摘要
本文从形态学、DNA分子标记、DNA序列三个方面,以无锡、南京、杭州和湖南浏阳的88个桂花(Osmanthus fragrans Lour.)栽培品种、野生桂花、木犀属(Osmanthus)2个对照种柊树(O. heterophyllus (G Don.) P. S. Green)和华东木犀(O.cooperi Hemsl.)为材料,对桂花种质资源的遗传多样性、亲缘关系、品种分类鉴定等问题进行了研究和探讨。建立并优化了桂花的SRAP扩增体系。利用SRAP和ISSR分子标记研究了桂花种质资源的遗传多样性和亲缘关系。测定了野生桂花及部分桂花品种的ITS序列和trnL-F序列,并用两个序列联合分析的方式,以木犀属的格树和华东木犀为外类群,初步探讨了桂花四个品种群之间的亲缘关系。依据形态特征编制了88个桂花品种的分类检索表,分别运用SRAP和ISSR分子标记构建了桂花的DNA指纹图谱,旨在为桂花品种的鉴定、培育和开发利用,提供形态和分子依据。研究的主要内容如下：
1.桂花SRAP扩增体系的建立及遗传多样性与亲缘关系研究
以早银桂为材料,通过对PCR扩增体系中模板DNA、Mg2+、dNTPs、引物、TaqDNA聚合酶进行单因子不同浓度梯度的比较试验,建立了适合桂花基因组DNA的SRAP-PCR优化体系。结果表明,在10μL反应体系中,SRAP-PCR优化体系中各因子适宜浓度为：模板DNA 30ng、Mg2+ 2.5 mmol·L-1、dNTPs 0.2 mmol·L-1、引物0.4μmol·L-1、Taq DNA聚合酶0.75U。
运用SRAP优化体系,以格树和华东木犀为对照种,研究了桂花88个品种和野生桂花的遗传多样性及亲缘关系。18对SRAP引物共获得296个位点,其中248个为多态性位点,多态性比率达83.78%。平均每对引物组合产生16.4个位点、13.8个多态性位点。其中,银桂品种群的Shannon信息指数(0.3412)、遗传多样性指数(0.2191)和位点多态性比率(84.46%)最高。桂花遗传分化系数为52.95%,说明大部分变异存在于品种群之间。聚类分析结果表明,以遗传相似系数0.762为截值,可将91份种质分成6类。各品种群的品种基本先聚在一起。四季桂品种群与秋季开花的三个品种群之间的遗传距离较远。色质较深的金桂往往与丹桂品种群的多个品种聚在一起。基于SRAP分子标记的聚类结果与基于形态的传统分类学的结果基本相符。说明将桂花的栽培品种根据开花季节、花序类型和花色等不同,划分为四季桂、银桂、金桂和丹桂四个品种群是可行的。
2.桂花遗传多样性及亲缘关系的ISSR分析
利用ISSR分子标记,以格树和华东木犀为对照种,研究了桂花88个品种和野生桂花的遗传多样性及亲缘关系。28个ISSR引物共获得286个位点,其中多态性位点269个,多态性比率达94.06%。在四个品种群中,银桂品种群的Shannon信息指数(0.3884)、遗传多样性指数(0.2532)和位点多态性比率(87.41%)最高。桂花遗传分化系数为56.84%,说明大部分变异存在于品种群之间。聚类分析结果表明,以遗传相似系数0.720为截值,可将91份种质分成7类。各品种群的品种基本先聚在一起。四季桂品种群与秋季开花的三个品种群遗传距离较远。色质较深的金桂往往与丹桂品种群的品种聚在一起。基于ISSR分子标记的聚类结果与基于形态的传统分类学的结果基本相符。说明将桂花的栽培品种根据开花季节、花序类型和花色等不同,划分为四季桂、银桂、金桂和丹桂四个品种群是可行的。
3.桂花SRAP与ISSR遗传多样性及亲缘关系比较分析
基于SRAP和ISSR两种分子标记的桂花遗传多样性有异同。结果表明,每对SRAP引物较每个ISSR引物扩增的位点数多(16.4个),而ISSR标记的位点多态性比率较高(94.06%)。两种标记皆表明：在四个品种群中,银桂品种群的观察等位基因数、有效等位基因数、Nei's遗传多样性指数、Shannon信息指数和位点多态性比率均最高,其次为金桂。位点多态性比率排序均为：银桂品种群>金桂品种群>丹桂品种群>四季桂品种群。两种标记均表明变异主要存在于品种群之间,四季桂品种群与秋季开花的银桂、金桂和丹桂品种群关系较远；丹桂品种群与四季桂品种群的遗传距离最远,与金桂品种群距离最近。基于SRAP和ISSR分子标记的聚类结果与基于形态的传统分类学的结果基本相符。利用SRAP标记得到的聚类结果与形态分类更为接近。本研究两个标记的聚类结果均表明,无锡梅园的几个桂花品种桃叶齿银桂、竹叶银桂、多裂银桂、五瓣银、银十字、圆瓣籽金、球橙和银丹,以及南京林业大学的冬香白,不但形态上有别于其他品种,在基因组水平上也与其他品种存在一定差异,因此从分子生物学角度进一步证实可以确立为新品种。
4.基于ITS和trnL-F序列的桂花亲缘关系的分子系统学初步研究
