Asins M J, Bernet G P, Ruiz C, Cambra M, Guerri J, Carbonell E A
Instituto Valenciano de Investigaciones Agrarias, Apdo. Oficial, 46113 Moncada ,Valencia, Spain.
Theor Appl Genet. 2004 Feb;108(4):603-11. doi: 10.1007/s00122-003-1486-7. Epub 2003 Nov 12.
Citrus tristeza virus (CTV) has caused the death of millions of trees grafted on sour orange ( Citrus aurantium). However, this rootstock is very well adapted to the Mediterranean, semi-arid conditions. The aim of the present research is to genetically analyze the accumulation of CTV in a progeny derived from the cross between C. aurantium and Poncirus trifoliata, both resistant to CTV isolate T-346. Graft propagation of 104 hybrids was done on healthy sweet orange as a rootstock. Three months later, each rootstock was graft inoculated with two patches of infected tissue (isolate T-346). One, 2, and sometimes, 3 and 4 years after inoculation, hybrids and infected patches were tested for CTV by tissue-blot immuno-assay. Additionally, CTV multiplication was evaluated every year as the optical density of double-antibody sandwich enzyme-linked immuno-sorbent assay reactions. Linkage maps for P. trifoliata based on 63 markers, and for C. aurantium based on 157 markers, were used. Most molecular markers were microsatellites and IRAP (inter-retrotransposon amplified polymorphisms). Some analogues of resistance and expressed sequences were also included for candidate gene analysis. Resistance against CTV was analyzed as a quantitative trait (CTV accumulation) by QTL (quantitative trait loci) analysis to avoid the assumption of monogenic control. Three major resistance QTLs were detected where the P. trifoliata resistance gene, Ctv-R, had been previously located in other progenies. Up to five minor QTLs were detected ( Ctv-A(1) to Ctv-A(5)). A significant epistatic interaction involving Ctv-R(1) and Ctv-A(1) was also found. An analogue of a resistance gene is a candidate for Ctv-A(3), and two expressed sequences are candidates for Ctv-A(1) and Ctv-A(5). Single-strand conformational polymorphism analysis of CTV genes QTL P20 and P25 (coat protein) in susceptible hybrids, was carried out to test whether or not any QTL accumulation was a defeated resistance gene. Since the same haplotype of the virus was visualized independently on the CTV titer, differences in the amount of virions are not explained through the selection of CTV genotypes by the host, but through differences among citradias in CTV replication and/or movement.
柑橘衰退病毒（CTV）已导致数百万株嫁接到酸橙（Citrus aurantium）上的树木死亡。然而，这种砧木非常适应地中海半干旱条件。本研究的目的是对酸橙和枳（Poncirus trifoliata）杂交后代中CTV的积累进行遗传分析，这两个亲本均对CTV分离株T - 346具有抗性。以健康甜橙为砧木对104个杂种进行嫁接繁殖。三个月后，每个砧木用两块感染组织（分离株T - 346）进行嫁接接种。接种后1年、2年，有时3年和4年，通过组织印迹免疫分析对杂种和感染组织块进行CTV检测。此外，每年通过双抗体夹心酶联免疫吸附测定反应的光密度评估CTV增殖情况。使用了基于63个标记构建的枳的连锁图谱以及基于157个标记构建的酸橙的连锁图谱。大多数分子标记是微卫星和IRAP（反转录转座子间扩增多态性）。还纳入了一些抗性类似物和表达序列用于候选基因分析。通过数量性状位点（QTL）分析将对CTV的抗性作为数量性状（CTV积累）进行分析，以避免单基因控制的假设。检测到三个主要抗性QTL，之前在其他后代中已定位到枳的抗性基因Ctv - R位于这些QTL处。还检测到多达五个次要QTL（Ctv - A(1)至Ctv - A(5)）。还发现了涉及Ctv - R(1)和Ctv - A(1)的显著上位性相互作用。一个抗性基因类似物是Ctv - A(3)的候选基因，两个表达序列是Ctv - A(1)和Ctv - A(5)的候选基因。对易感杂种中CTV基因QTL P20和P25（外壳蛋白）进行单链构象多态性分析，以测试是否任何QTL积累是一个失效的抗性基因。由于在CTV滴度上独立观察到病毒的相同单倍型，病毒粒子数量的差异不是通过宿主对CTV基因型的选择来解释，而是通过柑橘属植物在CTV复制和/或移动方面的差异来解释。
