배추와 무의 속간교배체에서의 배유 유래 교배 장벽에 관한 연구

학위논문 (석사)-- 서울대학교 대학원 : 농업생명과학대학 식물생산과학부(원예과학전공), 2019. 2. 허진회.Interspecific or intergeneric hybridization is vital to the evolution of flowering plants. However, interspecific or intergeneric hybridization often faces in hybridization barriers, resulting in seed abortion or sterility of F1 hybrids. In angiosperms, failure of endosperm development is one of the major causes of immediate hybridization barrier. To date, dysregulation of imprinted genes in developing endosperm has been proposed to be explain the endosperm-derived hybridization barrier. A general mechanism of hybridization barrier remains to be demonstrated. xBrassicoraphanus is an intergeneric hybrid of Chinese cabbage (Brassica rapa L.) and radish (Raphanus sativus L.). Newly synthesized xBrassicoraphanus F1 hybrids often display seed abortion accompanied with embryo arrest and underdeveloped endosperm. Here I report that intergeneric F1 hybrid between B. rapa and R. sativus showed endosperm-derived hybridization barrier. RNA-seq was conducted on intergeneric endosperm to identify imprinted genes related to hybridization barriers. Unexpectedly, the most of transcripts from intergeneric endosperm were maternal reads originated from B. rapa. Moreover, SCAR marker analysis revealed that intergeneric hybrid endosperm largely consisted of maternal genome derived from B. rapa. Therefore, these results suggested the possibility that the hybridization barrier in intergeneric F1 hybrids of B. rapa and R. sativus may be involved in the inhibition of fertilization between the central cell and sperm. These results provide further insights into the post-zygotic hybridization barrier between distantly related species in the Brassicaceae family.지구상의 많은 식물들은 종간이나 속간 교잡을 통하여 많은 종으로 분화가 되어 왔다. 일반적으로 교배 전 장벽 (Prezygotic hybridization barrier)과 교배 후 장벽 (Postzygotic hybridization barrier)에 의해 새로운 식물체를 얻는 것은 매우 힘들다고 알려져 있다. 특히 교배 후 장벽은 접합자 형성 이후 잡종의 발달과정이나 불임 등으로 생식적 격리가 일어나는 것을 말한다. 속간이질사배체 식물인 배무채 (xBrassicoraphanus)는 배추 (Brassica rapa L.)와 무 (Raphanus sativus L.) 사이에서 성공적으로 교잡되었다. 하지만, 배배양 (embryo rescue)을 통해서만 새롭게 배무채를 교잡할 수 있고, 이는 배유의 비정상적인 발달이 관련되어 있음을 시사한다. 본 연구에서는 배추와 무 사이의 이속간 교배에 있어서 배유 유래 교배 장벽을 연구하였다. 초기 배추와 무의 교잡 종자의 발달과정 관찰을 통해서 정상적인 배의 발달에 비해, 배유는 정상적으로 발달하지 못함을 확인하였다. 배유 유래 교배 장벽에 있어서 유전자 각인 현상이 관여한다는 최근 연구를 토대로 교잡 종자 배유의 전사체 분석을 하였다. 그 결과 모계의 전사체만을 확인하였다. 또한 SCAR 마커를 이용하여 교잡 종자의 배유에는 모계 유전체만이 존재함을 확인하였다. 정세포 (sperm)가 중심세포 (central cell)와는 수정되지 않고 난세포 (egg cell)와만 수정이 되어도 일시적인 배유 발달이 진행된다고 알려져 있다. 이와 비슷하게 배추와 무의 교잡 종자에서 수정된 배에 의해 일시적으로 미수정된 배유가 발달하는 현상을 보이고, 이후에는 정상적인 종자 발달을 하지 못하는 것으로 생각된다. 따라서 배추와 무의 이속간 교배에서는 배유의 미수정에 따른 교배장벽이 존재하는 것으로 추정된다.CONTENTS ABSTRACT ···························································· i CONTENTS ··························································· iii LIST OF TABLES ····················································· v LIST OF FIGURES ··················································· vi LIST OF ABBREVIATIONS ······································ viii INTRODUCTION ···················································· 1 LITERATURE REVIEWS ········································· 4 1. Hybridization barrier 2. Seed development 3. Autonomous endosperm MATERIAL AND METHODS ···································11 Plant materials Light microscopy RNA extraction for RNA-seq SNP calling Identification of imprinted genes DNA extraction and SCAR marker analysis RESULTS ·····························································16 Phenotypic analysis of intergeneric hybrid seeds Embryo and endosperm development in intergeneric hybrids Identification of imprinted loci in endosperm and embryo Double fertilization is disrupted by intergeneric hybridization barrier DISCUSSION·························································31 REFERENCES ······················································35 ABSTRACT IN KOREAN ········································44Maste