通过PCR直接测定法测定了分属四个品种群的4个桂花品种、桂花野生种的ITS序列和trnL-F序列,又在测序基础上,比较了银桂品种群内6个品种的ITS序列。以木犀属的格树、华东木犀为外类群,分析了两个序列的特点；将ITS序列和trnL-F序列进行联合分析,用UPGMA法构建了供试物种的分子系统树以探讨桂花四个品种群之间的亲缘关系。结果表明,所测物种的ITS序列全长在620-650bp之间,经排序后,两端切平,得到617bp的整齐序列；变异位点17个、信息位点2个,分别占全序列的2.76%、0.32%。所测物种的trnL-F序列全长在860-900bp之间,经排序后,两端切平,得到846bp的整齐序列；变异位点11个、信息位点4个,分别占总序列长度的1.28%和0.47%。6个银桂品种之间的ITS序列变异极少,推测ITS序列尚无法揭示桂花同一品种群内品种间的亲缘关系。用ITS和trnL-F序列联合分析的方式可在一定程度上揭示桂花四个品种群之间的亲缘关系。在秋桂类中,丹桂品种群与四季桂品种群亲缘关系最远,与金桂品种群亲缘关系最近；在四个品种群中,丹桂品种群与野生桂花亲缘关系最远,支持多位学者根据形态分类得出的丹桂品种群在秋桂中最进化的结论。ITS和trnL-F序列中皆有一些位点可用于区别格树、华东木犀与桂花(野生种和品种),或者野生桂花与部分栽培品种。
5.基于形态学和DNA指纹图谱的桂花品种鉴定
依据形态学构建的桂花品种检索表和依据分子标记构建的桂花DNA指纹图谱,可以分别作为桂花种质资源鉴定的形态依据和分子依据。
桂花的不同品种在形态上有明显区别。在花期、开花习性、花(花序、花型、花径、花色、花香、雌雄蕊的结实性)、果实和种子、叶、枝、树干等方面,都显著不同。有些性状非常稳定,可以作为桂花品种分类和鉴定的重要形态依据。根据开花季节、花序类型和花色等不同,将88个桂花品种划归四季桂、银桂、金桂和丹桂四个品种群。依据桂花四个品种群及各品种的主要鉴别特征,编制了桂花88个品种的分类检索表。并对无锡梅园和南京林业大学的7个待定新品种进行了形态描述。
分别运用SRAP-PCR体系和18对SRAP引物,以及ISSR-PCR体系和28个ISSR引物,以格树和华东木犀为对照种,研究了桂花88个品种和野生桂花特有的SRAP和ISSR分子标记,并将两对SRAP引物pm17-em10和sa15-em10结合起来构建了桂花的SRAP指纹图谱,将两个ISSR引物ISSR12和ISSR27结合起来构建了桂花的ISSR指纹图谱。结果表明,76个桂花品种、野生桂花及对照种格树和华东木犀均有特别的SRAP条带；64个桂花品种、野生桂花及对照种格树和华东木犀均有特别的ISSR条带,这些条带作为部分供试材料特有的SRAP和ISSR标记,可用于桂花种质的分子鉴定。构建的桂花SRAP指纹图谱和ISSR指纹图谱,均可将91份种质有效区分开,并可作为桂花种质鉴定的分子依据。将基于形态学的品种检索表与基于分子标记的DNA指纹图谱结合起来可以对桂花种质资源进行更准确有效的鉴定。
形态学、DNA分子标记、DNA序列三方面的研究结果均表明：桂花种质资源的遗传多样性丰富,桂花的不同品种和品种群不论在形态上还是分子水平都存在明显差异。丹桂品种群与四季桂品种群距离最远,与金桂品种群亲缘关系最近。基于SRAP和ISSR分子标记的品种聚类图均表明,将桂花的栽培品种根据开花季节、花序类型和花色等不同,划分为四季桂、银桂、金桂和丹桂四个品种群是可行的,但花色仅为分类的依据之一,还需考虑其他性状。
三方面的研究结果表明：桂花的品种分类、品种群演化及品种亲缘关系相当复杂,不同的手段往往得出不同结论。今后,应在传统的形态分类基础上,综合解剖学、孢粉学、数量分类、分子标记、DNA序列等多种分类方法,并采用充足且可靠的材料,相互印证,综合分类,以期更科学地解决相关问题。
This thesis mainly dealt with the genetic diversity, genetic relationships and cultivar identification of Osmanthus fragrans Lour. germplasm by morphology, DNA markers (SRAP and ISSR) as well as DNA sequences (nrDNA ITS and cpDNA trnL-F), with 88 cultivars, wild species of O. fragrans from Wuxi, Nanjing, Hangzhou and Liuyang (Hunan Province) and 2 contrast species in Osmanthus (O. heterophyllus and O. cooperi) as materials. SRAP amplification system suitable for O. fragrans genome was set up and optimized. Genetic diversity and genetic relationships of O. fragrans germplasm resources were studied and discussed with SRAP and ISSR markers. ITS sequence and trnL-F sequence of several cultivars, wild species of O. fragrans were measured, and relationships among the 4 groups of O. fragrans were discussed