The first intraspecific genetic linkage maps of wintersweet [Chimonanthus praecox (L.) Link] based on AFLP and ISSR markers

Scientia Horticulturae 124 (2010) 88–94

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

The first intraspecific genetic linkage maps of wintersweet [Chimonanthus praecox (L.) Link] based on AFLP and ISSR markers Dan-Wei Chen, Long-Qing Chen * Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 23 May 2009 Received in revised form 12 October 2009 Accepted 8 December 2009

Wintersweet [Chimonanthus praecox (L.) Link] (n = 11), a dichogamous species, is a famous traditional fragrant flower plant in China. We used AFLPs and ISSR to construct a genetic linkage map on a pseudo-F2 population of wintersweet derived from a cross between the individuals H4 and H29. A total of 370 polymorphism markers was generated from 12 AFLP primer combinations and 9 ISSR primers. Two separate female and male maps were constructed using 84 female-specific and 51 male-specific testcross markers, respectively. The maternal H4 map included 80 markers ordered in 12 linkage groups; while the paternal H29 map had 47 markers in 8 linkage groups. At a minimum logarithm of the odds (LOD) score of 3.0 and maximum map distance of 50 cM, the female map covered 2417.8 cM, with an average distance of 25.61 cM and maximum map distance of 48.2 cM between two loci. In contrast, the male map covered 1184.2 cM, with an average distance of 25.7 cM and maximum map distance of 49.0 cM between two loci. These genetic maps will serve as a framework for mapping QTLs and provide basic information for future molecular breeding studies. ß 2009 Published by Elsevier B.V.

Keywords: Intraspecific hybridization Pseudo-testcross Linkage map

1. Introduction Wintersweet [Chimonanthus praecox (L.) Link] belongs to the Calycanthaceae family. It is a famous traditional fragrant flower shrub, which has over 1000 years’ history of cultivation as an ornamental plant, native to southeastern China. It is a hardy, fastgrowing perennial, diploid (2n = 22) and dichogamous shrub species (Azuma et al., 2005), which blooms in winter from November to March in China. Wintersweet is mainly propagated through seeding and stem cutting, and the period from seed sowing to flowering takes 2–3 years. Due to the special flowering time and strong fragrance, Ch. praecox has been widely cultivated in China and was introduced to Korea, Japan, Europe, America, and Australia (Zhao et al., 2007). Wintersweet is generally used as garden, potted, cut-flower plants and traditional Chinese medicinal materials for the treatment of heatstroke, scald, bruise, cough and rheumatic arthritis (Zhao et al., 1993; Zhang and Liu, 1998). Furthermore, the chemicals of wintersweet flowers are rich in volatilized flavor (Zheng et al., 1990; Deng et al., 2004), being of great potential as a source of essential oil (Zhou et al., 2007). Although it is a traditional ornamental shrub, researches of

* Corresponding author. Tel.: +86 27 87281726; fax: +86 27 87282010. E-mail address: [email protected] (L.-Q. Chen). 0304-4238/$ – see front matter ß 2009 Published by Elsevier B.V. doi:10.1016/j.scienta.2009.12.008

wintersweet about genetics and breeding are limited (Zhao and Zhang, 2007). Genetic mapping provides a valuable tool in many fields of genetic studies and has been studied in some crops (Dida et al., 2007; Stallen et al., 2003; Capo-chichi et al., 2004; Ipek et al., 2005; Maughan et al., 2004) and trees (Grattapaglia and Sederoff, 1994; Arcade et al., 1996; Cervera et al., 2001; Venkateswarlu et al., 2006). A number of molecular markers have been used in genetic mapping including restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), inter-simple sequence repeat (ISSR) and simple sequence repeat (SSR). Linkage maps are also the framework for the use of genetic markers in breeding programs via marker-assisted selection (Mazur and Tingey, 1995). Most linkage maps in plants have been obtained from segregating population derived from crosses between inbred lines. However, such populations and the three-generation pedigrees are generally not available in trees due to a significant genetic load and time constraints (Grattapaglia and Sederoff, 1994). In two-way pseudotestcross mapping strategy, the F1 generation in woody plants can be assimilated to F2 or BC1 of an annual selfing crop (Tulsieram et al., 1992; Grattapaglia and Sederoff, 1994). Map construction in highly heterozygous plants can make use of informative genetic markers that segregate 1:1 for the presence or absence of a DNA fragment in F1 progeny (Ritter et al., 1990). Particularly, in the case

