海南龙血树种质资源的初步评价
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摘要
海南龙血树不仅是“血竭”的基原植物,也是一种园艺绿化树种,素有“平安树”、“千年不老松”等美誉。近年来,受花卉产业发展和产业结构调整的影响,海南龙血树野生资源蕴藏量急剧减少,为对海南龙血树种质资源的保护、开发和良种选育奠定基础,本研究从形态学、生化标记、ISSR分子标记对已收集的96份海南龙血树种质资源多样性和亲缘关系进行了研究,此外亦对海南龙树细胞学和传粉生物学特性进行了研究。具体实验结果如下：
1.初步建立了海南龙血树种质资源的植物性状描述规范,通过对海南龙血树的叶长、叶宽、叶厚、叶间距等11个植物学性状的测量,聚类分析将已收集的96份海南龙血树分为4类。
2.摸索出有效的海南龙血树基因组DNA提取方法。本研究优化了的CTAB法所提取的基因组DNA纯度高、杂质少、完整性好,A260/A280介于1.7～1.9之间,能够满足ISSR等后续实验的需要。
3.通过正交试验设计建立了海南龙血树ISSR反应体系及扩增程序.最适的20μl反应体系为：DNA模板量60ng,M2+浓度1.5 mmol/L,dNTPs浓度O.25 mmol/L,引物浓度1.Oμmol/L,Taq酶用量1U；最适PCR扩增程序为：94℃预变性5 min,94℃变性30s,52.0℃退火45 s,72℃延伸70s,35个循环,72℃延伸8min,4℃保存。
4.利用筛选出多态性好的10条ISSR引物对96份海南龙血树进行PCR扩增,共产生134条DNA条带,多态性条带133条,占总条带数的99.25%；遗传相似系数(GS)介于0.5896～0.9851,表明收集到的96份海南龙血树的遗传变异较小,在GS=0.71时,将96份种质分为4个类群。
5.通过对形态差异较大的11份海南龙血树种质,进行POD同工酶的遗传多样性聚类分析,可将其分为六类,分类结果和ISSR分子标记及形态学标记相接近。
6.采用染色体压片技术对海南龙血树的染色体数目和核型研究表明：在饱和对二氯苯和0.002mol·L—18羟基喹啉预处理中,后者预处理2h效果最好；海南龙血树核型为：2n=2x=42=22m+20sm,“2B”型,不对称系数为62.67%。主要由中部和近中部着丝粒染色体组成,未观察到随体染色体。
7.对海南龙血树的花部特征和访花昆虫及访花行为、访花频率进行了研究,并对海南龙血树的传粉方式、结实和种子萌发情况进行了检测。结果发现,海南龙血树具有虫媒花的典型花部特征,共观测到访花昆虫5目26种,其中传粉昆虫23种,螟蛾科和夜蛾科是主要的传粉昆虫。在自然条件下,海南龙血树的结实率差异较大,观测到的最低为4.01%,最高为14.99%,在实验室条件下种子萌发率可达71.2%。
D. cambodiana is famous for its important economic values for using as raw materials in ornamental, medicament, and chemical industry. Nowdays, with high speedy development of flower-plant industry and adjustment of industrial organization, the problems of dwindling resources becoming more and more serious. With the aim of a clear understanding of protection and utilization of D. cambodiana resources, genetic improvement for breeding, means of morphological characters, POD isozyme analysis and ISSR marker were used to determine the genetic diversity of D. cambodiana, also cytological markers and pollination biology of D. cambodiana were studied in this paper. The results are as follows:
1. Firstly established the morphological description standard of D. cambodiana.The length, width, thickness of leaf and etc,11 morphological characters of 96 germplasm resources of D. cambodiana can bedivided into four clusters.
2. Try to find out an effective genome DNA extraction of D.cambodiana, the mean of Improved CTAB is better than the others terms of DNA extraction method, the DNA extracted by Improved CTAB has high-purity, low levels of impurity and good integrity, and the A260/A280 ranged from 1.7 to 1.9, ISSR fragments amplified with DNA template extracted by Improved CTAB method showed clear bands, and good polymorphic, stability and repetition.
