Molecular Phylogenetics of the Wild Perennial Relatives of Soybean (Glycine Subgenus Glycine): Intron-Containing Nuclear Genes

Glycine亚属（Leguminosae科）的野生多年生种是亚洲已知的与大豆Glycine max及其野生祖先Glycine soja最近的近亲。该亚属包括16种原产于澳大利亚，其中两种广泛分布于太平洋地区。它们分布在各种栖息地，包括澳大利亚中部非常干燥的地区。某些品种具有潜在的经济利益，作为抗旱和抗病的来源，因此已在美国和澳大利亚的栽培大豆育种计划中使用。甘氨酸亚属物种间的关系已经用许多方法进行了研究，分组（“基因组组”）主要是在人工杂交的基础上假设的。Jeff Doyle教授几年前使用叶绿体DNA （cpDNA）进行的研究为该亚属提供了第一个严格的系统发育假说。尽管结果与早期的生物系统研究基本一致，但在基因组分组方面存在一些分歧。然而，在基因组群中，cpDNA数据要么无法解决关系，要么产生与传统分类学界限不一致的假设。叶绿体证据可能给出“正确”的答案，在这种情况下，分类界限应该重新评估和重新调整；另外，cpDNA数据的家谱模式可能无法追踪亚属中物种形成的历史模式，这可能是由于几种可能的原因。使用分子（遗传）数据测试这些替代品需要使用能够显示历史独立于叶绿体基因组的DNA序列。最明显的选择是核基因；然而，除了核糖体基因位点外，一个属内密切相关的物种的低水平分化所需的核基因序列还没有得到。这是分子系统学中的一个普遍问题，并非甘氨酸所独有。核基因的非编码区理论上应该提供适合在这个分类水平上进行系统发育重建的变异。在各种类型的非编码区中，中间序列（内含子）具有技术优势，因为它们的两侧是编码区（外显子），其序列保守性较高，有利于设计用于聚合酶链反应（PCR）扩增的寡核苷酸引物。在初步工作中，Doyle教授已经证明，组蛋白H3-D的内含子在甘氨酸中提供了有用的系统发育数据；他和他的澳大利亚同事布朗博士计划利用这个基因位点的DNA序列来测试亚属中广泛的叶绿体和非分子假设。这些包括二倍体基因组群之间和内部的关系，以及亚属中至少三种多倍体复合体之一的起源和多样化。基本方法包括PCR扩增，直接或经过分子克隆的PCR产物的DNA测序，以及系统发育分析。

9614984 Doyle The wild perennial species of Glycine subgenus Glycine (family Leguminosae) are the closest known relatives to the soybean, Glycine max, and its wild progenitor, Glycine soja, in Asia. The subgenus comprises 16 species native to Australia, two of which are widespread in the Pacific region. They occur in a variety of habitats including very dry areas of central Australia. Particular species are of potential economic interest as sources of drought and disease resistance, and consequently have been used in breeding programs with the cultivated soybean both in the U.S. and Australia. Relationships among the species of subgenus Glycine have been studied using many approaches, and groupings ("genome groups") have been hypothesized primarily on the basis of artificial hybridizations. Prof. Jeff Doyle's studies some years ago using chloroplast DNA (cpDNA) provided the first rigorous phylogenetic hypotheses for the subgenus. Although results were in substantial agreement with earlier biosystematic studies, some areas of disagreement were noted with the genome groupings. Within the genome groups, however, cpDNA data either failed to resolve relationships, or produced hypotheses that were incongruent with traditional taxonomic boundaries. The chloroplast evidence could be giving the "right" answers, in which case taxonomic boundaries should be reassessed and realigned; alternatively, the genealogical pattern of cpDNA data may not be tracking the historical pattern of speciation in the subgenus, for any of several possible reasons. Testing these alternatives using molecular (genetic) data requires the use of DNA sequences capable of showing historical independence from the chloroplast genome. The most obvious choices are nuclear genes; however, apart from the nuclear ribosomal gene locus, nuclear gene sequences appropriate for the low levels of divergence expected for closely related species within a genus have not been available. This is a general problem in molecular systematics that is not unique to Glycine. Non-coding regions of nuclear genes theoretically should provide variation suitable for phylogenetic reconstruction at this taxonomic level. Of the various types of non-coding regions, intervening sequences (introns) afford technical advantages because they are flanked by coding regions (exons) whose higher degree of sequence conservatism facilitates the design of oligonucleotide primers for amplification by the polymerase chain reaction (PCR). In preliminary work, Prof. Doyle has shown that introns of one such locus, histone H3-D, provide useful phylogenetic data in Glycine; and he and his Australian colleague, Dr. Brown, plan to utilize DNA sequences from this genetic locus to test a wide range of chloroplast and non-molecular hypotheses in the subgenus. These include relationships among and within the diploid genome groups as well as the origins and diversification of at least one of the three polyploid complexes in the subgenus. The basic methodology includes amplification by PCR, DNA sequencing of PCR products either directly or after molecular cloning, and phylogenetic analysis.