D.-W. Chen, L.-Q. Chen / Scientia Horticulturae 124 (2010) 88–94

of trees, genetic linkage maps would be a powerful tool for accelerating breeding through marker-assisted selection. The AFLP assay provided a new class of highly polymorphic markers for genetic fingerprinting, genome mapping and genetic variability studies (Zabeau and Vos, 1993; Vos et al., 1995; Marques et al., 1998). As a PCR-based technique, AFLPs can generate a large number of polymorphic markers without any prior knowledge of DNA sequences for the organism. Compared to other anonymous marker systems, AFLP markers detect more point mutations per reaction than RAPDs or RFLPs. AFLP markers are typically dominant, but codominant markers that vary in size and intensity can also be identified (Marques et al., 1998). AFLP fragments are related to unique positions in the genome and, hence, can be exploited as landmarks in genetic and physical mapping (Vos et al., 1995). The ISSR markers were used in mapping projects of several plant species (Venkateswarlu et al., 2006; Arcade et al., 2000) and have a higher level of polymorphism than RFLPs or RAPDs, and their developments are faster and easier than AFLP. In the present study, we used a first intraspecific pedigree to construct two genetic linkage maps for wintersweet, based on a two-way pseudo-testcross mapping strategy and AFLP, ISSR markers. These maps are the first reported genetic linkage maps for Ch. praecox. They can provide an overview of wintersweet genome structure for the development of high density genetic linkage maps, aiming to mapping quantitative trait loci (QTLs) and marker-assisted breeding.

89

each adapter having one selective nucleotide each, specifically EcoRI adaptor + A and MseI + C. Preamplification reactions were performed in a 20 mL volume containing 2.0 mL 10 PCR buffer, 1.5 mmol/L Mg2+, 0.1 mmol/L dNTP, both 40 ng EcoRI and MseI preamplified primer, 5.0 mL template DNA, and 1 U Taq DNA polymerase. PCR amplification consisted of 94 8C for 3 min and 28 cycles of 94 8C for 30 s, 56 8C for 60 s, 72 8C for 60 s. The preamplification products were diluted 30-fold and 4 mL was used in the selective amplification. Selective amplifications were conducted in 20 mL volume containing 2.0 mL 10 PCR buffer, 1.5 mmol/L Mg2+, 0.1 mmol/L dNTP, both 60 ng EcoRI and MseI primer with three selective nucleotides, 4.0 mL preamplified template DNA, and 0.6 U of Taq DNA polymerase. This selective amplification was carried out using a touch-down cycle profile (Vos et al., 1995) as follows: 94 8C for 50 s, 65 8C ( 0.7 8C/cycle) for 60 s, and 72 8C for 60 s during 13 cycles, until the optimal annealing temperature of 56 8C. Then 22 more cycles were used to complete the selective amplification with the extension step for 10 min at 72 8C. Electrophoresis was pre-run until an adequate temperature (45 8C) was reached. Approximately 3/4 (v/v) of sequence loading buffer was added to each selective amplified product and the mixture was denatured at 96 8C for 3 min and immediately chilled to 4 8C. About 7 mL mixture was loaded onto 6% (w/v) denaturing polyacrylamide DNA sequencing gel (7 M urea, 19:1 acrylamide:bisacrylamide, 0.5 TBE) and electrophoresed at 70 W for 2 h. Gel was then silver-stained following the protocol described by Zhou et al. (2007).

2. Materials and methods 2.4. ISSR protocol 2.1. Plant material A segregating mapping population of Ch. praecox (L.) Link (wintersweet) was derived from a cross between two elite unidentified cultivars (so far, there is no unique putative classification system for wintersweet) H4 and H29. H4, as the female parent, has yellow medium and inner tepals, big flower size and plate flower shape. While H29 as the male parent has yellow medium tepals and mauve inner tepals, small flower size and bell flower shape. Because of the dichogamy, wintersweet plants can be easily emasculated and pollinated by hand. The mapping population consisted of 64 F1 testcross (TC progenies) individuals. 2.2. DNA extraction Total genomic DNA for both AFLP and ISSR analyses was extracted from fresh young leaves following the cetyltrimethylammonium bromide (CTAB) procedure described by Loh et al. (1999) with minor modifications (Zhou et al., 2007). DNA concentration was estimated by the fluorescence intensities of ethidium bromide-stained samples on 0.8% agarose gels. 2.3. AFLP protocol AFLP analysis was performed according to Vos et al. (1995) with some modifications. Approximately 300 ng of genomic DNA was used for restriction digestion with EcoRI (MBI Fermantas) and MseI (New England Biolabs) and for the ligation of adapters. The digestions were carried out in a final volume of 40 mL at 37 8C for 3 h with 5 U each of EcoRI and MseI. Then all samples were heated to 65 8C for 30 min to inactivate the restriction enzymes. EcoRI and MseI adapters were subsequently ligated to the digested DNA fragments by adding 10 mL of ligation solution containing 0.5 mmol/L EcoRI, 5 mmol/L MseI adapters and 2 U T4 ligase. The ligation reactions were incubated at 4 8C overnight. A preamplification reaction was performed with primers complementary to

