Genetic and morphological variation and differentiation of South Korean natural populations of wild soybean, Glycine soja Sieb and Zucc

Genetic variation is the basis of crop improvement. As genetic background narrows in the cultivated germplasm, genes need to be introduced from new sources. Glycine soja is a wild relative of the cultivated soybean, glycine max (L.) Merr. It can be used in soybean breeding. Evaluation of wild soybean populations is not only necessary for use in cultivar improvement, will also provide information about origin, migration, evolution and natural selection of this species. Seeds were collected from six natural populations in South Korea to study the genetic variation and differentiation of wild soybean. The study was divided into two parts: (a) lab assay for 17 isozymes and one protein involving 35 loci; and (b) two-year greenhouse experiment, during which data for morphological traits were recorded. The average number of alleles per locus, 99% polymorphism and the expected heterozygosity in the total population were 2.1, 77.1% and 0.215, respectively. Nei\u27s gene differentiation (G\sb{\rm ST}) was 0.383. The average Nei\u27s genetic distance was 0.117. Populations were not significantly different in mean CV (coefficients of variation) for both years. However, 27 of the 33 individual morphological traits examined in 1989 and 31 of the 39 in 1990 differed significantly among populations. The average among-population variation per trait per degree of freedom was 84.38% in 1989 and 83.11% in 1990. Although there were no significant differences among populations in mean CV, those populations with high genetic variation also had high mean CV for morphological traits. There was no congruence between the isozyme and morphological data in terms of population relationships. Multilocus association analysis provided significant insight into the genetic structure of these natural populations. The analysis indicated that within each population, there were two to three dominant multilocus genotypes. The multilocus genotypes were biotypes at the morphological level. These different types might be the basic genetic division of mosaic self-pollinated plant populations, and the basic units in natural selection and evolution. The analysis also suggested that there was no migration among these six populations in recent history. The numbers of loci different between individuals were used as measures of genetic variation and genetic distance

Divergent northern and southern populations and demographic history of the pearl oyster in the western Pacific revealed with genomic SNPs

In the open ocean without terrain boundaries, marine invertebrates with pelagic larvae can migrate long distances using ocean currents, suggesting reduced genetic diversification. Contrary to this assumption, however, genetic differentiation is often observed in marine invertebrates. In the present study, we sought to explain how population structure is established in the western Pacific Ocean, where the strong Kuroshio Current maintains high levels of gene flow from south to north, presumably promoting genetic homogeneity. We determined the population structure of the pearl oyster, Pinctada fucata, in the Indo-Pacific Ocean using genome-wide genotyping data from multiple sampling localities. Cluster analysis showed that the western Pacific population is distinct from that of the Indian Ocean, and that it is divided into northern (Japanese mainland) and southern (Nansei Islands, China, and Cambodia) populations. Genetic differentiation of P. fucata can be explained by geographic barriers in the Indian Ocean and a local lagoon, and by environmental gradients of sea surface temperature (SST) and oxygen concentration in the western Pacific. A genome scan showed evidence of adaptive evolution in genomic loci, possibly associated with changes in environmental factors, including SST and oxygen concentration. Furthermore, Bayesian simulation demonstrated that the past population expansion and division are congruent with ocean warming after the last glacial period. It is highly likely that the environmental gradient forms a genetic barrier that diversifies P. fucata populations in the western Pacific. This hypothesis helps to explain genetic differentiation and possible speciation of marine invertebrates

