminaria in the future.

Results

Segregation analysis

Each of the twenty pairs of primer combinations with two orthree selective nucleotides could yield clear, unambiguous elec-trophoresis results displaying polymorphic at a special locus(Figure 1). A total of 333 polymorphic loci were scored from the60 individuals (Table 1), and an average of 17 polymorphic lociwas detected per primer. The polymorphic loci patterns yieldedby the twenty primer combinations are quite different. The codefor each primer set is given in Table 1 and corresponds to thenames of mapped loci on the genomic maps (Figures 2, 3). Ofthe 333 polymorphic loci, 173 segregated in a 1:1 ratio, whichmeans the corresponding DNA loci are heterozygous in a single

Figure 1. Electrophoresis patterns for the amplified fragment lengthpolymorphism (AFLP)products with primers combination A8 on 8%polyacrylamide gel.

p, paternal specie Laminaria japonica; m, maternal specie L.longissima; 1–30, 30 F1 progenies individuals from the cross familyof L. longissima× L. japonica. M, pUC18 DNA/MspI molecular marker.Segregating informative AFLP loci are indicated by arrows: “a” indi-cates loci segregated 1:1 in F1 progenies and “b” indicates locisegregated 3:1 in F1 progenies.

394 Journal of Integrative Plant Biology Vol. 49 No. 3 2007

parent, the other 58 loci existing in both parents followed a 3:1Mendelian segregation ratio, and the remained loci (30.6%) failedto follow either a 1:1 or a 3:1 ratio (Table 2). Of the 173 locisegregating 1:1, the heterozygous loci from the male and fe-male parents were 91 and 82, respectively.

The 173 loci segregating 1:1 were used to construct the

parental maps, representing 91 (male) and 82 (female) specificloci to each parent. Fourteen linkage groups (LG) were ob-tained for the male and female maps (Figures 2, 3) respectively.A summary of statistics for the maps is indicated in Table 3. Forthe paternal map, the total map distance is 648.6 cM, and 12unlinked loci (13.2%) were detected. For the maternal map, thetotal map distance is 601.9 cM, and there were 10 unlinked loci(12.2%).

Detection of homologies between the two parentalgenetic linkage maps

Marker loci being heterozygous in both parents should exhibit a3:1 segregation ratio. To identify homologies between the pa-rental linkage maps, we included 58 loci segregating 3:1 in theanalysis. Among them, 12 loci were tightly linked with existing1:1 segregating loci in either parental map (LOD = 3.0, h < 0.40).However, only seven loci were fixed on the two maps; amongthem, six loci were linked into LG5 of the paternal map and LG2of the maternal map with similar orientation, which indicatedpotential homology between the two linkage groups. The otherone was simply linked to single 1:1 segregating loci, so it cannot be regarded as a reference marker.

Discussion

Choice of parents in hybridization is important for genetic link-age mapping, which will directly determine whether geneticlinkage mapping is easy or not and the constructed geneticlinkage map is effective or not. If the parents are close in phy-logenies relation, the proportion of polymorphism loci from thehybrid progenies will be too low to construct high-density ge-netic linkage maps. Therefore, we selected two distant Lami-naria in phylogenies relation, L. japonica and L. longissima, asparents for hybridization. Obvious morphological and geneticdifferences between L. japonica and L. longissima (Wang etal. 2004) made them be suitable hybridization materials for high-density genetic linkage mapping. Considering maternal inherit-ance of length and width in Laminaria (Jiang et al. 1991), wechose L. longissima with prominent length character as a fe-male parent for potential loci localization of this trait. In addition,selection of population is crucial for the construction of high-density genetic linkage maps. To Laminaria with a relativelylonger life span and time consuming for obtaining F2 or BC1

progenies, it exhibited high heterozygosis through year-roundself-hybridization (Fang 1983), which made their F1 progeniesbe usable for the genetic linkage mapping in the “two-waypseudo-testcross” strategy.

Generally, the large mapping populations could yield morecreditable results. The reasons for choosing 60 sporophytes

Table 1. Code of the tested primer combinations and numbers ofpolymorphic amplified fragment length polymorphism (AFLP) am-plification products generated with 20 different primer combina-tions on the 60 individuals.Code Selective primer combinations Ntp

a Npsb Nnps

c

A1 P+AA/M+CAC 20 14 6A2 P+AA/M+CTA 12 11 1A3 P+AT/M+CAT 10 6 4A4 P+AT/M+CAG 12 6 6A5 P+AT/M+CTA 14 13 1A6 P+AT/M+CTT 9 8 1A7 P+TT/M+CAA 22 16 6A8 P+TT/M+CAC 31 18 13A9 P+TT/M+CTA 18 11 7A10 P+TT/M+CTT 16 8 8A11 P+AC/M+CTA 16 15 1A12 P+TG/M+CTA 23 13 10A13 P+AAC/M+CAC 23 17 6A14 P+AAC/M+CTA 14 10 4A15 P+AAC/M+CTG 22 14 8A16 P+ACA/M+CTA 13 3 10A17 P+ACC/M+CAC 16 15 1A18 P+ACC/M+CTA 14 13 1A19 P+ACG/M+CTA 13 10 3A20 P+AGG/M+CTA 15 10 5Total 333 231 102Percent 69.4 30.6aNtp represents total number of polymorphic AFLP amplificationproducts generated with different primer combinations on the 60individuals; bNps and cNnps respectively represents number of AFLPamplification products which are polymorphic or not in parents andsegregated in progenies of F1.