PCR reactions were used at 25 mL PCR solution contained 1 U of Taq DNA polymerase, with 4 ng template DNA, 0.2 mmol/L primer, 0.2 mmol/L dNTP, 2 mmol/L Mg2+, and 2.5 mL 10 PCR reaction buffer. The reaction was heated to 94 8C for 5 min and exposed to 38 cycles of 94 8C for 45 s, 51 8C for 45 s, 72 8C for 90 s, and final extension for 7 min at 72 8C. The PCR products were separated on 2% (w/v) agarose gel in 1 TAE buffer, stained with ethidium bromide, and photographed in Gel-Logic 200 image system (Eastman Kodak, Rochester, NY). 2.5. Primer screening and marker scoring ISSR, EcoRI and MseI primers were all purchased from Invitrogen. For the first screening, AFLP and ISSR were selected on the basis of the total number of bands and the level of polymorphism observed when analyzing on the two parents and five progeny individuals. Selected primers were used on the mapping population. AFLP and ISSR polymorphic markers were manually scored as numerical data corresponding to dominant parental type in two different data sets, only unambiguous markers were scored. Each segregating fragment was identified and labeled with consecutive numbers, starting from the top of the gel. They were scored as dominant and recessive markers for presence (1) and absence (0) respectively, ambiguous genotypes were resolved by assigning a blank score (–). Markers were named with the primer code, followed by a number reflecting relative position of fragment on the gel, and according to their parental origin, (f) represent male parent marker, (m) represent female parent marker, (b) represent both parents marker. 2.6. Data analysis and map construction The majority of AFLP and ISSR markers were scored in the 64 TC-progenies F1 individuals for their presence or absence in two

D.-W. Chen, L.-Q. Chen / Scientia Horticulturae 124 (2010) 88–94

90

different data sets according to their parental origin. In the pseudotestcross configuration, dominant markers should be heterozygous in one parent, absent in the other parent and are expected to segregate 1:1 in the progeny. Otherwise, marker loci that are heterozygous in both parents should yield a 3:1 segregation ratio. Each AFLP or ISSR marker was evaluated with a chi-square test of goodness-of-fit. All segregating fragments with 1:1 ratio (a = 0.05, 0.01) were used for the construction of both linkage maps and 3:1 ratio (a = 0.05, 0.01) were later integrated into the analysis to compare the female and male linkage maps. Linkage groups and map construction were accomplished using Mapmaker/Exp Version 3.0 (Lincoln et al., 1992) with the data type ‘‘F2 backcross’’ suited for the pseudo-testcross configuration. Linkage groups were determined by two-point linkage analysis with a minimum LOD score of 3.0 and maximum map distance of 50 cM. The most likely order of markers within a linkage group was determined by multipoint analysis. Distances between adjacent marker loci were calculated from recombination fractions using Kosambi’s mapping function. Finally, we used MapDraw (Liu and Meng, 2003) to draw genetic linkage maps based on given genetic linkage data. 2.7. Estimate genome length and map coverage The expected genome lengths (Ge) of the parental maps were estimated using the method No. 4 of Chakravarti et al. (1991) by multiplying the length of each linkage group a factor of (m + 1)/ (m 1), where m is the number of markers on each linkage group. G/Ge determined the observed genome coverage, where G is the length of the framework map. 3. Results 3.1. AFLP and ISSR marker segregations A total of 256 AFLP primer combinations and 38 ISSR primers were evaluated for the efficiency of amplification and detection of polymorphism based on the two parents and five progeny individuals (data not shown). Twelve AFLP primer combinations were polymorphism and highly reproducible and were used to assess the mapping population. The size of the AFLP fragments ranged from approximately 100 to 700 bp. These 12 primer combinations produced a total of 735 amplification products, of which 312 were polymorphic (Table 1). The number of polymorphic markers per primer combination varies from 15 to 48. On an average, 26 polymorphic markers were scored for each primer combination. Of the 135 markers segregating at 1:1 (a = 0.01), 51 Table 1 Numbers of AFLP amplification products generated with 12 different primer combinations. Primer combinations