초식 스트레스에 대한 쥐방울덩굴의 생태학적·분자생물학적 반응

학위논문 (박사) -- 서울대학교 대학원 : 사범대학 과학교육과(생물전공), 2021. 2. 김재근.Aristolochia contorta (Aristolochiaceae) is herbal vine species, which has the distinctive secondary metabolites of family Aristolochiaceae and the specialist herbivore Sericinus montela (swallowtail butterfly). To enhance the sustainability of co-existence of A. contorta and S. montela, interaction between the two species should be studied. To assess the response of A. contorta to the herbivory, I studied the ecological and molecular-biological aspects of A. contorta under the herbivory stress. First, I assessed genetic diversity of A. contorta populations to understand the long-term sustainability of A. contorta population. Genomic DNA samples of A. contorta leaf were used for analysis from four populations (CJ, GP, PT, and YJ) where the vigorous growth was observed in the South Korea. Intra-population genetic diversity and inter-population genetic distance were assessed using randomly amplified polymorphic DNA (RAPD). Overall intra-population genetic diversity was lower, compared to the other riparian plant species (h: 0.0607 ~ 0.1401; I: 0.0819 ~ 0.1759). Despite of the geographical distance, population GP showed the larger genetic distance from other populations. This result seemed to be caused by the fragmented habitat and lower sexual reproduction of A. controta. Secondly, I performed the mesocosm experiment to assess the phenotypic plasticity of A. contorta under the herbivory stress. Physical damage on the young leaf or mature leaf was applied to one-year-old A. contorta seedlings under two light availability conditions (daylight and shade condition). Light availability significantly affected the most of the morphological characteristics. Leaf damage seemed to induce the emergence of branch and new leaf. Biomass production also increased under leaf damage treatment. Compensatory growth effect of leaf, shoot, and biomass production seemed to be stronger when young leaves were damaged rather than mature leaves. The higher phenotypic plasticity to leaf damage was observed under the daylight treatment. These results indicate that A. contorta could show the vigorous growth under the moderate leaf damage stress with sufficient light. Subsequently, I tried to assess the transcriptomic response of the A. contorta under herbivory stress by de novo transcriptome assembly. Transcriptome of the A. contorta leaves under control, simple wounding (W+DW), and simulated herbivory with oral secretion of S. montela (W+OS) treatment were compared. In addition, systemic response was also assessed from the upper leaves (systemic leaf). Total 92,323 contigs were filtered, and 28,231 contigs could be annotated under Gene Ontology (GO) database. Over half of the total DEGs (1,875 of 3,177 contigs) differentially expressed only by W+OS treatment. Secondary cell wall seemed to be reinforced under both W+DW and W+OS treatments from the cell wall related terms and lignin biosynthesis pathway. Both W+DW and W+OS treatments seemed to trigger the reactive oxygen species (ROS), ethylene, and jasmonic acid related signaling pathway. Contigs which are predicted to be involved in general herbivory response such as polyphenol oxidase, chitinase, MYB transcription factors, and jasmonate O-methyltransferase were up-regulated under W+OS treatment. Biosynthesis of some secondary metabolites including alkaloids were predicted to be induced by herbivory, which could affect the generalist herbivores rather than the specialist herbivores. However, specific secondary metabolite biosynthesis of Aristolochia such as aristolochic acids seemed to be not induced by herbivory. This results suggest the major defense mechanism against specialist herbivore of basal angiosperms could be similar to the previously studied eudicots. From my study, A. contorta seemed to be able to co-exist with the specialist herbivore S. montela even under the herbivory stress with the compensatory growth and defense mechanism. On the other hand, genetic diversity of A. contorta population was relatively low. To enhance the sustainability of the co-existence of S. montela and A. contorta, proper environmental condition should be provided. Results from this study could contribute to the integrative understanding of plant response to herbivory as well as the conservation of plant-herbivore interaction.쥐방울덩굴(Aristolochia contorta)은 쥐방울덩굴과(Aristolochiaceae)에 속하는 덩굴성 초본식물로, 쥐방울덩굴과 특유의 이차 대사산물을 생산한다. 또한 쥐방울덩굴은 꼬리명주나비(Sericinus montela)의 유일한 기주식물이다. 쥐방울덩굴과 꼬리명주나비의 상호작용에 관한 이해는 두 종의 공존의 지속가능성을 이해하는 데 있어 반드시 필요하다. 본 연구에서는 초식에 대한 쥐방울덩굴의 반응을 파악하기 위하여 초식 스트레스 조건에서 쥐방울덩굴의 반응에 관한 생태학, 분자생물학적 현상에 관하여 알아보았다. 