锦带花表型、花粉形态及ISSR分子标记比较研究
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摘要
锦带花(Weigela florida (Bunge) A. DC)是珍贵的观赏植物种质资源,在河北省主要集中分布在承德雾灵山地区,有粉色和白色两种花色。本研究以承德雾灵山区野生锦带花为材料,从枝、叶、花等器官的表型形态,孢粉形态和DNA分子标记3个方面分别对白色和粉色锦带花两个群体进行了分析研究。研究结果如下:
1.不同花色锦带花叶1/4宽、叶3/4宽、叶宽距叶基处的距离、叶尖角、叶基角以及花冠长等形态因子均有极其显著差异。
2.锦带花白花花粉粒为三突起不规则圆形,三个形状较规则萌发孔盖分布在对称的三个部位;粉花花粉粒为较规则圆形,有一个花粉萌发孔盖,形状不规则。两花色花粉粒大小差异明显,白花的花粉粒直径为80.9±2.9μm,粉花的花粉粒直径为77.7±1.2μm;花粉粒表面有锥状突出物,锥状突出物分布较白花分布密集。
3.建立了锦带花ISSR-PCR反应体系,并对体系中各个影响因素进行了优化,即在20μL的反应体系中, DNA模板20.00 ng/μL,0.20mmol/L dNTP,稀释10倍体积高保真PCR缓冲液含(Mg2+)3.00μL;1.00μmol/L引物,1.25UTaq酶。扩增程序为:94℃预变性5min;94℃变性30s,52℃退火45s,72℃延伸90s,45个循环;最后72℃延伸5min,4℃保温。用此反应体系扩增得到了锦带花的ISSR图谱。
4.采用ISSR技术,对2个花色锦带花群体的DNA进行扩增,每个引物扩增出谱带2-7条,平均扩增出4.5条谱带,产物大小均在200bp-2000bp之间,多态位点的数量为12,多态位点的百分比为100.00 %。利用POPGENE32软件分析得到,片段H上的等位基因频率在白色和粉色群体间有较大差异,白色群体出现的少,粉色群体出现的多;白色群体在不同的基因位点上A值变异大,粉色群体的Shannon’s信息指数高于白色群体,粉色群体的基因多样性较高;两个花色群体的无偏差遗传相似度(I)为0.6709,说明群体间的遗传变异较大;群体间的无偏差遗传距离为0.3991,群体间有一定的距离;群体之间Nm为1.5767,两种花色间存在基因流动。
Weigela florida is the precious genetic resources which is mainly distributed in the Wuling mountain in Hebei province with pink and white colors. In this study, the wild Weigela florida in the Wuling mountain was taken as the experiments materials. The comparative research had been conducted from the morphological phenotype including branches, leaves, flowers, the pollen morphology and DNA molecular markers between the two groups of wild Weigela florida species. The results showed as follows:
1. The 1/4 leaf width, the 3/4 leaf width, the distance from the leaf width to stem base, sharp leaf angle, basic leaf angle and corolla long between different color flowers were significantly different.
2. The white flowers pollen grains were three protuberant irregular circle and three germinal apertures distributed in three symmetrical parts. Pink flower pollen grains were rules circle and it had one pollen germination hole cover with irregular shape. The pollen grain size differences were also significant. The diameter of the white pollen grains was 80.9±2.9μm and pink is 77.7±1.2μm. Besides there were many cone protrusions in the pollen grain surface which were distributed more intensively in the pink flower compared with white population.
3. At the same time, the impacting factors of ISSR-PCR reaction system were optimized to establish the best reaction system for Weigela florida. That was in 20.μL reaction system, DNA template with 20.00 ng/μL, 0.20mmol/L dNTP, 10×Pfu PCR Buffer(Mg2+)3.00μl, 1.00μmol/L primer and 1.25U Taq enzyme.The optimal PCR amplification process was:5 minutes at 94℃for predenaturation, then followed by 45 cycles, each with 30 seconds at 94℃for denaturation, 45 seconds at 52℃for annealing, 90 seconds at 72℃for extension, finally extension at 72℃for 5 minutes and holding the samples at 4℃.
4. DNA sequences between different color groups were amplified by 12 primers which were candidate ISSR marker, and 2 to 7 bands could be amplified by each primer,an average of 4.5 per primer, product size were 200 bp to 2000 bp.The number of polymorphic loci was 12, polymorphic loci percentage for 100.00% . Using POPGENE 32 software, fragments of H allele frequency in white and pink between populations were quite different, white group was less. White flowers in different genetic loci group on A value had great variability.Shannon's information indexes of pink group were higher than white group, which was higher of the genetic diversity in pink group. Unbiased genetic similarity (I) of two color groups was 0.6709, which means that two color groups had higher genetic variation. Unbiased genetic distance between two groups was 0.3991. According to the result mentioned above, there was a distance between two populations. Two kinds of color populations existed the gene flow,1.5767 Nm between groups.