Primer code

E-AAA + M-CGA E-AAA + M-CGT E-AAT + M-CTA E-AAT + M-CTC E-AAG + M-CAG E-ATT + M-CTA E-ATT + M-CAT E-ATT + M-CAC E-AGA + M-CAT E-AGG + M-CCA E-ACA + M-CAA E-ACA + M-CAT

E1M9 E1M10 E2M5 E2M8 E3M3 E6M5 E8M2 E8M4 E9M2 E11M13 E13M1 E13M2

Total Average

– –

Total bands 52 71 64 68 43 49 59 55 72 46 92 64 735 61.25

Polymorphism bands 20 48 23 23 15 15 25 24 24 26 48 21 312 26.00

Polymorphism (%) 38.46 67.61 35.94 33.82 34.88 30.61 42.37 43.64 33.33 56.52 52.17 32.81 42.45 –

Table 2 Numbers of ISSR amplification products generated with 9 different primers. Primer code

Repeat motifa

Optimal Total annealing bands temperature(8C)

Polymorphism Polymorphism bands (%)

IR1 IR3 IR5 IR7 IR9 IR11 IR15 IR17 IR19

(AG)8T (AG)8YT (AG)8GC (AG)8YA (AC)8YG (AC)8YT (GA)8YC (GA)8C (GA)8YT

56 53 55 53 55 54 53 55 55

7 9 8 6 10 3 4 5 6

Total – Average – a

– –

10 12 15 9 14 9 12 11 11

103 58 11.44 6.44

70.00 75.00 53.33 66.67 71.43 33.33 33.33 45.45 54.55 – 55.90

R: A,G; H: A,C,T; B: C,G,T; V: A,C,G; D: A,G,T; Y: C,T.

were specific to male parent H29 and 84 specific to female parent H4. Thirty markers were present in both parents and showed 3:1 segregation, and 144 markers displayed skewed segregation ratios of 36.52% (Table 2). Of the 38 ISSR primers, 9 have clear patterns at the suitable anneal temperature (Table 3). The 9 primers produced a total of 103 amplification products, of which 58 were polymorphic. Fragment size ranged from 250 to 2500 bp. 3.2. Linkage analysis A total of 165 markers were finally used to build the parental maps, including 139 AFLPs and 26 ISSRs. The female parent H4 map had a total of 80 markers distributed in 12 linkage groups (Fig. 1) with 34 markers unlinked (29.8%). This maternal framework map covers 2471.8 cM of total map distance. The number of markers per linkage group ranged from 2 to 50. Linkage distance spanned by individual linkage groups ranged from 1.6 cM (LM10) to 2066 cM (LM1). The linkage groups had an average length of 205.9 cM and the average spacing of 36.4 cM. In contrast, the male parent map involves 47 markers distributed in 8 linkage groups (Fig. 2) with 34 markers unassigned (42.0%). This paternal framework map covered 1184.2 cM of total map distance with 2–23 markers in each linkage group. Linkage distance spanned by individual linkage groups ranged from 8.5 cM (LF5) to 799.7 cM (LF8). The average length of linkage groups is 148.0 cM and the average spacing is 30.3 cM. 3.3. Map comparison Using method No. 4 of Chakravarti et al. (1991), we estimated that the total genome size of 2974.5 and 1581.9 cM for the female and male, respectively. Therefore, the coverage of female and male maps, based on these estimates genome length, was 83.1% and 74.9%, respectively. Sixty-six markers were found to be heterozygous in both parents and 30 of them segregated in a 3:1 ratio in the progeny (a = 0.01). In principle, such markers could be helpful to define homologies between linkage groups in the two maps. So, we included these markers in the analysis. Twenty markers revealed straightforward homologies between linkage group 8 (LF8) in H29 and linkage group 1 (LM1) in H4. 4. Discussion For the Ch. praecox (2n = 22), 11 LGs are expected to be mapped corresponding to the chromosomes. In this study, we obtained 8 linkage groups in the male map and 12 in the female map.

