Chattopadhyay Tirthartha, Sangam Surabhi, Akhtar Shirin
Department of Plant Breeding and Genetics, Bihar Agricultural College, Bihar Agricultural University, Sabour, Bhagalpur, Bihar 813210 India.
Department of Horticulture (Vegetable and Floriculture), Bihar Agricultural College, Bihar Agricultural University, Sabour, Bhagalpur, Bihar 813210 India.
3 Biotech. 2023 Mar;13(3):85. doi: 10.1007/s13205-023-03499-x. Epub 2023 Feb 14.
Insertion/deletion (InDel) markers are second most abundant polymerase chain reaction (PCR)-based molecular markers having enormous applications in genotyping and molecular breeding in different crops. Although standard polymerase chain reaction (PCR) for DNA amplification generally takes ~ 1.5 to 2 h, small amplicons can be effectively generated using dynamic heating and cooling through PCR with "V"-shaped thermal profile (VPCR) in ~ 15 to 20 min. Here, we evaluated the applicability of a partly modified VPCR method for amplifying InDels of tomato genome. Out of the 31 InDel markers tested in 15 diverse tomato genotypes, 29 markers resulted in sharp amplicons, where 26 markers were found to be polymorphic. Using this method, the individual DNA amplification reactions could be completed within ~ 30 min. The method was effective for primers varying in melting temperature ( ) and GC contents. Furthermore, the need for empirically determining suitable annealing temperature could be bypassed using this generalised thermal profile. Through our results, we advocate the use of this method of DNA amplification in other plants to achieve rapid genotyping using standard molecular biology equipments and procedures.
The online version contains supplementary material available at 10.1007/s13205-023-03499-x.
插入/缺失（InDel）标记是第二丰富的基于聚合酶链反应（PCR）的分子标记，在不同作物的基因分型和分子育种中具有广泛应用。虽然用于DNA扩增的标准聚合酶链反应（PCR）通常需要约1.5至2小时，但使用具有“V”形热曲线（VPCR）的PCR通过动态加热和冷却可在约15至20分钟内有效产生小扩增子。在此，我们评估了一种部分改良的VPCR方法用于扩增番茄基因组InDels的适用性。在15种不同番茄基因型中测试的31个InDel标记中，29个标记产生了清晰的扩增子，其中26个标记被发现具有多态性。使用该方法，单个DNA扩增反应可在约30分钟内完成。该方法对熔解温度（）和GC含量不同的引物有效。此外，使用这种通用的热曲线可以绕过凭经验确定合适退火温度的需求。通过我们的结果，我们提倡在其他植物中使用这种DNA扩增方法，以利用标准分子生物学设备和程序实现快速基因分型。
在线版本包含可在10.1007/s13205-023-03499-x获取的补充材料。
