白术四倍体试管苗形态学及AFLP分析(1)
[摘要]为探明白术四倍体试管苗形态变异的遗传基础，以白术二倍体试管苗为试验材料，采用抽滤灭菌的秋水仙素浸泡0.5～1.0 cm的白术幼芽，在获得白术四倍体的基础上，比较其与二倍体在外观形态和气孔方面的差异，并采用AFLP技术探究两者基因组DNA的多态性。结果表明用0.1%的秋水仙素处理白术幼芽36 h，为获得白术同源四倍体的最适条件，四倍体诱导率高达32.0%;四倍体与二倍体植株在叶面积指数、叶长、叶宽等形态指标及保卫细胞叶绿体数、气孔大小、气孔密度等方面均存在显著差异;筛选出24对引物获得451条位于80～500 bp的DNA片段，多态性位点占总检测位点的32.59%。白术染色体加倍前后DNA碱基序列在 AFLP 水平上发生了改变，二倍体和四倍体试管苗在形态上的差异是DNA多态性反映的结果。
[关键词]白术;四倍体;基因组;AFLP
[收稿日期]2014-08-23
[基金项目]中央高校基本科研业务费基金项目(YZ201210)
, 百拇医药
[通信作者]*向增旭，副教授，主要从事药用植生物技术育种研究，E-mail：zxxiang@njau.edu.cn
[作者简介]王红娟，硕士研究生，E-mail：2012104201@njau.edu.cnMorphology and AFLP analysis of tetraploid plantlets of
Atractylodes macrocephala
WANG Hong-juan， LI Ya-ting， XIANG Zeng-xu*
(College of Horticulture， Nanjing Agriculture University， Nanjing 210095， China)
[Abstract]In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala， diploid plantlets were taken as experimental material， sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared， and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index， leaf length and width、the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers， the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala， and the differences in morphology of them were reflected in the DNA polymorphism.
, http://www.100md.com
[Key words]Atractylodes macrocephala; tetraploid; genome; amplified fragment length polymorphism
doi：10.4268/cjcmm20150307
白术是菊科苍术属多年生草本植物白术Atractylodes macrocephala Koidz.的干燥根茎[1]。近代药理研究发现白术具有抗肿瘤、抗衰老、免疫调节及抑制子宫平滑肌等作用[2-3]。目前我国白术人工栽培品种退化、产量低下、病虫害严重、药材品质越来越差，无法满足市场需求。因此，白术新品种的选育是目前生产中亟待解决的问题。多倍体育种是所有新品种选育方法中最有可操作性的一种手段，药用植物多倍体在自然界中表现出明显优势，其抗逆性强、营养部位大、活性成分含量高，长期以来受到广大育种专家的重视[4-5]。白术四倍体诱导虽有文献报道[6-7]，但前人的研究诱导率低(13.3%～15%)，新品种选育风险大，难以形成规模化生产。鉴于此，本文在前人研究基础上，对诱导剂的灭菌、诱导浓度及时间等进行了新的尝试，以期获得高产、优质、高抗的白术新品种。
, http://www.100md.com
近年来，国内外对白术的研究主要集中在化学成分[8-9]、药理作用[10-11]、炮制及鉴别[12-14]等方面，对白术四倍体试管苗形态、细胞及遗传学等方面的系统研究尚未报道。四倍体药用植物随着染色体的加倍，其核质比例发生变化，互作效应、基因的剂量效应等都会打破原有二倍体生理生化功能的平衡，从而使植株在形态指标、器官大小及活性成分含量等方面发生一系列变化[15]。为了阐明白术二倍体染色体加倍前后的DNA变化情况及四倍体试管苗外观形态呈现巨大性的变异机制，本文在研究获得白术同源四倍体的基础上，利用多态性检出较强的AFLP(DNA 扩增片段长度多态性)分子标记技术探究白术二倍体及其同源四倍体的DNA碱基序列变化，为阐述白术四倍体遗传变异机制奠定基础，以期为进一步开展白术四倍体优势品种选育及其大面积推广提供理论依据。, 百拇医药(王红娟 李雅婷 向增旭)
[关键词]白术;四倍体;基因组;AFLP
[收稿日期]2014-08-23
[基金项目]中央高校基本科研业务费基金项目(YZ201210)
, 百拇医药
