与大豆田间老化抗性连锁的分子标记的发掘及辅助选择应用研究

大豆种子成熟至收获期间如遇高温高湿天气,种子活力及活性会急剧下降,这就是所谓的田间老化(field weathering).田间老化是热带、亚热带地区大豆生产的主要限制因素之一.本研究旨在寻找与田间老化性状相连锁的DNA标记并将其应用于辅助选择育种.为此,利用修改的培养箱老化法和人工控制老化法对大豆品种Chiangmai 60(敏感),GC10981(抵抗)及其F2群体(139个体)进行了鉴定.在两种处理条件下,F2代群体的种子发芽率及活性均为正态分布,说明大豆种子田间老化抗性受多个基因控制.根据F2代个体的种子发芽率及活性,6个高抗个体及7个高感个体的DNA分别被混合为抗性池和感性池,并利用AFLP标记进行了混合群体分析(Bulk Segregant Analysis).从扩增的2162个标记中,发现了5个可能于大豆种子田间抗性相连锁的片段.通过DNA克隆和测序,设计了5对引物用于从大豆总DNA中扩增相应的片段.其中3对引物扩增的片段差异太小或未能扩增正确大小的片段,没能用于F2群体.引物Eaag/Mcac-233和Eact/Mctt-157能扩增出差异明显的多态性,通过对F2代群体的分析,这2个标记属同一连锁群,遗传距离为25.8cM.QTL分析结果显示有一个QTL位于这两个标记之间,距Eaag/Meac-233约14cM,可以解释29.7%的变异.用这两对引物对整个F2群体进行筛选,20个个体属于抗性群体,结合抗性鉴定的结果,7个个体被用于与Chiangmai 60进行回交.18个BC1F1个体(41.9%)的抗性高于其亲本的平均值.说明这些标记进行可以被用于大豆田间老化抗性的辅助筛选研究.

Developing DNA Markers for Assisting Selection of Field Weathering Resistance in Soybean

The deterioration of seed vigor as well as viability，due to high temperature and high relative humidity during the stages of the post-maturation and pre-harvest period is referred to as field weathering.Field weathering is the main limitation for producing high quality soybean seeds in the tropics and subtropics.The purpose of this study is to identify DNA markers linked to the field weathering resistant genes，and to develop markers for assisting selection in breeding program.The field weathering resistance of soybean variety Chiangmai 60(susceptible)，GC10981 (resistant)and 139 F2 progenies derived from the cross of CM60/GC10981 was tested by modified incubator weathering and the controlled deterioration treatment.The seeds germination and viability of F2 progenies showed normal distribution under both treatments.It hinted that the field weathering resistance was controlled by polygene.According to the seeds germination and viability of the F2 progenies，six extremely resistant plants and seven extremely susceptible plants were pooled for bulk segregant analysis by AFLP markers.Five field weathering resistance linked polymorphism were identified from 2162 AFLP markers.The 5 DNA fragments were cloned and sequenced.PCR primers were designed from the sequences to amplify the related DNA fragment from the genomic DNA of F2 progenies.It was found that marker Eaag/Mcac-233 and Eact/Mctt-157 were in the same linkage group with a genetic distance of 25.8 cM.A major QTL controlling the field weathering resistance was identified between these two markers.The QTL located at 14 cM from marker Eaag/Mcac-233 and explained 29.7% of the Cariation in field weathering resistance.These two DNA markers have been used for assisting selection in breeding program as an attempt.Seven F2 progenies were selected and backcrossed to CM60 using the developed markers in combination with field weathering resistance characters.The germination and viability of 18 BC1F1 progenies(41.9%)were higher than the mean of CM60 and GC10981 by controlled deterioration test.It is potentially possible to use these markers for assisting selection in breeding programs that focus on seed quality in the tropics.