D.-W. Chen, L.-Q. Chen / Scientia Horticulturae 124 (2010) 88–94

91

Table 3 Numbers of segregation types of AFLP and ISSR amplification products. Primer code

E1M9 E1M10 E2M5 E2M8 E3M3 E6M5 E8M2 E8M4 E9M2 E11M13 E13M1 E13M2 IR1 IR3 IR5 IR7 IR9 IR11 IR15 IR17 IR19 Total

Bands segregating 1:1

Bands segregating 3:1

Bands showing segregation-ratio distortion

a = 0.05

a = 0.05

a = 0.05

a = 0.01

a = 0.01

Bands showing abnormal segregation

a = 0.01

6 17 9 8 1 8 8 4 7 10 19 7 0 2 3 1 1 1 0 1 1

6 20 9 8 3 9 8 9 8 12 25 8 0 2 3 1 1 1 0 1 1

0 1 1 1 2 0 1 0 3 0 3 1 1 3 1 3 2 0 2 1 2

0 1 1 1 2 0 0 0 3 0 4 2 1 3 2 3 2 0 2 1 2

8 12 6 13 3 4 10 7 9 11 13 3 6 0 0 3 6 1 0 3 1

8 15 6 13 5 5 10 13 10 13 20 5 6 0 1 3 6 1 0 3 1

6 15 7 1 7 2 6 7 4 3 6 8 0 4 3 0 1 1 2 0 2

114

135

28

30

119

144

85

Therefore, our linkage groups were in good agreement with (in female) or close to (in male) the haploid chromosome numbers. The genome coverage for both parents is 83.1% and 74.9%, respectively. The presence of unlinked markers also indicated that the true map length would be larger than what we have got. It is clear from the results that there are big differences in the number of polymorphic markers and map size between the male and female parents. Specific biological differences in the levels or location of DNA polymorphisms, rates of recombination, variation in copy number of specific genomic sequences (van Eck et al., 1995) or sampling error are factors that could affect the distribution of markers. The observed differences on female and male maps are most probably due to the relatively limited size of the mapping population. In addition, small group (less than 5 markers) in the maps, suggested that the markers are sparse in a few regions of genome and dense in others (Kesseli et al., 1994; Becker et al., 1995). Additional markers are required to fill the gaps, condense the existing maps and identify homologous female and male linkage groups. The high proportion of unlinked markers (35.6%) also reported in other linkage mapping studies. The existence of the minor linkage groups and the unlinked markers indicates that there are many large gaps with few markers (Kesseli et al., 1994). These small linkage groups and unlinked markers are related to small linkage groups and small population size, as well as the low map saturation. The small linkage groups will be merged into large groups and the unlinked markers would be very valuable when more markers are assigned in further studies. The requirement for large numbers of markers or integrated mapping populations to reduce the linkage group numbers to haploid chromosome numbers has been seen in many mapping experiments (Jeuken et al., 2001; Sharma et al., 2002). The proportion of segregation-ratio distorted fragments in this study was 34.8% (a = 0.01). Distorted segregation could be caused by gametic and zygotic and affected by genetic, physiological, and environmental factors (Burt et al., 1991; Ky et al., 2000). The transmission of genetic markers and chromosomes from one to the next generation could be distorted by lethal genes or chromosomal rearrangements that alter segregation ratios (Causse et al., 1994; Foolad et al., 1995).

AFLP analysis is a powerful technique in terms of its ability to identify a large number of polymorphic bands without any prior knowledge of DNA sequences in the organism. The relatively high yield of information achieved with AFLP markers makes it an efficient tool for mapping in wintersweet. AFLP markers detect more point mutations (5–45 in our experiment) per reaction than RAPDs or ISSRs. The overall detection rate of polymorphisms from 12 AFLP primer combinations in this study is slightly higher than that reported by Marques et al. (1998). We obtained an average of 26 markers polymorphism per primer combination, approximately 4 times more than the number of ISSR markers per primer in this study. ISSR primers are easy to design as they require no prior knowledge of sequence. Due to the primer length (Kojima et al., 1998) and a more stringent temperature at the annealing step, ISSR have a greater repeatability than RAPD markers. In our experiment, only 15.8% of ISSR markers were used to build the map, which is surprisingly low in comparison with other mapping studies. The lack of polymorphism in the case of ISSR markers could then be attributed to a non-optimal protocol rather than to a biological cause, such as a reduced heterozygosity of the parents or a low genetic divergence between species. However, we assayed only 38 primers this time and many other ones are available (Tsumura et al., 1996). Moreover, the separation of ISSR fragments on nondenaturing polyacrylamide gels instead of agarose gels could enhance the resolution of ISSR bands. Another interest of this type of marker lies in its linkage to SSR loci. Indeed, microsatellite markers were shown to be linked to coding regions (Echt et al., 1996). Genetic linkage maps have been constructed in the past for many crop species and forest trees using intraspecific and/or interspecific mating systems. It is generally believed that the degree of polymorphism is lower in an intraspecific population than in an interspecific population. This may be explained by the high level of genetic variability within an interspecific population or the level of heterozygosity of the parents. However, intraspecific crosses have several advantages over interspecific crosses for breeding programs, particularly for polygenic characteristics, and also for the reduction of the problems of low fertility and low recombination rates associated with interspecific crosses

