Zheng X Y, Wolff D W, Baudracco-Arnas S, Pitrat M
Department of Horticulture Science, Texas Agricultural Experiment Station, The Texas A&M University System, 2415 East Highway 83, Weslaco, TX 78596, USA, US.
Theor Appl Genet. 1999 Aug;99(3-4):453-63. doi: 10.1007/s001220051257.
Fusarium wilt, caused by Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans, is a worldwide soil-borne disease of melon (Cucumis melo L.). Resistance to races 0 and 1 of Fusarium wilt is conditioned by the dominant gene Fom-2. To facilitate marker-assisted backcrossing with selection for Fusarium wilt resistance, we developed cleaved amplified polymorphic sequences (CAPS) and restriction fragment length polymorphisms (RFLP) markers by converting RAPD markers E07 (a 1.25-kb band) and G17 (a 1.05-kb band), respectively. The RAPD-PCR polymorphic fragments from the susceptible line 'Vedrantais' were cloned and sequenced in order to construct primers that would amplify only the target fragment. The derived primers, E07SCAR-1/E07SCAR-2 from E07 and G17SCAR-1/G17SCAR-2 from G17, yielded a single 1.25-kb fragment (designated SCE07) and a 1.05-kb fragment (designated SCG17) (the same as RAPD markers E07 and G17), respectively, from both resistant and susceptible melon lines, thus demonstrating locus-specific associated primers. Potential CAPS markers were first revealed by comparing sequence data between fragments amplified from resistant (PI 161375) and susceptible ('Vedrantais') lines and were then confirmed by electrophoresis of restriction endonuclease digestion products. Twelve restriction endonucleases were evaluated for their potential use as CAPS markers within the SCE07 fragment. Three (BclI, MspI, and BssSI) yielded ideal CAPS markers and were subsequently subjected to extensive testing using an additional 88 diverse melon cultigens, 93 and 119 F(2) individuals from crosses of 'Vedrantais' x PI 161375 and 'Ananas Yokneam'×MR-1 respectively, and 17 families from a backcross BC(1)S(1) population derived from the breeding line 'MD8654' as a resistance source. BclI- and MspI-CAPS are susceptible-linked markers, whereas the BssSI-CAPS is a resistant-linked marker. The CAPS markers that resulted from double digestion by BclI and BssSI are co-dominant. Results from BclI- and MspI-CAPS showed over 90% accuracy in the melon cultigens, and nearly 100% accuracy in the F(2) individuals and BC(1)S(1) families tested. This is the first report of PCR-based CAPS markers linked to resistance/susceptibility for Fusarium wilt in melon. The RFLP markers resulting from probing with a clone-derived 1.05-kb SCG17 PCR fragment showed 85% correct matches to the disease phenotype. Both the CAPS and RFLP markers were co-dominant, easier to score, and more accurate and consistent in predicting the melon phenotype than the RAPD markers from which they were derived.
尖孢镰刀菌黄瓜专化型（Fusarium oxysporum Schlecht f. sp. melonis Snyder & Hans）引起的枯萎病是一种在全球范围内发生的甜瓜（Cucumis melo L.）土传病害。对枯萎病0号和1号生理小种的抗性由显性基因Fom-2控制。为便于在回交育种中利用标记辅助选择甜瓜枯萎病抗性，我们分别将RAPD标记E07（1.25 kb条带）和G17（1.05 kb条带）转化为酶切扩增多态性序列（CAPS）和限制性片段长度多态性（RFLP）标记。对感病品系‘Vedrantais’中的RAPD-PCR多态性片段进行克隆和测序，以便构建仅扩增目标片段的引物。从E07衍生的引物E07SCAR-1/E07SCAR-2和从G17衍生的引物G17SCAR-1/G17SCAR-2，分别从抗病和感病甜瓜品系中扩增出一条单一的1.25 kb片段（命名为SCE07）和一条1.05 kb片段（命名为SCG17）（与RAPD标记E07和G17相同），从而证明了位点特异性关联引物。通过比较从抗病（PI 161375）和感病（‘Vedrantais’）品系扩增的片段之间的序列数据，首次揭示了潜在的CAPS标记，然后通过限制性内切酶消化产物的电泳进行确认。评估了12种限制性内切酶在SCE07片段内作为CAPS标记的潜力。其中三种（BclI、MspI和BssSI）产生了理想的CAPS标记，随后使用另外88个不同的甜瓜栽培品种、分别来自‘Vedrantais’×PI 161375和‘Ananas Yokneam’×MR-1杂交组合的93个和119个F2个体，以及以育种系‘MD8654’为抗性来源的回交BC1S1群体中的17个家系进行了广泛测试。BclI-CAPS和MspI-CAPS是感病连锁标记，而BssSI-CAPS是抗病连锁标记。由BclI和BssSI双酶切产生的CAPS标记是共显性的。BclI-CAPS和MspI-CAPS在甜瓜栽培品种中的准确率超过90%，在测试的F2个体和BC1S1家系中的准确率接近100%。这是关于甜瓜枯萎病抗性/感病性相关的基于PCR的CAPS标记的首次报道。用克隆的1.05 kb SCG17 PCR片段进行探针杂交得到的RFLP标记与病害表型的正确匹配率为85%。CAPS和RFLP标记都是共显性的，比其衍生的RAPD标记更易于评分，并且在预测甜瓜表型方面更准确、更一致。
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