林业科学  2003, Vol. 39 Issue (2): 162-167   PDF    
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文章信息

Gan Siming, Shi Jisen, Bai Jiayu, Wu Kunming, Wu Juying
甘四明, 施季森, 白嘉雨, 吴坤明, 吴菊英
DNA POLYMORPHISM AND HETEROZYGOSITY IN SEVEN PARENTAL EUCALYPT CLONES AS REVEALED BY RAPD MARKERS
7个桉树杂交亲本RAPD位点多态性和杂合性的研究
Scientia Silvae Sinicae, 2003, 39(2): 162-167.
林业科学, 2003, 39(2): 162-167.

文章历史

Received date: 2000-04-13

作者相关文章

甘四明
施季森
白嘉雨
吴坤明
吴菊英

7个桉树杂交亲本RAPD位点多态性和杂合性的研究
甘四明1, 施季森2, 白嘉雨1, 吴坤明1, 吴菊英1     
1. 中国林业科学研究院热带林业研究所 广州 510520;
2. 南京林业大学林木遗传与基因工程实验室 南京 210037
摘要:利用RAPD分子标记技术对桉树杂交亲本的RAPD位点多态性和杂合性进行了比较研究。研究材料包括不同起源的3个尾叶桉母本和4个细叶桉父本及其杂交产生的5个全同胞家系,每家系10株子代。RAPD为显性标记,其检测一个杂交群体中分离位点的概率可以用分离位点至少在一个个体中出现的概率来表示,即1-(1/2) n-1(Aa ×aaaa ×Aa)或1-(3/4) n-1(Aa ×Aa) (n为群体内个体数),则利用10个个体的杂交群体,其概率分别为99.8%和92.5%,检测的有效性较高。各样品RAPD扩增结果统计中,以1代表一条谱带出现,以0代表不出现。9个随机引物共扩增出58条谱带,亲本间呈多态性的谱带42条,占72. 4%,多态性较高。利用亲本的RAPD数据矩阵,计算了亲本间的遗传距离。根据亲本出现的谱带在子代中是否分离判断该位点是否为杂合位点,如果某谱带出现于亲本且在子代中分离,则亲本在该位点上的基因型为Aa,即杂合位点,从而统计出不同亲本的杂合位点数。以亲本的杂合位点数占其总位点数的百分比表示亲本的杂合性,检测各亲本的杂合性水平。7个亲本的杂合性平均为28. 0%,杂合性较高。但不同亲本的杂合性有差异,介于16. 2%和39. 5%之间。并且,同一无性系在不同的家系中检测到的杂合位点数和计算的杂合性有差异,这主要是由于以下两个原因; 一是RAPD为显性标记技术,不能有效探测样品本身的杂合位点,因此当另一亲本在该位点上基因型为AA时,则亲本的基因型即使为Aa,子代在该位点上的RAPD谱带也没有分离; 二是10个个体的群体偏小,有些实际分离的位点需要更大的群体才能检测到(尤其是偏分离的位点)。林木中,常用F1谱系和显性标记进行遗传连锁图谱构建,相应的作图策略为拟测交策略(Pseudo-testcross strategy),即可用于图谱构建的标记为只出现于一个亲本、而在子代中按1:1分离的标记,类似于测交的分离方式。因此,亲本的较高杂合性表明其杂交时有大量的拟测交位点出现,这种位点在子代中的分离可以用显性标记有效检测。所以,本研究中亲本杂合性均较高的家系可以用作遗传图谱构建的材料(合适大小的群体,如100个以上的子代),这对增加图谱的标记数量和提高作图的效率具有重要意义。
DNA POLYMORPHISM AND HETEROZYGOSITY IN SEVEN PARENTAL EUCALYPT CLONES AS REVEALED BY RAPD MARKERS
Gan Siming1, Shi Jisen2, Bai Jiayu1, Wu Kunming1, Wu Juying1     
1. Research Institute of Tropical Forestry, Chinese Academy of Forestry Guangzhou 510520;
2. Laboratory of Forestry Genetics and Gene Engineering, Nanjing Forestry University Nanjing 210037
Abstract: Five controlled crosses of Eucalyptus urophylla S.T. Blake × E. tereticornis Smith were used for RAPD analysis on DNA polymorphism and heterozygosity of the seven parental clones. A total of 58 fragments were amplified with 9 single arbitrarily chosen primers,and 42 fragments(72.4%)were scored as polymorphic among the parents. The genetic distance (GD)between parents was calculated with RAPD data matrix. High levels of heterozygosity were revealed in the parents studied,with an average 28.0%,but the percentage of heterozygous loci varied with parent from 16.2% to 39.5%. The implications for material selection in genetic linkage map construction were discussed.
Key words: RAPD    多态性    杂合性    桉树    

Eucalyptus species(family Myrtaceae) are native to Australia, Indonesia and the adjacent islands and largely introduced for plantation establishment in global tropics and subtropics due to their fastness in growth, multiplicity of uses and plasticity of adaptations (Jacobs, 1981; Eldridge et al., 1993). E. urophylla S.T. Blake and E. tereticornis Smith are two of the most important species in the genus. In China, substantial genetic gain in such traits as growth and wood properties has been demonstrated in field trials through hybridization and recurrent selection in the two trees (Wu et al., 1996; Xu et al., 1996). The two species along with their hybrids are of great potential in establishing short-rotation and industry-oriented (e. g. pulp and paper making)plantations in worldwide context, and great efforts have been placed on their multiple objective improvement, especially with a focus on integrating modern biological techniques.