92

D.-W. Chen, L.-Q. Chen / Scientia Horticulturae 124 (2010) 88–94

Fig. 1. Genetic linkage map of female parent showing the location of 80 loci distributed over 12 linkage groups. Segregation data is based on 67 AFLP loci and 13 ISSR loci. Locus nomenclature for AFLP markers is shown in the form as follows: e.g. IR9_6b where IR9 refers to the primer/primer combination code (Tables 1 and 2), 6 refers to the fifth polymorphic band to be scored in the order of descending molecular weight, f (male) and m (female) represent the dominant parental types, b refers to present in both parents (see Section 2).

(Capo-chichi et al., 2004). Consequently, the use of intraspecific crosses allows better map resolution than interspecific crosses (Lefebvre et al., 1995). Since the populations we studied were derived from an intraspecific cross, we expected a low level of polymorphism compared to that found in interspecific crosses. As expected, the polymorphism rate was indeed low. This was not surprising, because except for flower size and color of inner tepals they were morphologically very similar. The genetic maps of wintersweet were not saturated, because not all markers could be mapped. The existence of the minor linkage

groups and the unlinked markers indicates that there are many large gaps with few markers (Kesseli et al., 1994). These small linkage groups and unlinked markers would be likely to associate with the small population size and the low map saturation. An common disadvantage of tree breeding is their long life cycle. Wintersweet is a perennial species, generation of a mapping population would normally take more than 6 years. The pseudotestcross segregating populations have been proved to be an effective and efficient approach for genetic and QTL mapping in tree species (Pearl et al., 2004). In this study, we took only 1 year to

D.-W. Chen, L.-Q. Chen / Scientia Horticulturae 124 (2010) 88–94

93

Fig. 2. Genetic linkage map of male parent showing the location of 47 loci distributed over 8 linkage groups. Segregation data is based on 35 AFLP loci and 12 ISSR loci. Locus nomenclature for AFLP markers is shown in the form as follows: e.g. E1M9_5f where E1M9 refers to the primer combination/primer code (Tables 1 and 2), 5 refers to the fifth polymorphic band to be scored in the order of descending molecular weight, f (male) and m (female) represent the dominant parental types, b refers to present in both parents (see Section 2).

construct a mapping population by pseudo-testcross mapping strategy. Marker-assisted selection has the potential to improve the efficiency of the breeding programs because it allows inferior seedlings to be removed early in the breeding cycle, thus, it can reduce the cost in field maintenance (Venkateswarlu et al., 2006). In the long term, linkage between molecular markers and valuable genes is the first step towards the positional cloning of these genes, and genetic map will be a useful tool to find the linkage. The present maps of wintersweet are the first reported one in Chimonanthus species. They provide a basic tool for identifying and localizing QTLs. The results of further QTL studies will provide a new insight into the genetic architecture of complex traits such as color or fragrance. In the future, the maps will be saturated with

more wintersweet-specified SSRs and other markers, thus, morphological traits or other single gene traits could possibly be placed on these maps. Acknowledgements We thank Dr. Xingguo Li (CSIRO Plant Industry, BlackMoutain Laboratory) for his critical reading. This work was supported by the National Natural Science Foundation of China (No. 30571310). References Arcade, A., Faivre, R.P., Le Guerroue´, B., Paˆques, L.E., Prat, D., 1996. Heterozygosity and hybrid performance in larch. Theor. Appl. Genet. 93, 1274–1281.