1.不同花色锦带花叶1/4宽、叶3/4宽、叶宽距叶基处的距离、叶尖角、叶基角以及花冠长等形态因子均有极其显著差异。
2.锦带花白花花粉粒为三突起不规则圆形,三个形状较规则萌发孔盖分布在对称的三个部位;粉花花粉粒为较规则圆形,有一个花粉萌发孔盖,形状不规则。两花色花粉粒大小差异明显,白花的花粉粒直径为80.9±2.9μm,粉花的花粉粒直径为77.7±1.2μm;花粉粒表面有锥状突出物,锥状突出物分布较白花分布密集。
3.建立了锦带花ISSR-PCR反应体系,并对体系中各个影响因素进行了优化,即在20μL的反应体系中, DNA模板20.00 ng/μL,0.20mmol/L dNTP,稀释10倍体积高保真PCR缓冲液含(Mg2+)3.00μL;1.00μmol/L引物,1.25UTaq酶。扩增程序为:94℃预变性5min;94℃变性30s,52℃退火45s,72℃延伸90s,45个循环;最后72℃延伸5min,4℃保温。用此反应体系扩增得到了锦带花的ISSR图谱。
4.采用ISSR技术,对2个花色锦带花群体的DNA进行扩增,每个引物扩增出谱带2-7条,平均扩增出4.5条谱带,产物大小均在200bp-2000bp之间,多态位点的数量为12,多态位点的百分比为100.00 %。利用POPGENE32软件分析得到,片段H上的等位基因频率在白色和粉色群体间有较大差异,白色群体出现的少,粉色群体出现的多;白色群体在不同的基因位点上A值变异大,粉色群体的Shannon’s信息指数高于白色群体,粉色群体的基因多样性较高;两个花色群体的无偏差遗传相似度(I)为0.6709,说明群体间的遗传变异较大;群体间的无偏差遗传距离为0.3991,群体间有一定的距离;群体之间Nm为1.5767,两种花色间存在基因流动。
Weigela florida is the precious genetic resources which is mainly distributed in the Wuling mountain in Hebei province with pink and white colors. In this study, the wild Weigela florida in the Wuling mountain was taken as the experiments materials. The comparative research had been conducted from the morphological phenotype including branches, leaves, flowers, the pollen morphology and DNA molecular markers between the two groups of wild Weigela florida species. The results showed as follows:
1. The 1/4 leaf width, the 3/4 leaf width, the distance from the leaf width to stem base, sharp leaf angle, basic leaf angle and corolla long between different color flowers were significantly different.
2. The white flowers pollen grains were three protuberant irregular circle and three germinal apertures distributed in three symmetrical parts. Pink flower pollen grains were rules circle and it had one pollen germination hole cover with irregular shape. The pollen grain size differences were also significant. The diameter of the white pollen grains was 80.9±2.9μm and pink is 77.7±1.2μm. Besides there were many cone protrusions in the pollen grain surface which were distributed more intensively in the pink flower compared with white population.
3. At the same time, the impacting factors of ISSR-PCR reaction system were optimized to establish the best reaction system for Weigela florida. That was in 20.μL reaction system, DNA template with 20.00 ng/μL, 0.20mmol/L dNTP, 10×Pfu PCR Buffer(Mg2+)3.00μl, 1.00μmol/L primer and 1.25U Taq enzyme.The optimal PCR amplification process was:5 minutes at 94℃for predenaturation, then followed by 45 cycles, each with 30 seconds at 94℃for denaturation, 45 seconds at 52℃for annealing, 90 seconds at 72℃for extension, finally extension at 72℃for 5 minutes and holding the samples at 4℃.
4. DNA sequences between different color groups were amplified by 12 primers which were candidate ISSR marker, and 2 to 7 bands could be amplified by each primer,an average of 4.5 per primer, product size were 200 bp to 2000 bp.The number of polymorphic loci was 12, polymorphic loci percentage for 100.00% . Using POPGENE 32 software, fragments of H allele frequency in white and pink between populations were quite different, white group was less. White flowers in different genetic loci group on A value had great variability.Shannon's information indexes of pink group were higher than white group, which was higher of the genetic diversity in pink group. Unbiased genetic similarity (I) of two color groups was 0.6709, which means that two color groups had higher genetic variation. Unbiased genetic distance between two groups was 0.3991. According to the result mentioned above, there was a distance between two populations. Two kinds of color populations existed the gene flow,1.5767 Nm between groups.
引文
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