Table 2. Average number of segregation types for amplified frag-ment length polymorphism (AFLP) amplification products over theprimer combinations testedTotal loci Loci Loci Loci showingscored segregating segregating segregation-ratio

1:1* 3:1* distortion333 173 58 102100% 52.0% 17.4% 30.6%*Significant at p = 0.05 level.


for the analysis were that it required over 30 individuals for alldominant alleles’ detection probability reaching 95% (Sjögrenand Wyöni 1994). Because the sporophytes of L. japonica

were highly heterozygous (Fang 1983), more segregation lociwould be detected even with fewer samples of F1 progenies.In addition, time and costs were another consideration in our

Figure 2. Laminaria japonica (male) linkage map.

For each linkage group, the marker distance (cM) is shown on the left of the group, and the marker names indicated on the right.


study. With the 60 progenies applied, the 333 polymorphismAFLP loci were yielded with the 20 selective primer combina-tions and 69.4% of them followed the Mendelian inherence andsegregation ratios. This indicated our sample selection couldreflect the genotype inheritance in the F1 progenies.

With the 173 loci segregated 1:1, we obtained 14 linkage

groups for the male and female maps respectively, which wereless than the reported haploid chromosome numbers of 30 forL. longissima (Funano 1980) and 16–32 for L. japonica (Funano1980; Yabu and Yashui 1991; Lewis et al. 1993). The propor-tions of unlinked loci were 13.2% and 12.2%, respectively forL. japonica and L. longissima. This is probably due to the small

Figure 3. Laminaria longissima (female) linkage map.

For each linkage group, the marker distance (cM) is shown on the left of the group, and the marker names indicated on the right.


size of the mapping population, which is one of the main causesfor a large number of loci unlinked (Liu 1998). In addition, thehigh number of Laminaria chromosomes (2n = 32–64) and therelatively low number of usable loci in genetic linkage mappingwill result in differences between the number of linkage groupsand that of the haploid chromosome. It is expected that high-density linkage maps be constructed with the F2 progenies,which can be supplements to the maps constructed with F1

progenies.It is crucial to choose a valid method for genetic linkage

mapping. Comparing with other molecular marker systems,AFLP is reliable, stable, rapid and reproducible (Meksem et al.1995; Vos et al. 1995). Our study indicated that AFLP detectionyielded higher polymorphism (16.5 loci per primer combination)than RAPD (1.7 loci per primer; data not showed) and ISSR (0.2 loci per primer; data not showed). Restriction endonucleasedigestion is a key step of AFLP analysis. Whether the genomicDNA was digested completely or not will directly affect thefollowed reactions. EcoR I and Mse I are commonly appliedrestriction endonucleases in AFLP analysis for plants. However,in our study, among the six kinds of tested restriction endonu-cleases of Mse I, EcoR I, Pst I, BamH I, Hind III, and Pvu II, onlyMse I and Pst I could digest the extracted DNA completely, theothers could only partially or not digest the extracted DNA at all.This was consistent with our previous work (Wang et al. 2005a),so we finally selected endonucleases of Pst I and Mse I todigest the extracted Laminaria genomic DNA.

The proportion of skewed loci occupied about 30.6% for

Table 3. Summary statistics for the linkage map of a cross family (F1

from Laminaria longissima × L. japonica) Male Female

Total number of loci scored 218 217Number of loci mapped(linkage to at least one other marker) 79 72Linkage groups 14 14Average number of loci on one genetic group 5.6 5.1Minimum number of loci on one genetic group 2 2Maximum number of loci on one genetic group 12 10Average marker distance (cM) 8.2 8.4Minimum length of linkage group (cM) 10.5 10.5Maximum length of linkage group (cM) 96.2 79.4Total genome coverage (cM) 648.6 601.9

both Laminaria species, which were higher than that of com-monly estimated values for other plants (Jenczewski et al. 1997).There are several factors influencing segregation distortion,such as close linkage of loci to genes or chromosomal regionsaffecting gametogenesis, fertilization or embryogenesis (Abeand Tsuda 1988), genetic drift and natural selection (Jung et al.1996), chromosomal rearrangements (Gebhardt et al. 1991;Kianian and Quiros 1992), and loci sampling error or low popu-lation size (Lu et al. 1998). The mechanism of segregation dis-tortion in Laminaria needs to be explored in future.

Materials and Methods

Materials

The parent strains applied in the hybridization experiments wereLaminaria longissima and L. japonica, which are from Insti-tute of Oceanology, Chinese Academy of Sciences (Table 4).The hybridization experiments were conducted according tothe procedures: Laminaria gametophytes were gently grindedin a mortar to produce suspensions of male and female fila-ments 1–10 cells. Then the suspensions were equally (maleand female) poured into a beaker containing seedling coiledrope under the bottom. The incubation was processed at 10ºC, 40 μmol.m−2.s−1 and the culture mediums were refreshedevery week. When the juvenile sporophytes grew into 1 cmlong in 2 months, the Laminaria seedlings were transferredand hung on the cultivating rope on a raft in the sea. The 60sporophytes of F1 cross family (L. longissima × L. japonica)were collected when they grew to 7–12 cm in length.