3. The optimized ISSR reaction system was worked out by orthogonal design. Based on the result of PCR products tested on the agarosegel,an optimum reaction system obtained in the test was as follows:60 ng DNA template,1.5 mmol/L Mg2+,0.25 mmol/L L dNTPs,1.0μamol/L primer,1 U Taq polymerase,total volume of reaction system being 20μl. The PCR reaction procedure for ISSR analysis was as the follow:pre-denatuizing at 94℃for 5min, denaturizing at 94℃for 30s, annealing at 52.0℃for 45s, extension at 72℃for 70s, reaction of 35 cycles and re-extension at 72℃for 8 min and holding the samples at 4℃.
4. A total of 134 bands was obtained by 10 ISSR primers,133 were polymorphic bands, the average percentage of polymorphic bands was 99.25%. Genetic similarity coefficients were changed form 0.5896 to 0.9851. And 4 groups were classified by UPGMA method.
5.The POD isozyme of 11 accessions of D. cambodiana were analyzed by polyacrylamide electrophoresis, POD isozyme can be used the genetic diversity of D. cambodiana, and it is a valuable complement to other markers.
6.The chromosome number and karyotype of Dracaena cambodiana Pierre ex Gag nep was studied by applying fingertip pressing method.The effect was best when root tips were treated with 0.002mol·L-1 8-hydroxyquinoline for 2 hours among 2 types of pre-treatments. The karyotypes belonged to "2B" type.The karyotype formula was 2n=2 x=42=22m+20sm and the index of the karyotypic asymmetry 62.67%. The diploid kar yotype mainly consisted of metacentic and submetacentric chormosomes,but not found satellites.
7.The floral characteristics of Dracaena cambodiana Pierre ex Gagnepain, species of insect visitors and their visiting behavior, visiting frequency were investigated. The pollination type and status of fruit and seed germination were also tested. The rusults show that the floral characteristics of Dracaena cambodiana Pierre ex Gagnepain belong to typical entomophilous flower. Twenty-six species of visiting insect which belong to five orders are observed, and twenty-three are the pollination insects, in which one species of Pyralididae and one species of Noctuidae are the main pollination insects. In natural condition, the seed setting rate were difference of all Dracaena cambodiana Pierre ex Gagnep,the observations were changed form 4.01%to 14.99%, under laboratory conditions, the seed germination rate reached 71.2%.
1.初步建立了海南龙血树种质资源的植物性状描述规范,通过对海南龙血树的叶长、叶宽、叶厚、叶间距等11个植物学性状的测量,聚类分析将已收集的96份海南龙血树分为4类。
2.摸索出有效的海南龙血树基因组DNA提取方法。本研究优化了的CTAB法所提取的基因组DNA纯度高、杂质少、完整性好,A260/A280介于1.7～1.9之间,能够满足ISSR等后续实验的需要。
3.通过正交试验设计建立了海南龙血树ISSR反应体系及扩增程序.最适的20μl反应体系为：DNA模板量60ng,M2+浓度1.5 mmol/L,dNTPs浓度O.25 mmol/L,引物浓度1.Oμmol/L,Taq酶用量1U；最适PCR扩增程序为：94℃预变性5 min,94℃变性30s,52.0℃退火45 s,72℃延伸70s,35个循环,72℃延伸8min,4℃保存。
4.利用筛选出多态性好的10条ISSR引物对96份海南龙血树进行PCR扩增,共产生134条DNA条带,多态性条带133条,占总条带数的99.25%；遗传相似系数(GS)介于0.5896～0.9851,表明收集到的96份海南龙血树的遗传变异较小,在GS=0.71时,将96份种质分为4个类群。
5.通过对形态差异较大的11份海南龙血树种质,进行POD同工酶的遗传多样性聚类分析,可将其分为六类,分类结果和ISSR分子标记及形态学标记相接近。
6.采用染色体压片技术对海南龙血树的染色体数目和核型研究表明：在饱和对二氯苯和0.002mol·L—18羟基喹啉预处理中,后者预处理2h效果最好；海南龙血树核型为：2n=2x=42=22m+20sm,“2B”型,不对称系数为62.67%。主要由中部和近中部着丝粒染色体组成,未观察到随体染色体。
7.对海南龙血树的花部特征和访花昆虫及访花行为、访花频率进行了研究,并对海南龙血树的传粉方式、结实和种子萌发情况进行了检测。结果发现,海南龙血树具有虫媒花的典型花部特征,共观测到访花昆虫5目26种,其中传粉昆虫23种,螟蛾科和夜蛾科是主要的传粉昆虫。在自然条件下,海南龙血树的结实率差异较大,观测到的最低为4.01%,最高为14.99%,在实验室条件下种子萌发率可达71.2%。