primarily based on the combination of two sequences, with O. heterophyllus and O. cooperi as outgroups. The keys to 88 cultivars were made based on their major morphological characteristics, and two fingerprinting maps in DNA of O. fragrans were constructed based on SRAP and ISSR markers, and they can be used as morphological and molecular traits respectively for identification, cultivation, development and utilization of O. fragrans germplasm. The main results were as follows:
1. Establishment of SRAP-PCR Amplification System and Analysis of Genetic Diversity and Genetic Relationships for O. fragrans
With O. fragrans'Zao Yingui'as material, concentration gradients of template DNA, Mg2+, dNTPs, primer and Taq DNA polymerase enzyme in SRAP-PCR system were compared for genomic DNA of O. fragrans by monofactorial experiment, and the optimal SRAP-PCR amplification system was established. The optimal concentrations of each factor in the total 10μL system are 30 ng DNA,2.5 mmol·L"1 Mg2+,0.2 mmol·L-1 dNTPs, 0.4μmol·L-1 primer and 0.75U Taq polymerase enzyme.
Genetic diversity and cultivar classification of 88 cultivars and wild species of O. fragrans were evaluated by using sequence-related amplified polymorphism (SRAP) marker, with O. heterophyllus and O. cooperi as contrast species.18 primer pairs produced 296 loci, out of which 248 were polymorphic, the percentage of polymorphic loci was 83.78%.16.4 loci and 13.8 polymorphic loci were amplified by each pair of primers. The Shannon information index (0.3412), genetic diversity (0.2191) and polymorphic loci rate (84.46%) of Albus Group were the highest among the 4 groups of O. fragrans. The measurement of genetic variation showed that the coefficient of genetic differentiation of O. fragrans was 52.95%, most of the genetic variation existed among groups. The cluster analysis showed that six groups could be clustered when genetic similarity coefficient was given as 0.762. The genetic distance between Asiaticus Group and other 3 groups was far. Cultivars with deep color in Luteus Group often clustered together with several cultivars in Aurantiacus Group. Cluster result based on SRAP marker was approximately consistent with taxonomic result based on morphology.