DNA extraction

Sporophytes genomic DNA of F1 progenies and their parentswere extracted with a MiniBEST Plant Genomic DNA Extractionkit (Takara Biotechnology, Dalian, China) according to themanufacturer’s instructions.

AFLP analysis

Amplified fragment length polymorphism protocol wasadapted from the edition described by Vos et al. (1995).About 50 ng DNA was digested with 5U Pst I, 5U Mse I in 15

Table 4. Gametophytes applied in the experimentsGametophyte Sporophytic characteristicsLaminaria longissima (female) Little flourishing holdfast, thin and long stipe and dark brown blade with no vertical channel and

narrower edge.L. japonica (male) Flourishing holdfast, dark brown narrower blade with obvious vertical channel and circular base.


μL restriction-ligation buffer (10 nmol/L Tris-HAc (pH 7.5), 10mmol/L MgAc, 50 mmol/L KAc, 5 mmol/L DTT, and 50 ng/μLBSA) (NEB) at 37 °C for 12 h. After restriction digestion, 50pmol of MseI oligonucleotide adapter, 5 pmol of PstI oligonucle-otide adapter (Table 5) (Sangon, co. Shanghai), 0.2 mmol/LATP, and 1U T4 DNA-ligase (TaKaRa Biotechnology, Dalian) in10 μL restriction-ligase buffer were added to the template DNAand incubated at 20 °C for 6 h. Then 5μL template DNA wasmixed with 30 ng of preamplification primers which are comple-mentary to the PstI and MseI adapters (P+0 and M+0 primers,Table 5), 0.2 mmol/L dNTPs, and 1U Taq DNA polymerase(Promega, USA) in 20 μL 1×AFLP buffer (10 mmol/L Tris–HClpH 8.0, 1.5 mmol/L MgCl2, and 50 mmol/L KCl ). The PCR proce-dure for preamplification consisted of 30 cycles of 30 s at94 °C, 30 s at 56 °C, and 1 min at 72 °C. Preamplification prod-ucts were diluted 20× and 1 μL dilution was used as templateDNA for selective amplification. The selective amplification wasconducted using primers with two or three selective nucle-otides (Table 1) (Sangon). Finally, the products of selectiveamplification were separated on 8% polyacrylamide gels anddetected with silver staining method (Xu et al. 2002).

Scoring and segregation analysis of AFLP loci

Segregating AFLP loci were obtained by visual scoring analy-sis from the gels with “1” representing presence and “0” repre-senting absence of the amplified bands. The parental origin ofthe locus was also recorded. Two separate data sets wereobtained, one for each parent. It presumed that the genotypesof parents with strip in one locus were indicated as “AA” or“Aa”, while absent in the locus as “aa”. Fitness of observed-to-expected Mendelian allelic ratios on all scored loci was ana-lyzed using χ2 test (p = 0.05).

Linkage analysis and construction of genetic maps

Loci without significant variation from Mendelian ratios at the α= 0.05 level were used in the linkage analyses. Only heterozy-gous genetic loci presented in a single parent and segregated

1:1 in F1 progenies were used for constructing separate ge-netic linkage map in a two-way pseudo-testcross strategy.

Data were processed with the software Mapmaker/Exp3.0(Whitehead Institute, ftp://ftp-genome.wi.mit.edu/distribution/software/mapmaker3/, F2 backcross model) (Lander et al. 1987).The data set was duplicated and recoded to allow the detec-tion of linkage of loci in the repulsion phase. Initially, the resultsof AFLP loci were transformed to the letters according to therequirement of the software Mapmaker/Exp3.0 with “A” forrecessive loci “aa”, “H” for heterozygous loci “Aa” and “-” forambiguous or absent data. The linkage threshold for groupingloci was set as the following: the LOD score was 5.0 and themaximum recombination fraction (h) was lower to 0.25, and thegroups were speculated by implementing the “group” command.Then the relatively less stringent criteria (LOD = 3.0 and h = 0.40) were applied to test whether or not there were any addi-tional loci which could be mapped to the speculated groups.The most likely loci orders with a significance value (p = 0.00001) were determined by implementing “compare” and “try”commands. The framework of linkage groups was establishedwith multipoint mapping functions by implementing the “map”command. After mapping, error was detected by the “errordetection” command. Map distance in centiMorgans was cal-culated using Haldane’s mapping function. Double recombina-tion events were examined in each linkage group to ensure thatthere was no potential genotyping error for data process. AFLPloci were named using codes of Mse I/Pst I selective primercombinations and the approximate size (bp) determined by apUC18 DNA/Msp I molecular marker.

Acknowledgements

We are grateful to Dr. Yehao Liu (Nanjing University) for helpand suggestions in this study.

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Documents

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