Zaleski-Cox Marysia, Miklas Phillip N, Soler-Garzón Alvaro, Hoyos-Villegas Valerio
Department of Plant Science, McGill University, Montreal, QC, Canada.
Grain Legume Genetics and Physiology Research Unit, USDA-ARS, Prosser, WA, USA.
Plant Methods. 2023 Oct 3;19(1):102. doi: 10.1186/s13007-023-01071-5.
Common beans (Phaseolus vulgaris L.) provide important protein and calories globally. Anthracnose (Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889) is a major disease in common bean and causes significant yield losses in bean production areas. Screening for markers linked to known disease resistance genes provides useful information for plant breeders to develop improved common bean varieties. The Kompetitive Allele Specific PCR (KASP) assay is an affordable genetic screening technique that can be used to accelerate breeding programs, but manual DNA extraction and KASP assay preparation are time-consuming. Several KASP markers have been developed for genes involved in resistance to bean anthracnose, which can reduce yield by up to 100%, but their usefulness is hindered by the labor required to screen a significant number of bean lines. Our research objective was to develop publicly available protocols for DNA extraction and KASP assaying using a liquid handling robot (LHR) which would facilitate high-throughput genetic screening with less active human time required. Anthracnose resistance markers were used to compare manual and automated results.
The 12 bean anthracnose differential cultivars were screened for four anthracnose KASP markers linked to the resistance genes Co-1, Co-3 and Co-4 both by hand and with the use of an LHR. A protocol was written for DNA extraction and KASP assay thermocycling to implement the LHR. The LHR protocol reduced the active human screening time of 24 samples from 3h44 to 1h23. KASP calls were consistent across replicates but not always accurate for their known linked resistance genes, suggesting more specific markers still need to be developed. Using an LHR, information from KASP assays can be accumulated with little active human time.
Results suggest that LHRs can be used to expedite time-consuming and tedious lab work such as DNA extraction or PCR plate filling. Notably, LHRs can be used to prepare KASP assays for large sample sizes, facilitating higher throughput use of genetic marker screening tools.
普通菜豆（Phaseolus vulgaris L.）在全球范围内提供重要的蛋白质和热量。炭疽病（Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, 1889）是普通菜豆的一种主要病害，在菜豆产区会导致显著的产量损失。筛选与已知抗病基因连锁的标记可为植物育种者培育改良的普通菜豆品种提供有用信息。竞争性等位基因特异性PCR（KASP）分析是一种经济实惠的基因筛选技术，可用于加速育种计划，但手动DNA提取和KASP分析准备工作耗时较长。已经开发了几种针对参与菜豆炭疽病抗性的基因的KASP标记，炭疽病可使产量降低高达100%，但其实用性受到筛选大量菜豆品系所需劳动力的限制。我们的研究目标是开发使用液体处理机器人（LHR）进行DNA提取和KASP分析的公开可用方案，这将有助于以较少的人工操作时间进行高通量基因筛选。使用炭疽病抗性标记来比较手动和自动化结果。
通过手工和使用LHR对12个菜豆炭疽病鉴别品种进行了与抗性基因Co-1、Co-3和Co-4连锁的4个炭疽病KASP标记的筛选。编写了一份用于DNA提取和KASP分析热循环的方案以实施LHR。LHR方案将24个样品的人工筛选时间从3小时44分钟减少到1小时23分钟。KASP调用在重复实验中是一致的，但对于其已知的连锁抗性基因并不总是准确的，这表明仍需要开发更特异的标记。使用LHR，KASP分析的信息可以在很少的人工操作时间内积累起来。
结果表明，LHR可用于加快DNA提取或PCR板填充等耗时且繁琐的实验室工作。值得注意的是，LHR可用于为大量样品准备KASP分析，便于更高通量地使用基因标记筛选工具。
