茭白筍黑穗菌Ustilago esculenta交配型系統及其基因多型性

Abstract

茭白（Zizania latifolia Turcz.），為禾本科多年生水生植物，受到茭白筍黑穗菌（Ustilago esculenta）侵染，造成莖基部膨大，而成為常見且供食用的茭白筍。茭白主要分布於中國、日本、臺灣等地，在臺灣以青殼及赤殼品種為主。目前，茭白與共生黑穗菌的相關研究有限，而其近緣模式物種玉米黑穗病菌（Ustilago maydis）的研究指出，其感染與有性生殖有關，入侵菌絲的形成及病害的發展，受到交配型基因所調控，因此本研究首先探討U. esculenta 的交配型及其基因，與有性生殖之誘導。本研究收集臺灣茭白筍主要產區的品種，包括埔里的青殼種、三芝及金山之赤殼種與青殼種，在144株小生子分離株中，59株菌株來自筍體中發芽之厚膜冬孢子，其餘85株則直接分離自植物組織。由誘導厚膜冬孢子發芽，分離減數分裂產生的4個單倍體小生子菌株，經由有性生殖配對試驗，觀察菌絲之生成，確認其交配型為兩兩相同，並且其生殖菌絲與U. maydis型態相似，部份橫隔間細胞具有細胞質中空化的現象，以及前端生殖菌絲部分細胞具有雙核之特性。而為分析茭白筍黑穗菌交配型基因之組成及結構，利用PCR及NGS技術，找到U. esculenta交配型基因位點，目前發現臺灣菌株中，具有三種交配型對偶基因：a1b1、a2b2、a3b3，並且與Sporisorium reilianum黑穗菌的交配型基因位點序列具有高度共線性，其中基因包含2個費洛蒙基因、1個費洛蒙基因接收蛋白質，以及bE與bW基因。另外，在部分基因位點中，亦有Fot1 Family 之跳躍子基因插入。目前，利用PCR技術，大量篩選有性生殖後代菌株及田間分離株的交配型基因，發現子代間或菌株中，交配型對偶基因間並無重組現象，顯示其交配系統可能非原預期之四極異宗型式。而為了進行茭白品種選育工作，亦嘗試進行茭白筍黑穗菌的接種試驗，目前發現青殼品種植株之接種成功率較赤殼植株為高。

Zizania latifolia is a perennial aquatic plant. With the infection of smut fungus Ustilago esculenta, swollen stem is developed in Z. latifolia and becomes an edible vegetable, so called water bamboo. Z. latifolia is mainly distributed in China, Japan, and Taiwan. Green shell and red shell are two major cultivars currently grown in Taiwan. Although this crop has been grown for hundreds years, researches related to Z. latifolia and its smut fungus are limited. It is known that the infection process of related smut model fungus, Ustilago maydis, is coupled with sexual differentiation. Development of mating/infectious filaments and disease progression are under control of the mating type locus. Therefore, we aimed to characterize its mating system, mating type loci and induction of sexual development in U. esculenta. In this study, we collected plant materials from the major production areas in Taiwan, including green shell from Puli, red and green shells from Sanchi and Jinshan. In total of 144 U. esculenta strains collected, 59 were isolated from teliospores and 85 were directly isolated from stem tissues. By mating assay, we determined the mating compatibility and defined the mating type. In addition, morphological features of mating hyphae in U. esculenta were characterized. The features such as empty sections in its mating filaments and dikaryons at the front ends of hyphae, which are similar to those in U. maydis, were observed. To determine the mating system and genetic diversity of mating type in U. esculenta, we employed PCR and NGS approaches and identified the mating type locus. Three idiomorphs, including 2 mating pheromones, 1 pheromone receptor, and bW and bE genes were identified from the mating type loci of Taiwan isolates and they shared great synteny conservation with the mating type locus of Sporisorium reilianum. Besides, the Fot1 family transposons were inserted in some idiomrphs of U. esculenta. Idiomorph PCR screening among meiotic offsprings and field isolates found no recombination between idiomorphs, suggesting the heterothallic mating system of U. esculenta may not be tetrapolar as previously thought. In order to breed new variety of water bamboo, we conducted inoculation experiment. Preliminary data showed that green shell showed higher successful rate than red shell.