基于流式细胞术对27种石斛的倍性鉴定和基因组大小分析

石斛属（Dendrobium）是兰科（Orchidaceae）一个大属，我国植物志记载有76种，主要分布于我国海南省、西南和两广地区，石斛属植物种类繁多，花朵鲜艳，颜色丰富，花期长，具有极高观赏价值。目前，石斛属植物野外资源濒临灭绝，面临自交异交不亲和、观赏种数量少、缺乏优良育种亲本的困境，为保护现有良种、培育新物种及为建立基因分子库奠定基础，以石斛属植物幼嫩叶片为材料，采用MGb解离液制备细胞核悬浮液，利用玉米（Zea mays, ‘B73’）和番茄（Lycopersicon esculentum）作为内标，建立了基于流式细胞术测定石斛倍性和基因组大小的方法，用流式细胞仪对兰科（Orchidaceae）石斛属（Dendrobium）27种石斛属植物的倍性和基因组大小进行测定。研究表明：以玉米为内标区分度较好，且没有重叠峰，峰型清晰集中，对27种石斛属植物的倍性和基因组大小能准确估测；27种石斛中二倍体19种，三倍体5种，四倍体3种；27种石斛分属于8组，分别是禾叶组1种，顶叶组2种，石斛组16种，瘦轴组1种，叉唇组1种，距囊组1种，草叶组4种，基肿组1种，各个组间基因组大小不同，各组内基因组大小也存在差异；27种石斛预估基因组大小在0.98~2.41 pg之间，预估的平均基因组大小为1.44 pg，27种石斛的基因组大小均大于0.7 pg，主要集中在0.7~1.4 pg之间，其中草石斛的预估基因组最大，梳唇石斛最小，二者的预估基因组大小相差近2.5倍；27种石斛的基因组可归为极小基因组或小基因组。该研究为石斛属植物的人工杂交授粉配置、 杂交育种、多倍体诱导提供便利，同时，为石斛属植物基因分子库的建立以及石斛属植物基因组学、遗传变异、进化生物学与全基因组测序等研究奠定基础。

Estimation Ploidy and Genome Size of 27 Dendrobium Species by Flow Cytometry

Dendrobium is a large genus within Orchidaceae, and there are 76 species recorded in China, mainly distributed in Hainan, southwest China, and Guangdong, Guangxi. Dendrobium species are highly ornamental because of the wide variety, bright, colorful flowers and long flowering period. At present, Dendrobium plant resources in the field are facing the dilemma of extinction, self-incompatibility, rare ornamental species and lack of good breeding parents. In order to protect the existing varieties, cultivate new species and establish a gene molecular library, the nuclear suspension was prepared by MGb dissociation solution from the young leaves of Dendrobium speceis, and maize (Zeamays, ‘B73’) and tomato (Lycopersicone culentum) were used as internal standard. At the same time, to establish a method for determining the ploidy and genome size of Dendrobium species based on flow cytometry, the ploidy and genome size of 27 Dendrobium species in Orchidaceae were determined by flow cytometry. The results show that it is better differentiation by using maize as internal standard, clear and concentrated which can accurately estimate the ploidy and genome size of Dendrobium species. Among 27 Dendrobium species, 19 species were diploid, 5 species were triploid and 3 species were tetraploid. The 27 Dendrobium species belong to 8 sections, including 1 species of sect. Grastidium, 2 species of sect. Chrysotoxae, 16 species of sect. Dendrobium, 1 species of sect. Breviflores, 1 species of sect. Stuposa, 1 species of sect. Pedilonum, 4 species of sect. Stachyobium and 1 species of sect. Crumenata. The estimated genome size of 27 Dendrobium species ranged from 0.98 pg to 2.41 pg, and the estimated average genome size was 1.44 pg. The estimated genome size of 27 dendrobium species were all greater than 0.7 pg, mainly between 0.7-1.4 pg. The estimated genome size of Dendrobium compactum was the largest, and that of Dendrobium strongylanthum was the smallest, and the difference between them was nearly 2.5 times; the genomes of 27 Dendrobium species can be classified as minimal genome or small genome. This study provides convenience for artificial cross pollination configuration, cross breeding and polyploid induction of Dendrobium species, lays a foundation for the establishment of gene molecular library, genetic variation, evolutionary biology and whole genome sequencing for Dendrobium species.