첫째로, 장기적인 쥐방울덩굴 개체군의 지속 가능성을 파악하기 위하여 개체군의 유전적 다양성을 조사하였다. 쥐방울덩굴 생육이 활발한 것으로 알려진 국내의 네 개체군에서 잎을 채집하여 유전체 DNA를 추출하여 분석에 사용하였다. 개체군 내 유전적 다양성과 개체군간 유전적 다양성은 5개 무작위 프라이머를 이용한 RAPD (randomly amplified polymorphic DNA) 방법을 사용하여 분석하였다. 수변 경계종임을 고려했을 때, 다른 습지식물들에 비하여 유전적 다양성이 상대적으로 낮은 것으로 나타났다 (h: 0.0607 ~ 0.1401; I: 0.0819 ~ 0.1759). 또한 지리적 거리와 무관하게 개체군 GP는 다른 개체군들로부터 유전적 거리가 큰 것으로 나타났다. 이러한 결과는 쥐방울덩굴의 파편화된 서식지 범위와 낮은 유성생식 비율에 기인한 것으로 보인다. 둘째로, 초식 스트레스에 의한 쥐방울덩굴의 표현형적 가소성을 알아보고자 메조코즘 실험을 수행하였다. 두 가지 빛 가용성 조건(일광 조건 및 그늘 조건) 하에서 생육 중인 일년생 개체의 어린 잎 혹은 성숙 잎을 손상시킨 후 생육 반응을 조사하였다. 상대광도는 대부분의 형태적 특성에 영향을 미쳤다. 잎 손상은 새로운 가지와 잎의 생성을 유도하는 효과를 보였다. 또한 잎 손상이 일어난 개체에서 생물량 또한 높은 것으로 나타났다. 이러한 잎 손상에 따른 보상 생장은 성숙 잎보다 어린 잎을 손상시켰을 때 더 크게 나타났다. 또한 표현형적 가소성의 경우 일광 조건에서 더 큰 것으로 나타났다. 이러한 결과는 적절한 빛이 주어졌을 때 쥐방울덩굴은 적정 수준의 초식 스트레스 하에서도 활발한 생장을 보일 수 있음을 시사한다. 마지막으로 쥐방울덩굴의 전사체의 de novo assembly를 수행하여 초식 스트레스 하에서 유전자 발현 양상 변화를 파악하고자 하였다. 대조군, 단순 잎 손상(W+DW), 손상 부위에 꼬리명주나비 유충 구강 분비물을 처리한 모의 초식(W+OS) 세 처리 조건에서 쥐방울덩굴 잎의 전사체 변화를 비교하였으며, 전신 반응(systemic response) 또한 비교하였다. 총 3,177개 DEG (differentially expressed genes) 중 절반 이상인 1,875개 DEG가 W+OS 처리가 가해진 조건에서만 다르게 발현되는 contig들에 해당하였다. 세포벽 혹은 리그닌 생합성 관련 term들의 발현 양상으로 미루어 보아, 두 처리조건 모두에서 세포벽 보강이 유도됨을 유추할 수 있었다. 또한 두 처리조건에서 모두 활성산소종(reactive oxygen species), 에틸렌, 자스몬산 등과 관련된 신호전달 경로를 활성화시키는 것으로 추정되었다. W+OS 조건에서 일반적인 초식 반응에 관여하는 것으로 알려진 polypenol oxidase, chitinase, MYB transcription factors, jasmonate O-methyltransferase 등의 발현량이 증가하는 것으로 나타났다. 알칼로이드계 물질을 포함한 일부 이차 대사산물들의 합성량이 초식에 의해 유도되는 것으로 보였으나, 아리스톨로크산과 같은 Aristolochia속 특이적인 이차 대사산물 발현과 관련된 유전자 발현은 유도되지 않는 것으로 나타났다. 이러한 결과로부터 기저 속씨식물에서 특이적 초식동물에 대한 방어기작은 일반적인 방어기작을 증가시키는 방향으로 유도되는 것으로 유추하였다. 본 연구로부터, 쥐방울덩굴은 특이적 초식동물인 꼬리명주나비의 초식에 대한 보상 생장과 방어 기작을 통해 공존할 수 있음을 알 수 있었다. 이러한 연구 결과는 식물-곤충 상호작용 관점에서의 보전과 관련된 기초적인 자료를 제공함과 더불어 초식에 대한 식물의 반응에 대한 다각적 이해에 기여할 수 있을 것이다.Abstract i LIST OF FIGURES vii LIST OF TABLES x Chapter 1. Introduction 1 1.1. Study Background 1 1.1.1. Plant-herbivore interaction and herbivory 1 1.1.2. Plant population under herbivory stress 1 1.1.3. Plant defense to herbivory 2 1.1.4. Secondary metabolites of basal angiosperms 3 1.1.5. Aristolochia contorta and its specialist herbivore Sericinus montela 3 1.2. Purpose of Research 4 Chapter 2. An analysis of the genetic diversity of a riparian marginal species, Aristolochia contorta 5 2.1. Introduction 5 2.2. Methods 7 2.2.1. Study site 7 2.2.2. Analysis of genetic diversity 7 2.3. Results and Discussion 9 2.3.1. Intraspecific genetic diversity 9 2.3.2. Genetic distance among populations 11 2.3.3. Genetic diversity of A. contorta and implications to the conservation 12 2.4. Conclusion 14 Chapter 3. Different growth response to the leaf damage under different relative light intensity in herbal vine Aristolochia contorta 15 3.1. Introduction 15 3.2. Methods 17 3.2.1. Growth condition and treatment 17 3.2.2. Measurement of growth characteristics 19 3.2.3. Phenotypic plasticity 19 3.2.4. Statistical analysis 20 3.3. Results 20 3.3.1. Growth characteristics under different light condition and herbivory 20 3.3.2. Phenotypic plasticity against leaf damage under different light condition 26 3.4. Discussion 30 3.4.1. Shade tolerance of Aristolochia contorta 30 3.4.2. Daylight enhance the phenotypic plasticity against leaf damage 30 3.4.3. Possible response of Aristolochia contorta against herbivory 32 3.5. Conclusion 33 Chapter 4. De novo transcriptome assembly of Aristolochia contorta reveals the defense strategy against its specific herbivore, Sericinus montela 34 4.1. Introduction 34 4.2. Methods 37 4.2.1. Plant materials and treatment 37 4.2.2. mRNA library construction 39 4.2.3. De novo assembly and functional annotation 39 4.2.4. Differentially expressed gene analysis 40 4.2.5. Ordination of differentially expressed genes 40 4.2.6. Validation of gene expression 41 4.3. Results 43 4.3.1. Overview of the A. contorta transcriptome 43 4.3.2. Gene expression among experimental conditions 44 4.3.3. Gene Ontology enrichment analysis 48 4.3.4. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis 56 4.3.5. Difference in the expression profile on the herbivory defense 61 4.3.6. Validation of gene expression by qRT-PCR 77 4.4. Discussion 83 4.4.1. Response to the special herbivory in the wounded leaf 83 4.4.2. Systemic response against special herbivory 86 4.4.3. Defense of the Aristolochia contorta against its specific herbivore 87 Chapter 5. General conclusion 93 Reference 95 Abstract in Korean 104 Appendix A. Detailed methods for RNA extraction of Aristolochia contorta 106 Appendix B. GO (gene ontology) barplot of enriched contigs under wounding or artificial herbivory treatment in A. contorta 108Docto

