Blake N K, Sherman J D, Dvorák J, Talbert L E
Plant Sciences Department, Montana State University, Bozeman, MT 59717, USA.
Theor Appl Genet. 2004 Oct;109(6):1295-1302. doi: 10.1007/s00122-004-1743-4.
Common wheat (Triticum aestivum L.,2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat-rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence.Exon-anchored primers, which amplify across introns,allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT),and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (Ssl). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms(SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of theseSNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.
普通小麦（Triticum aestivum L.，2n = 6x = 42）是一种异源六倍体，由三个密切相关的基因组组成，分别命名为A、B和D。小麦的遗传分析很复杂，因为大多数基因以三倍体形式存在于同源染色体的相同染色体区域。本报告的目的是利用从小麦-水稻序列比较中收集的基因组信息，开发针对五个参与淀粉生物合成基因的基因组特异性引物组。通过将小麦cDNA序列与水稻基因组序列比对来推断小麦中的内含子位置。跨越内含子进行扩增的外显子锚定引物，使得能够对每个基因的三个基因组的内含子进行测序。三个小麦基因组内含子中的序列变异为基因组特异性引物设计提供了基础。对于三个基因，即ADP-葡萄糖焦磷酸化酶（Agp-L）、蔗糖转运蛋白（SUT）和蜡质（Wx），为所有三个基因组开发了基因组特异性引物组。针对Agp-S和淀粉合酶I（Ssl）这三个基因中的两个基因组开发了基因组特异性引物。因此，使用该策略开发了15个可能的基因组特异性引物组中的13个。七个基因组特异性引物组合用于扩增六倍体小麦品系中的等位基因以进行序列比较。在至少十个小麦种质的5093 bp比较中鉴定出三个单核苷酸多态性（SNP）。其中两个SNP可转化为酶切扩增多态性序列（CAPS）标记。我们的结果表明，利用基于内含子的序列差异设计基因组特异性引物组成功的可能性很高，而在一个基因组内等位基因间多态性的鉴定可能是一项挑战。