A successful breeding program should take full advantage of broad genetic base, for such poorly improved species as forest trees in particular, and genetic polymorphism and heterozygosity are always important indices used to monitor the genetic diversity (Lanham, 1996). Isozymes were initially sought in plant for such purpose (Hamrick et al., 1990), but their use was limited at some extent by the low amount of variability. Recent development of molecular marker technology has made it possible to conduct such studies with more powerful assays, and random amplified polymorphic DNA (RAPD)is preferred in this term because it is easy to analyze, requires very little DNA and does not need radioactivity handling facilities(Williams et al., 1990; Welsh et al., 1990; Gan et al., 1998; Martin et al., 1993). Though the reproducibility of RAPD markers are sometimes questioned, reliable results have been verified in many cases of plants (Xiao et al., 1996; Lanza et al., 1997). Now DNA polymorphism and heterozygosity have been potently identified with RAPD markers in quite a few species, such as intraspecific crossing of crop (Brassica napus)(Marshall et al., 1994), cultivars of tree (Ribes nigrum L.) (Lanham, 1996), and sibs of a single urediniospore-derived culture of fungus (Cronartium quercuum f. sp. fusiforme) (Doudrick et al., 1993).

In this study, we use RAPD markers and 5 full-sib families to detect the levels of polymorphism and heterozygosity in seven parental clones of E. urophylla S. T. Blake (3 females) and E. tereticornis Smith (4 males). The implications for material selection in linkage mapping studies are discussed.

1 Materials and Methods 1.1 Plant materials

Three maternal clones of E. urophylla and four paternal ones of E. tereticornis were employed in this study, which seedlings were raised in 1989 with seeds collected in natural forests (by courtesy of CSIRO, Australia). Details of origin and code of the parental clones are shown in Table 1. The trees were conserved in the gene pool in Research Institute of Tropical Forestry, CAF, and grafted in 1992. Controlled pollination was made in 1996 to produce five full-sib families A, B, C, D and E(Table 1), and more than ten sib seedlings per family were then raised in nursery with seeds harvested. For each family, both parents and ten sibs were used for RAPD analysis.

Tab.1 Parental clones of E. urophylla and E. tereticornis employed in this study
1.2 DNA extraction and RAPD assays

Fresh tender leaves about 1 g per parent and 5-month-old sib seedling were sampled and stored at -20 ℃ for subsequent DNA extraction. Preparation of DNA was followed with the procedure describe in Doyle and Doyle (1990), amended by the addition of 5% polyvinylpyrrolidone (PVP) and 2% 2-mercaptoethanol to the extraction buffer. DNA concentration was estimated by electrophoresis of agarose gel stained with ethidium bromide (EB)basing on the fluorescence intensities of the sample.

RAPD was performed in thin-walled microcentrifuge tubes with Idaho Rapidcycler (Idaho Technologies). The amplification program and RAPD reaction mixture were in accordance with Gan et al(2000). A total of 20 candidate primers (Operon Technologies Inc.), namely OPE01~OPE20, were used for primer screening against the seven parental DNA templates, and 9 primers that could produce polymorphic, clear and stable fragements were eventually selected for formal amplification (Table 2).

Tab.2 Primers used in formal amplification and the number of fragments amplified
1.3 Polymorphism and heterozygosity tests

RAPD genotypes of 7 parents were scored for the presence absence of amplification fragments, with 1 representing presence and 0 absence of a fragment. Polymorphic markers among parents were recorded for polymorphism estimation. Genetic distance of different parent pair was then calculated according to Nei's method (1978). The number of heterozygous loci per parent was evaluated on the basis of the segregation across the sibs, and heterozygosity was calculated as the percentage of heterozygous loci taking of the total number of fragments per parental clone.

2 Results and Discussion 2.1 DNA polymorphism among parental clones

Totally 58 fragments were amplified with 9 primers selected out of 20 candidates, including 42(72. 4%)being polymorphic and 16 persistent (non-polymorphic) among the 7 parental clones (Table 2). The number of fragments varied with primer. Two unique primers OPE09 and OPE13 could discriminate clearly between all the parents. The high level of polymorphism might be indicative of a relatively high level of polymorphism for the parents studied (Nei, 1978). Figure 1 shows the RAPD profiles of 7 parents amplified with primer OPE02 and OPE09.

Fig.1 Amplification profiles of 7 parents with primer OPE02 and OPE09 The arrows show the polymorphic fragments. M is the 100bp DNA ladder size standard(MBI) (with fragments representing 3 000, 2 000, 1 500, 1 200, 1 031, 900, 800, 700, 600, 500, 400, 300, and 200bp from the top to the bottom)

Pairwise comparisons of RAPD data between parental clones resulted in a matrix of genetic distance (GD)(Table 3). The five families followed in parental GD a descending order A>E>B>C >D. This order was roughly the same as clone fingerprints previously amplified with other primers (Gan et al., 1999).