Morphometric, genetic and reproductive characteristics of mud crabs (genus Scylla de Haan, 1833) from Southeast Asia

The edible mud crab, genus Scylla, is important to fisheries and aquaculture throughout the Indo Pacific region, but its taxonomic status has been confused for decades and a new classification has only recently been proposed. This project was undertaken to investigate the species status of mud crabs in Southeast Asia, with a view to deciding whether two sympatric morphs of Scylla found in Ban Don Bay, Surat Thani Province, Thailand, are two separate species. A further aim was to elucidate any possible pre-zygotic reproductive isolating mechanisms (RIMs) and ecological features that maintain the apparent sympatry between these two morphs. Mud crabs were collected from a primary site (Surat Thani, Thailand) as well as from six other locations in Thailand, Vietnam, Malaysia and Bangladesh. Crab samples from the latter sites were used selectively to provide a comparison to the primary study site. Descriptive taxonomy, multivariate morphometrics and allozyme electrophoresis were used to a) determine the number of species present within the crab samples collected; b) to ascertain which species they represent; c) to discover any geographical variation between locations sampled; d) to produce a possible phylogeny that summaries the relationship between Scylla species; and e) to look for pre-zygotic RIMs to explain the sympatry of the two morphs in Surat Thani. Findings from the present study reinforce the recent revision of the taxonomy of the genus Scylla into four species, S. serrata, S. olivacea, S. tranquebarica and S. paramamosain and provides new information on two of the four species which are dominant within Southeast Asia, including Ban Don Bay, Surat Thani Province, S. paramamosain and S. olivacea. Population studies showed both genetic and morphological differentiation between conspecific populations of S. paramamosain and S. olivacea, indicating stock structure for each species, although there is some disparity between morphological and genetic distances for S. paramamosain. This is discussed in relation to the effects of larval dispersal mechanisms and the subsequent recruitment of juvenile crabs. Phylogenetic interpretation of both genetic and morphological characters revealed that both S. serrata and S. olivacea are the most diverged of the four Scylla species; however, the direction of evolution is open to interpretation and the evidence for either S. olivacea or S. serrata as the more primitive species are discussed. Reproductive studies on the two mud crab species found in Surat Thani revealed no physical barrier to hybridization. Both species have a protracted breeding season which continues throughout the year. However, the size at first sexual maturity was significantly smaller for S. olivacea when compared to S. paramamosain. This and other potential mechanisms that may maintain these two species sympatrically are discussed. The clarification of four Scylla species, and the establishment of diagnostic genetic and morphological characters that can be used to identify them, means that research can now focus on both the ecology and life history of these closely related species. Such information is needed urgently with respect to fisheries management as well as to understanding the environmental requirements of each species in order to develop their potential for aquaculture