2. Analysis of the Genetic Diversity and Genetic Relationships of O. fragrans Based on ISSR Markers
Cultivar classification of 88 cultivars and wild species of O. fragrans were evaluated by using ISSR markers, with O. heterophyllus and O. cooperi as contrast species.28 primers produced 286 loci, out of which 269 were polymorphic, the percentage of polymorphic loci was 94.06%. The Shannon information index (0.3884), genetic diversity (0.2532) and polymorphic loci rate (87.41%) of Albus Group were the highest among the 4 groups of O. fragrans. The measurement of genetic variation showed that the coefficient of genetic differentiation of O. fragrans was 56.84%, most of the genetic variation existed among groups. The cluster analysis showed that seven groups could be clustered when genetic similarity coefficient was given as 0.720. Cultivars within the same group often clustered together first. The genetic distance between Asiaticus Group and other 3 groups was far. Cultivars with deep color in Luteus Group often clustered together with several cultivars in Aurantiacus Group. Cluster result based on ISSR marker was approximately consistent with taxonomic result based on morphology.
3. Comparison of Analysis on Genetic Diversity and Genetic Relationships Based on SRAP and ISSR Markers
Genetic diversity and genetic relationships based on SRAP and ISSR markers were both similar and different. The result showed that more (16.4) loci were amplified by each pair of SRAP primers than that by each ISSR primer, whereas the percentage of polymorphic loci produced by ISSR marker was higher (94.06%) than that by SRAP marker. Results based on two markers both showed that observed number of alleles, effective number of alleles Nei's gene diversity, Shannon information index, genetic diversity and polymorphic loci rate of Albus Group were the highest among the 4 groups of O. fragrans, and those indexes of Luteus Group were the second. The sequence of polymorphic loci rate was: Albus Group> Luteus Group> Aurantiacus Group>Asiaticus Group. The genetic distance between Asiaticus Group and other 3 groups was far; the genetic distance between Aurantiacus Group and Asiaticus Group was the farthest while the genetic distance between Aurantiacus Group and Luteus Group was the nearest. Two markers also showed that most of the genetic variation existed among groups. Cluster results based on SRAP and ISSR markers were approximately consistent with taxonomic result based on morphology, and cluster result based on SRAP marker was more similar to morphological classification result. O. fragrans cultivars'Taoye Chi Yin','Zhuye Yin','Duolie Yin','Wuban Yin','Yin Shizi','Yuanban Zi Jin','Qiu Cheng'and'Yin Dan'from Wuxi Plum Garden, were different not only on morphological level but also on gene level compared with other cultivars, so they could be further classified as new cultivars.
4. Primary Phylogeny Study of O. fragrans Based on nrDNA ITS and cpDNA trnL-F Sequences
The ITS and trnL-F sequences of 4 cultivars representing 4 groups, wild species of O. fragrans were measured by using direct sequencing of PCR product, and sequences of 6 cultivars from Albus group were compared based on the measured sequences. The sequences of ITS and trnL-F were analyzed with O. heteropyllus and O. cooperi as outgroups, and genetic relationships of the 4 groups of O. fragrans was discussed primarily by setting up UPGMA tree based on the combination of two sequences. The length of ITS sequence of all tested materials was 620-650bp and an regular 617bp sequence was obtained by sequence comparison and sequence cutting on both ends. There were 17 variable sites, out of which 2 were parsim-info ones, accounting for 2.76% and 0.32% of the total length. The length of trnL-F sequence of all tested materials was 860-900bp and an regular 846 bp sequence was obtained by sequence comparison and sequence cutting on both ends. There were 11 variable sites, out of which 4 were parsim-info ones, accounting for 1.28% and 0.47% of the total length. ITS sequences of 6 cultivars from Albus Group varied slightly, so it was deduced that genetic relationships of cultivars in the same group can not be revealed by their ITS sequences.