94

D.-W. Chen, L.-Q. Chen / Scientia Horticulturae 124 (2010) 88–94

Arcade, A., Anselin, F., Rampant, P.F., Lesage, M.C., Paˆques, L.E., Prat, D., 2000. Application of AFLP, RAPD and ISSR markers to genetic mapping of European and Japanese larch. Theor. Appl. Genet. 100, 299–307. Azuma, H., Toyota, M., Asakawa, Y., 2005. Floral scent chemistry and stamen movement of Chimonanthu preacox (L.) Link (Calycanthaceae). Acta Phytotax. Geobot. 56 (2), 197–201. Becker, J., Vos, P., Kuiper, M., Salami, F., Heun, M., 1995. Combined mapping of AFLP and RFLP markers in barley. Mol. Gen. Genet. 249, 65–73. Burt, A., Bell, G., Harvey, P.H., 1991. Sex differences in recombination. J. Ecol. Biol. 4, 259–277. Capo-chichi, L.J.A., Morton, C.M., Weaver, D.B., 2004. An intraspecific genetic map of velvetbean (Mucuna sp.) based on AFLP markers. Theor. Appl. Genet. 108, 814– 821. Causse, M.A., Fulton, T.M., Cho, Y.G., Ahn, S.N., Chunwongse, J., Wu, K., Xiao, J., Yu, Z., Ronald, P.C., Harrington, S.E., Second, G., McCouch, S., Tanksley, S.D., 1994. Saturated molecular map of the rice genome based on an interspecific backcross population. Genetics 138, 1251–1274. Cervera, M.T., Storme, V., Ivens, B., Gusma˜o, J., Liu, B.H., Hostyn, V., Slycken, J.V., Montagu, M.V., Boerjan, W., 2001. Dense genetic linkage maps of three populus species (Populus deltoides, P. nigra and P. trichocarpa) based on AFLP and microsatellite markers. Genetics 158, 787–809. Chakravarti, A., Lasher, L.A., Reefer, J.E., 1991. A maximum-likelihood method for estimating genome length using genetic linkage data. Genetics 128, 175–182. Deng, C.H., Song, G.X., Hu, Y.M., 2004. Rapid determination of volatile compounds emitted form Chimonanthus praecox flowers by HS–SPME–GC–MS. Z. Naturforsch. C 59, 636–640. Dida, M.M., Ramakrishnan, S., Bennetzen, J.L., Gale, M.D., Devos, K.M., 2007. The genetic map of finger millet, Eleusine coracana. Theor. Appl. Genet. 114, 321– 332. Echt, C.S., May-Marquardt, P., Hseih, M., Zahorchak, R., 1996. Characterization of microsatellite markers in eastern white pine. Genome 39, 1102–1108. Foolad, M.R., Arulsekar, S., Becerra, V., Bliss, F.A., 1995. A genetic map of Prunus based on an interspecific cross between peach and almond. Theor. Appl. Genet. 91, 262–269. Grattapaglia, D., Sederoff, R., 1994. Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo-testcross: mapping strategy and RAPD markers. Genetics 137, 1121–1137. Ipek, M., Ipek, A., Almquist, S.G., Simon, P.W., 2005. Demonstration of linkage and development of the first low-density genetic map of garlic, based on AFLP markers. Theor. Appl. Genet. 110, 228–236. Jeuken, M., van Wijk, R., Peleman, J., Lindhout, P., 2001. An integrated interspecific AFLP map of lettuce (Lactuca) based on two L. sativa  L. saligna F2 populations. Theor. Appl. Genet. 103, 638–647. Kesseli, R.V., Paran, I., Michelmore, R.W., 1994. Analysis of a detailed genetic linkage map of Lactuca sativa (Lettuce) constructed from RFLP and RAPD markers. Genetics 136, 1435–1446. Kojima, T., Nagaoka, T., Noda, K., Ogihara, Y., 1998. Genetic linkage map of ISSR and RAPD markers in einkorn wheat in relation to that of RFLP markers. Theor. Appl. Genet. 96, 37–45. Ky, C.L., Barre, P., Lorieux, M., Trouslot, P., Akaffou, S., Louarn, J., Charrier, A., Hamon, S., 2000. Interspecific genetic linkage map, segregation distortion, and genetic conversion in coffee (Coffea sp.). Theor. Appl. Genet. 101, 669–676. Lefebvre, V., Palloix, A., Caranta, C., Pochard, E., 1995. Construction of an intraspecific integrated linkage map of pepper using molecular markers and doublehaploid progenies. Genome 38, 112–121. Lincoln, S., Daly, M., Lander, E., 1992. Constructing genetic Maps With MAPMAKER/ EXP 3.0, 3rd ed. Whitehead Institute Technical Report. Liu, R.H., Meng, J.L., 2003. MapDraw: a Microsoft Excel macro for drawing genetic linkage maps based on given genetic lingkage data. Hereditas 25 (3), 317–321.