Tab.3 Nei's genetic distance between the seven parents
2.2 Heterozygosity tests of seven parents

According to the dominant nature of RAPD markers, we could deduce the genotype AA or Aa at a locus from the presence of a RAPD fragment and recessive homologous aa of the absence. Thus, the fragment-specific parent could be genotyped as heterozygous Aa if the fragment segregated in the offspring in a bi-parental diploid mode. In this way the number and percentage of putative heterozygous RAPD loci (RAPDs)of each parent were calculated (Table 4). The average of heterozygosity was calculated as the percentage that the total heterzygous loci took of the total number of fragments amplified for all the 7 clones studied. Figure 2 showed the segregating markers among the sibs of each of the families amplified with primer OPE13.

Tab.4 Parental variations of heterozygous RAPDs in five families tested
Fig.2 RAPD profiles of five families amplified with primer OPE13 The arrows show the fragments that are polymorphic between parents and segregate among the sibs per family. In each family panel, M is the 100bp DNA ladder size standard(MBI)(order as Figure 1), P1 the female parent, P2 the male parent, and the rest 01~10 are sibs.

Heterozygosity was defined as the percentage that heterozygous individuals took at a specific locus of the total individuals in a population (Nei, 1978). It was originally a concept of population genetics and later extended to refer the percentage of heterozygous loci at individual level (Lanham, 1996; Marshall et al., 1994). In our observations, high levels of heterozygosity were revealed in the parental eucalypt clones, with an average of 28. 0%(72 257). The heterozygosity varied with parent, as PB1(PC1, Clone 03) ranked the first in percentage of heterozygous loci (39.5%) but PD1(Clone 10) the last (16.2%). The levels of heterozygosity of eucalypts in this study were much higher than those estimated in nearly isogenic line of canola (Brassica napus)(3.6% at the most) (Marshall et al., 1994), and similar to those of Ribes nigrum(about 20%) (Lanham, 1996)

Interestingly, heterozygous loci or the number varied in the same parental clone with family (Table 4). For example, clone 03 showed 14 heterozygous loci in family B, but 9(one being exclusive) in family C. This might be caused by: (1) the disability of RAPD technology in detecting heterozygous loci in case of AA ×Aa, where the locus would not segregate in the sibs, and (2) the utilization of not-large-enough size of F1 population as some factual segregating loci could not be observed in a small population. The ideal solutions could rely on utilization of codominant markers, e. g. RFLP, and or selfed populaions in this term. However, dominant markers were employed on many occasions, and selfed crosses were difficult to be obtained due to self-incompatibility in most tree species (Marshall et al., 1994; Dudley et al., 1991). The usual populations used were single outcross pedigree in related studies. In addition, the probability for detecting a heterozygous loci in a population could be given by the probability of a fragment being present in at least one sib as 1-(1/2)n-1 when Aa ×aa (or aa ×Aa) and 1-(3/4)n-1 when Aa ×Aa, where n was the size of population. For a population of 10 sibs, the probability was 99.8% and 92.5% respectively. So a population size of 10 sibs could be effective for detecting possible segregating RAPD loci as was the case in this study.

In genetic mapping of highly heterozygous species, such as forest trees, the usual mapping population was F1 pedigree and the strategy was followed in a pseudo-testcross configuration. In such a strategy, the marker available for linkage map construction should be present in the mapping parent (genotype Aa)but absent in another (genotype aa) and segregate 1:1 in the sibs in a Mendelian fashion (Carlson et al., 1991; Grattapaglia et al., 1994). The high level of heterozygosity in eucalypt clones studied may indicate that testcross cases for RAPD markers were so common in eucalypt and the lack of codominance with RAPD might not represent such a disadvantage in genetic map construction. We could suggest that preliminary screening of small samples of progeny be conducted to determine the parental polymorphism and heterozygosity prior to fullscale segregating analysis. In this respect, Nps(Table 2) could be referable in choosing a rational F1 population and those fragments polymorphic between parents and segregant among sibs be potentially useful for genetic mapping in E. urophylla and E. tereticornis. Therefore, family E (Nps = 19), followed with families C and D (Nps=15), would be probably sound candidates of plant materials for genetic mapping (with an appropriate number of sibs, e. g. 100 or more). It might be inferred from this study that more than 300 primers could be selected out of 1 000 candidate primers and about 500 markers be produced for genetic mapping of a typical eucalypt species. If these primers were utilized in combination, even more markers could be mapped (Carlson et al., 1991).

In summary, the results from the present study indicate that high levels of polymorphism ad heterozygosity are reliably revealed in parental eucalypt clones with RAPD markers and thus facilitate the construction of genetic maps for plant material preparation considerations.

Acknowledgements

Thanks are due to (China) National Science Foundation Committee (grant No. 39870619)and International Foundation for Science (grant No. D 2947-1) for financial support in this study.

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