Tang Shunxue, Kishore Venkata K, Knapp Steven J
Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331-3002, USA.
Theor Appl Genet. 2003 Jun;107(1):6-19. doi: 10.1007/s00122-003-1233-0. Epub 2003 Mar 25.
Simple sequence repeat (SSR) and other DNA sequence-tagged site markers can be genotyped more rapidly and cost efficiently by simultaneously amplifying multiple loci (multiplex PCR). The development of PCR-multiplexes for a nearly genome-wide framework of 78 SSR marker loci in cultivated sunflower ( Helianthus annuus L.) is described herein. The most outstanding single-locus SSR markers in the public collection (300 out of 1,089) were identified and screened for polymorphisms among 24 elite inbred lines, preparatory to selecting SSR markers for testing in multiplex PCRs. The selected SSR markers produced robust PCR products, amplified a single locus each, were polymorphic among elite inbred lines (minimum, mean and maximum heterozygosities were 0.08, 0.53 and 0.85, respectively), and supply a dense genome-wide framework of predominantly or completely codominant, single-locus DNA markers for molecular breeding and genomics research in sunflower. Thirteen six-locus multiplex PCRs were developed for 78 SSR marker loci strategically positioned throughout the sunflower genome (three to five per linkage group) by identifying compatible SSR primer combinations and optimizing multiplex PCR protocols. The multiplexed SSR markers, when coupled with 17 complementary SSR marker loci, create a 'standard genotyping' set ideal for first-pass scans of the genome, as are often needed when screening bulked-segregant DNA samples or mapping phenotypic trait loci. The minimum, mean and maximum heterozygosities of the multiplexed SSR markers were 0.38, 0.62 and 0.83, respectively. The PCR-multiplexes increase genotyping throughput, reduce reagent costs, and are ideal for repetitive genotyping applications where common sets of SSR marker loci are required or advantageous.
通过同时扩增多个位点（多重PCR），简单序列重复（SSR）和其他DNA序列标签位点标记能够更快速且经济高效地进行基因分型。本文描述了用于栽培向日葵（Helianthus annuus L.）中近全基因组框架的78个SSR标记位点的PCR多重体系的开发。在公共数据库中的1089个SSR标记中，鉴定并筛选出了最优秀的单一位点SSR标记（共300个），用于检测24个优良自交系之间的多态性，为选择用于多重PCR检测的SSR标记做准备。所选的SSR标记产生了稳定的PCR产物，每个标记扩增一个单一位点，在优良自交系中具有多态性（最小、平均和最大杂合度分别为0.08、0.53和0.85），并为向日葵的分子育种和基因组学研究提供了一个密集的全基因组框架，主要或完全是共显性的单一位点DNA标记。通过鉴定兼容的SSR引物组合并优化多重PCR方案，为分布于向日葵全基因组（每个连锁群3至5个）的78个SSR标记位点开发了13个六位点多重PCR。当多重SSR标记与17个互补的SSR标记位点结合时，可创建一个“标准基因分型”集，非常适合对基因组进行首次扫描，这在筛选混合分离DNA样本或定位表型性状位点时经常需要。多重SSR标记的最小、平均和最大杂合度分别为0.38、0.62和0.83。这些PCR多重体系提高了基因分型的通量，降低了试剂成本，非常适合需要或有利于使用常见SSR标记位点集的重复性基因分型应用。