[通信作者]*向增旭，副教授，主要从事药用植生物技术育种研究，E-mail：zxxiang@njau.edu.cn
[作者简介]王红娟，硕士研究生，E-mail：2012104201@njau.edu.cnMorphology and AFLP analysis of tetraploid plantlets of
Atractylodes macrocephala
WANG Hong-juan， LI Ya-ting， XIANG Zeng-xu*
(College of Horticulture， Nanjing Agriculture University， Nanjing 210095， China)
[Abstract]In order to investigate the genetic basis of morphological variation of tetraploid plantlets of Atractylodes macrocephala， diploid plantlets were taken as experimental material， sterile filtration colchicine was used to soak 0.5-1.0 cm long buds. The difference between morphology and stomatal of diploid and tetraploid of A. macrocephala was compared， and genome polymorphism was explored by AFLP. The results showed that the buds dipped in 0.1% colchicine solution for 36 h was optimal conditions to induce tetraploid of A. macrocephala with induction rate of 32.0%. Morphological indexes such as leaf area index， leaf length and width、the density of stomas and the number of chloroplast of tetraploid were distinctly different from diploid. Four hundred and fifty-one bands ranging with 80-500 bp were amplified with 24 pairs of primers， the rate of polymorphism was 32.59%. These amplification sites of diploid were different from tetraploid of A. macrocephala， and the differences in morphology of them were reflected in the DNA polymorphism.
, http://www.100md.com
[Key words]Atractylodes macrocephala; tetraploid; genome; amplified fragment length polymorphism
doi：10.4268/cjcmm20150307
白术是菊科苍术属多年生草本植物白术Atractylodes macrocephala Koidz.的干燥根茎[1]。近代药理研究发现白术具有抗肿瘤、抗衰老、免疫调节及抑制子宫平滑肌等作用[2-3]。目前我国白术人工栽培品种退化、产量低下、病虫害严重、药材品质越来越差，无法满足市场需求。因此，白术新品种的选育是目前生产中亟待解决的问题。多倍体育种是所有新品种选育方法中最有可操作性的一种手段，药用植物多倍体在自然界中表现出明显优势，其抗逆性强、营养部位大、活性成分含量高，长期以来受到广大育种专家的重视[4-5]。白术四倍体诱导虽有文献报道[6-7]，但前人的研究诱导率低(13.3%～15%)，新品种选育风险大，难以形成规模化生产。鉴于此，本文在前人研究基础上，对诱导剂的灭菌、诱导浓度及时间等进行了新的尝试，以期获得高产、优质、高抗的白术新品种。
, http://www.100md.com
近年来，国内外对白术的研究主要集中在化学成分[8-9]、药理作用[10-11]、炮制及鉴别[12-14]等方面，对白术四倍体试管苗形态、细胞及遗传学等方面的系统研究尚未报道。四倍体药用植物随着染色体的加倍，其核质比例发生变化，互作效应、基因的剂量效应等都会打破原有二倍体生理生化功能的平衡，从而使植株在形态指标、器官大小及活性成分含量等方面发生一系列变化[15]。为了阐明白术二倍体染色体加倍前后的DNA变化情况及四倍体试管苗外观形态呈现巨大性的变异机制，本文在研究获得白术同源四倍体的基础上，利用多态性检出较强的AFLP(DNA 扩增片段长度多态性)分子标记技术探究白术二倍体及其同源四倍体的DNA碱基序列变化，为阐述白术四倍体遗传变异机制奠定基础，以期为进一步开展白术四倍体优势品种选育及其大面积推广提供理论依据。, 百拇医药(王红娟 李雅婷 向增旭)