Genetic relationships of the 4 groups of O. fragrans could be revealed to some extent based on the combination analysis of two sequences, with O. heterophyllus and O. cooperi as outgroups. Among autumn flowering cultivars, the genetic relationships between Aurantiacus Group and Asiaticus Group was the farthest, the genetic relationships between Aurantiacus Group and Luteus Group was the nearest. Among the 4 groups, the genetic relationships between Aurantiacus Group and wild species of O. fragrans was the farthest, which agreed with the conclusion drawn by several scholars that Aurantiacus Group is the most developed among autumn flowering cultivars based on morphological classification. Both ITS and trnL-F sequences had some variable sites that could be used for distinguishing O. heterophyllus and O. cooperi with O. fragrans (wild species and cultivars), or wild species with some cultivars of O. fragrans.
5. Cultivar Identification of O. fragrans Based on Morphology and DNA Fingerprinting Maps Based on Molecular Markers
Keys of O. fragrans cultivars based on morphology and DNA fingerprinting maps based on molecular markers can be used as morphological and molecular traits respectively for identification of O. fragrans germplasm.
Different cultivars of O. fragrans vary distinctively in morphology, in aspects like flowering season, flower character (inflorescence type, flower type, flower size, flower color, flower smell, stamen, pistil and fertility), fruit and seed, stem, bark, branch, leaf, etc., Some of these morphological characteristics are stable enough to serve as important taxonomic traits of morphology. The 88 cultivars belong to 4 groups, based on different flowering seasons, inflorescence types and flower colors. They are O. fragrans Asiaticus Group, O. fragrans Albus Group, O. fragrans Luteus Group and O. fragrans Aurantiacus Group. The keys to 88 cultivars were made based on their major morphological characteristics.
Special SRAP and ISSR markers of 88 cultivars, wild species of O. fragrans were obtained by 18 SRAP primer pairs and 28 ISSR primers, with O. heterophyllus and O. cooperi as contrast species. SRAP fingerprinting map in DNA of O. fragrans was constructed by two primer combinations pm17-em10 and sa15-em10; ISSR fingerprint map in DNA of O. fragrans was constructed by the combination of two primers ISSR12 and ISSR27. The result showed that 76 cultivars, wild species of O.fragrans, O. heterophyllus and O. cooperi had special SRAP bands, whereas 64 cultivars, wild species of O. fragrans, O. heterophyllus and O. cooperi had special ISSR bands, and these bands could serve as special SRAP and ISSR markers for molecular identification of O. fragrans germplasm. The SRAP and ISSR fingerprinting maps of O.fragrans constructed both can identify each of 91 germplasm and can be used as molecular marker traits for identification of O. fragrans germplasm. Identification of O. fragrans germplasm can be conducted more effectively and accurately by combining keys of O.fragrans cultivars based on morphology with DNA fingerprinting maps based on molecular markers.
The results from morphology, DNA markers and DNA sequences showed that O. fragrans germplasm is rich in genetic biodiversity. Different cultivars and groups vary not only on morphological level but also on molecular level. Genetic relationships between Aurantiacus Group and Asiaticus Group is the farthest, while the genetic relationships between Aurantiacus Group and Luteus Group is the nearest. Cluster results based on SRAP and ISSR markers showed that it is rational to divide cultivars of O. fragrans into four groups (Asiaticus Group, Albus Group, Luteus Group and Aurantiacus Group), but flower color is only one criterion, other characteristics also should be considered.
Analysis based on three aspects showed that cultivar classification, group evolution and genetic relationships of O.fragrans is rather complicated, and different methods might draw different conclusions. In order to solve above problems more scientifically, more methods like anatomy, palynology, quantity research, DNA molecular markers and DNA sequences should be combined and verified with each other, with sufficient and reliable materials.
引文
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