山核桃MITE序列识别和MITE-PCR反应体系的建立  

Identification of MITE-containing Sequences and Establishment of a MITE-PCR Protocol in Hickory

作　　者：夏晓庆 董昂 曾燕如[1] Xia Xiaoqing;Dong Ang;Zeng Yanru(The State Key Laboratory of Subtropical Silviculture,Zhejiang A&F University,Hangzhou,311300)

机构地区：[1]浙江农林大学,省部共建亚热带森林培育国家重点实验室,杭州311300

出　　处：《分子植物育种》2020年第19期6424-6432,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31370678);浙江农林大学省部共建亚热带森林培育国家重点实验室项目(ZY20180208);浙江省自然科学基金重点项目(LZ13C160002)共同资助。

摘　　要：微型反向重复转座元件(miniature inverted repeat transposable elements,MITEs)在植物中广泛分布,可产生适应性的遗传变异,尤其可在非重组无性繁殖物种中产生变异,可用于遗传多样性分析。为促进山核桃的遗传学研究,本研究运用MITE Digger软件对山核桃基因组测序数据进行扫描,共识别得到739条山核桃MITE序列。基于MITE序列,使用Primer-BLAST在线设计引物680对,占MITE序列的92.02%。基于小麦的MITE-PCR体系,通过降低退火温度5℃优化了山核桃的反应体系。随机挑选300对引物进行筛选,其中108对引物具有稳定的扩增产物。108对引物中100对引物在不同单株山核桃个体中扩增呈现单条带,占92.6%,8对引物扩增呈现双条带,占7.4%。PCR产物测序结果发现,呈现单条带的PCR扩增产物均包含MITE序列,但呈现双条带的PCR扩增产物中大多不含有MITE序列。研究结果为山核桃遗传多样性研究提供了科学依据。Miniature inverted repeat transposable elements(MITEs)are widely distributed in plants,which could lead to adaptively genetic variations a nd are associated with variation in non-recombinant,asexually propagated species.For genetic study of hickory(Carya cathayensis),the genomic sequencing data were scanned with MITE Digger and 739 MITE-containing sequences were identified,based on which 680 primer pairs(accounting for92.02%of the total MITE sequences identified)were designed with the online Primer-BLAST.A MITE-PCR protocol in wheat was optimized for hickory only by reducing an annealing temperature by 5℃.108 pairs of primers with stably amplified PCR(polymerase chain reaction)products were screened from 300 primer pairs selected at random.Out of these 108 primer pairs,100 pairs presented a single band,accounting for 92.6%and the rest(7.4%)presented double bands.Sequencing of some PCR products showed that single-band PCR products contained MITE sequences,but this was not the case with double-band PCR products,most of which didn’t contain MITE sequences.The results provide scientific basis for the genetic diversity study in hickory.

关 键 词：山核桃(Carya cathayensis) 转座子 MITE 

分 类 号：S664.1[农业科学—果树学]