D. cambodiana is famous for its important economic values for using as raw materials in ornamental, medicament, and chemical industry. Nowdays, with high speedy development of flower-plant industry and adjustment of industrial organization, the problems of dwindling resources becoming more and more serious. With the aim of a clear understanding of protection and utilization of D. cambodiana resources, genetic improvement for breeding, means of morphological characters, POD isozyme analysis and ISSR marker were used to determine the genetic diversity of D. cambodiana, also cytological markers and pollination biology of D. cambodiana were studied in this paper. The results are as follows:
1. Firstly established the morphological description standard of D. cambodiana.The length, width, thickness of leaf and etc,11 morphological characters of 96 germplasm resources of D. cambodiana can bedivided into four clusters.
2. Try to find out an effective genome DNA extraction of D.cambodiana, the mean of Improved CTAB is better than the others terms of DNA extraction method, the DNA extracted by Improved CTAB has high-purity, low levels of impurity and good integrity, and the A260/A280 ranged from 1.7 to 1.9, ISSR fragments amplified with DNA template extracted by Improved CTAB method showed clear bands, and good polymorphic, stability and repetition.
3. The optimized ISSR reaction system was worked out by orthogonal design. Based on the result of PCR products tested on the agarosegel,an optimum reaction system obtained in the test was as follows:60 ng DNA template,1.5 mmol/L Mg2+,0.25 mmol/L L dNTPs,1.0μamol/L primer,1 U Taq polymerase,total volume of reaction system being 20μl. The PCR reaction procedure for ISSR analysis was as the follow:pre-denatuizing at 94℃for 5min, denaturizing at 94℃for 30s, annealing at 52.0℃for 45s, extension at 72℃for 70s, reaction of 35 cycles and re-extension at 72℃for 8 min and holding the samples at 4℃.
4. A total of 134 bands was obtained by 10 ISSR primers,133 were polymorphic bands, the average percentage of polymorphic bands was 99.25%. Genetic similarity coefficients were changed form 0.5896 to 0.9851. And 4 groups were classified by UPGMA method.
5.The POD isozyme of 11 accessions of D. cambodiana were analyzed by polyacrylamide electrophoresis, POD isozyme can be used the genetic diversity of D. cambodiana, and it is a valuable complement to other markers.
6.The chromosome number and karyotype of Dracaena cambodiana Pierre ex Gag nep was studied by applying fingertip pressing method.The effect was best when root tips were treated with 0.002mol·L-1 8-hydroxyquinoline for 2 hours among 2 types of pre-treatments. The karyotypes belonged to "2B" type.The karyotype formula was 2n=2 x=42=22m+20sm and the index of the karyotypic asymmetry 62.67%. The diploid kar yotype mainly consisted of metacentic and submetacentric chormosomes,but not found satellites.
7.The floral characteristics of Dracaena cambodiana Pierre ex Gagnepain, species of insect visitors and their visiting behavior, visiting frequency were investigated. The pollination type and status of fruit and seed germination were also tested. The rusults show that the floral characteristics of Dracaena cambodiana Pierre ex Gagnepain belong to typical entomophilous flower. Twenty-six species of visiting insect which belong to five orders are observed, and twenty-three are the pollination insects, in which one species of Pyralididae and one species of Noctuidae are the main pollination insects. In natural condition, the seed setting rate were difference of all Dracaena cambodiana Pierre ex Gagnep,the observations were changed form 4.01%to 14.99%, under laboratory conditions, the seed germination rate reached 71.2%.
引文
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