Laroche A, Demeke T, Gaudet D A, Puchalski B, Frick M, McKenzie R
Agriculture and Agri-Food Canada, Lethbridge Research Centre, AB.
Genome. 2000 Apr;43(2):217-23.
In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) is effective against all the known races of common bunt caused by Tilletia tritici and T laevis. The genotypes of 199 F2 plants, originated from a cross between BW553 containing Bt-10 and the susceptible spring wheat cultivar 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bunt reaction, indicating that Bt-10 was expressed in a partially dominant fashion. A polymorphic DNA fragment, amplified using RAPD, and previously shown to be linked to Bt-10 was sequenced and SCAR (sequence characterized amplified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a polymorphic fragment of 490 bp resulting from a single base pair difference between lines possessing Bt-10 and those lacking the gene. As per the base pair difference, FSD and RSA primers were designed to generate a 275-bp polymorphic DNA fragment. Both 275- and 490-bp polymorphic fragments were present in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cultivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F2 population. Using Southern analysis, the 490-bp fragment was effective in differentiating homozygous resistant plants from those heterozygous for Bt-10, based on its presence and the hybridization signal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was also observed for the molecular marker and corresponded to 88% of the phenotypes deduced from the original F2 population. The molecular marker was estimated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, based on the segregation analysis. Segregation analyses of Bt-10 and the 275-bp marker, evaluated in three different Canada Prairie Spring (CPS) wheat populations, demonstrated a segregation ratio of 3:1 for the molecular marker in two of the populations. These results demonstrated that the PCR marker system using the FSD and RSA primer pair permitted a rapid and reliable identification of individual lines carrying the Bt-10 gene for resistance to common bunt.
在加拿大西部，小麦（普通小麦）中的Bt - 10抗性基因对由小麦网腥黑穗病菌和光腥黑穗病菌引起的所有已知小麦腥黑穗病生理小种均有效。通过温室和田间接种研究，确定了199株F2植株的基因型，这些植株源自含有Bt - 10的BW553与感病春小麦品种‘尼帕瓦’的杂交后代。对于腥黑穗病反应，观察到抗性∶杂合∶感病的比例为1∶2∶1，这表明Bt - 10以部分显性方式表达。利用随机扩增多态性DNA（RAPD）扩增出的一个多态性DNA片段，此前已证明其与Bt - 10连锁，对该片段进行了测序并设计了序列特异性扩增区域（SCAR）引物。然而，SCAR引物未能扩增出该多态性片段。用DraI酶切PCR产物，揭示出一个490 bp的多态性片段，它源于含有Bt - 10的株系与缺乏该基因的株系之间的单个碱基对差异。根据碱基对差异，设计了FSD和RSA引物以产生一个275 bp的多态性DNA片段。在已知携带Bt - 10的所有22个品种中均存在275 bp和490 bp的多态性片段，而在所有16个缺乏Bt - 10的品种中均不存在。在F2群体中，观察到275 bp标记的存在∶缺失比例为3∶1。通过Southern分析，基于490 bp片段的存在及其杂交信号强度，该片段可有效区分纯合抗性植株与Bt - 10杂合植株。对于分子标记，也观察到抗性∶杂合∶感病的比例为1∶2∶1，且与从原始F2群体推断出的表型的88%相符。基于分离分析，估计该分子标记与Bt - 10抗性基因的距离在1.1厘摩至6.5厘摩之间。在三个不同的加拿大草原春小麦群体中对Bt - 10和275 bp标记进行分离分析，结果表明在其中两个群体中该分子标记的分离比例为3∶1。这些结果表明，使用FSD和RSA引物对的PCR标记系统能够快速、可靠地鉴定携带Bt - 10基因以抗小麦腥黑穗病的个体株系。