Loh, J.P., Kiew, R., Kee, A., Gan, L.H., Gan, Y.Y., 1999. Amplified fragment length polymorphism (AFLP) provides molecular markers for the identification of Caladium bicolor cultivars. Ann. Bot. 84, 155–161. Marques, C.M., Arau´jo, J.A., Ferreira, J.G., Whetten, R., O’Malley, D.M., Liu, B.H., Sederoff, R., 1998. AFLP genetic maps of Eucalyptus globulus and E. tereticornis. Theor. Appl. Genet. 96, 727–737. Maughan, P.J., Bonifacio, A., Jellen, E.N., Stevens, M.R., Coleman, C.E., Ricks, M., Mason, S.L., Jarvis, D.E., Gardunia, B.W., Fairbanks, D.J., 2004. A genetic linkage map of quinoa (Chenopodium quinoa) based on AFLP, RAPD, and SSR markers. Theor. Appl. Genet. 109, 1188–1195. Mazur, B.J., Tingey, S.V., 1995. Genetic mapping and introgression of genes of agronomic importance. Curr. Opin. Biotechnol. 6, 175–182. Pearl, H.M., Nagai, C., Moore, P.H., Steiger, D.L., Osgood, R.V., Ming, R., 2004. Construction of a genetic map for arabica coffee. Theor. Appl. Genet. 108, 829–835. Ritter, E., Gebhardt, C., Salami, F., 1990. Estimation of recombination frequencies and construction of linkage maps from crosses between heterozygous parents. Genetics 125, 645–654. Sharma, R., Aggarwal, R.A.K., Kumar, R., Mohapatra, T., Sharma, R.P., 2002. Construction of an RAPD linkage map and localization of QTLs for oleic acid level using recombinant inbreds in mustard (Brassica juncea). Genome 45, 467–472. Stallen, N.V., Vandenbussche, B., Verdoodt, V., Proft, M.D., 2003. Constuction of a genentic linkage map for witloof (Cichorium intybus L. var. foliosum Hegi). Plant Breed. 122, 521–525. Tsumura, Y., Ohba, K., Strauss, S.H., 1996. Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica). Theor. Appl. Genet. 92, 40–45. Tulsieram, L.K., Glaubitz, J.C., Kiss, G., Carlson, J.E., 1992. Single tree genetic linkage mapping in conifers using haploid DNA from megagametophyte. Biotechnology (NY) 10, 686–690. van Eck, H.J., van der Voort, J.R., Draaistra, J., van Zandvoort, P., van Enckevort, E., Segers, B., Peleman, J., Jacobsen, E., Helder, J., Bakker, J., 1995. The inheritance and chromosomal localization of AFLP markers in a non-inbred potato offspring. Mol. Breed. 1, 397–410. Venkateswarlu, M., Urs, S.R., Nath, B.S., Shashidhar, H.E., Maheswaran, M., Veeraiah, T.M., Sabitha, M.G., 2006. A first genetic linkage map of mulberry (Morus spp.) using RAPD, ISSR and SSR markers and pseudotestcross mapping strategy. Tree Genet. Genomes 3, 15–24. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Fritjters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Zabeau, M., Vos, P., 1993. Selective restriction fragment amplification: a general method for DNA fingerprinting. European Patent Application 92402629.7, Publ. no. EP 0534858 A1. Zhang, R.H., Liu, H.E., 1998. Wax Shrubs in World (Calycanthaceae). China Science and Technology, Beijing. Zhao, B., Zhang, Q.X., 2007. The advancement of germplasm on Chimonanthus. J. Northwest Forest. Univ. 22 (4), 57–61. Zhao, K.G., Zhou, M.Q., Chen, L.Q., Zhang, D.L., Gituru, W.R., 2007. Genetic diversity and discrimination of Chimonanthus praecox (L.) Link germplasm using ISSR and RAPD markers. Hortscience 42 (5), 1144–1148. Zhao, T.B., Chen, Z.X., Gao, B.Z., Li, Z.Q., Song, L.G., 1993. Chinese Wintersweet. Henan Science and Technology Press, Zhengzhou. Zheng, Y.Q., Zhu, Y., Zhang, R.Y., Sun, Y.L., Wu, Z.P., Liu, M.X., 1990. Studies on the natural flavor components of Chimonanthus praecox L. flower. Acta Scicentiarum Naturalum Universitis Pekinesis 26 (6), 667. Zhou, M.Q., Zhao, K.G., Chen, L.Q., 2007. Genetic diversity of Calycanthaceae accessions estimated using AFLP markers. Scientia Horticulturae 112, 331–338.