Assessing genetic identity of sporophytic offspring of the brown alga Undaria pinnatifida derived from mono-crossing of gametophyte clones by use of amplified fragment length polymorphism and microsatellite markers

The haploid stage of gametophytes of the subtidal brown alga Undaria pinnatifida can be vegetatively propagated under favorable conditions. This unique characteristic makes it possible to establish independent gametophyte cell lines that are zoospore-derived. Sporophytic offspring can be generated through hybridizing the male and female gametophytes, which are derived from different cell lines. Accumulated experiences in this and other species in Laminariales demonstrated the applicability of this novel way to breed desired strains for open-sea cultivation. Sporophytic offspring originated from mono-crossing of male and female gametophyte clones were shown to have similar morphological characteristics under identical ambient conditions. However, there has been no report to relate this similarity on molecular levels. In this report, amplified fragment length polymorphism (AFLP) and microsatellite markers were used to analyze the genetic identity of sporophytic offspring of U. pinnatifida originated from two mono-crossing lines (M1 and M2), two self-breeding lines (S1 and S2) and one wild population (W). Totally 318 AFLP loci were revealed by use of 11 primer sets, of which 4.7%, 0.3%, 17.9%, 16.4% and 36.5% were polymorphic in M1, M2, S1, S2 and W, respectively. The pairwise genetic identity among the individuals of the same line was assessed. It was shown that offspring from mono-crossing lines had a higher degree of identity (95.6-100%) than self-breeding lines (87.7-98.4%) and the wild population (81.5-92.1%). Analysis by use of six microsatellite loci also revealed a higher genetic identity among individuals of the mono-crossing line, further confirming the results of AFLP analysis. Results from this investigation support, on molecular levels, the novel way to produce and maintain strains in U. pinnatifida by use of different gametophyte cell lines.

缘毛鸟足兰的生殖生态学研究

缘毛鸟足兰是在具有近20,000种的兰科中迄今为止报导过的雄性不育类型与两性类型共存的唯一例子。深入研究缘毛鸟足兰中雄性不育类型和两性类型共存的现象有助于丰富对兰科生殖系统演化的理解。本文通过生殖生态学研究，得出如下结论。 1. 证实了缘毛鸟足兰中只存在两性类型和雄性不育类型。雄性不育类型始终与两性类型出现在同一居群中，没有单独的雄性不育类型居群。雄性不育类型在调查的16个居群中，在6个居群中出现，约占38%。两种类型共存的居群中雄性不育类型的比率变化很大，从1%到78%，但只在一个居群中超过50%。 2. 通过交配系统实验发现，共存于同一居群中的短距雌性类型和两性类型都存在无融合生殖方式，种子的形成无需授粉。本文第一次报导了在兰科中存在同一无融合生殖种中两性类型和雄性不育类型共存于同一居群的现象。 3. 缘毛鸟足兰花部结构变异很大，结合性别和花的形态特征，将缘毛鸟足兰划分为6类生物型，分别是小花两性、大花两性、短距两性、短距雌性、长距雌性和三瓣雌性。 4. 在同一居群中短距雌性型和小花两性型在生殖和生态上存在差异：1)短距雌性型更能耐受不利的环境条件;2) 短距雌性型的花期早于两性型;3) 短距雌性型平均单株结实较多，但从平均单株产生的胚总数上看，两种类型之间却没有显著差异;4)在资源分配策略上，两种类型各有利弊。

甘蓝型油菜（Brassica napus L.）花粉和柱头的发育及其与自交不亲和性的关系

作者用扫描电镜、透射电镜和组织化学方法研究了甘蓝型油菜(Brassica napus L．)花粉和柱头的发育及相互作用,得到了如下结果： 一．甘蓝型油菜具有典型的同型孢子体自交不亲和性(Homomorphic and sporophytic self-incompatibility)。自花授粉后，部分花粉粘住在乳突细胞表面，随后萌发出花粉管，花粉管生长受阻于乳突细胞，花粉管表现出各种异常形态，缠绕卷曲(Coiled pattern),顶端膨大成基足状(pad-Like swelling),花粉管二叉分枝（dichotomous branching Pollen tubes），花粉管相互连接(connections of pollen tubes），有的花粉粒还形成两个花粉管。 二．不同时期的乳突细胞的扫描电镜观察表明： 开花前6—7天 乳突细胞壁内陷，柱头中央有一沟槽; 开花前4—5天 乳突细胞被有孔的块状物覆盖; 开花前2—3天 覆盖物消失，壁表面只剩下波状纹或小凹; 开花前l - 2天 乳突细胞充分吸水膨胀，呈指状，排列疏松，细胞壁上有一些小颗粒。此时的乳突细胞已发育成熟。 三.不亲和授粉时，花粉和乳突细胞均有强烈的胼胝质荧光。 四.乳突细胞的组织化学特点：缺乏淀粉积累，细胞壁和细胞质中有过氧化物酶活性。 五.乳突细胞的超微结构特点：细胞壁分为三层，蜡质层、角质层和纤维素层。粗面内质网成群分布在细胞壁附近，并以及泡形式向细胞壁分泌物质。缺乏质体，细胞核位于乳突细胞基部，细胞中央为大液泡占据。 六.花粉的超微结构发育特点：单核花粉已发育出内壁和外壁，外壁内层不明显。细胞核位于中央。细胞质浓厚，缺乏层膜结构而积累大量淀粉粒的质体存在于细胞质中。其他细胞器不发达。两细胞花粉时期，花粉壁接受乌氏体转运的孢粉素和含油体转运的脂类物质。生殖细胞没有壁，悬浮在营养细胞的细胞质中。细胞核大，细胞质稀薄，只有一些嵴不明显的线粒体。营养细胞的核显著，细胞质浓厚，线粒体发达，质体内部的淀粉消失，转变成嗜饿小体。内质网短而粗，遍布于细胞质中，高尔基体缺乏。 七.绒毡层积极参与了花粉外壁的建成。首先，它通过分泌作用把物质（可能是蛋白质）转移到单核花粉的腔隙中或在它后期滚解后，由分布在二细胞花粉间的粗面内质网合成蛋白质，转移到花粉壁内。其次，绒毡层细胞的质体层膜形成许多造油小体，至二胞花粉时，造油小体进入壁的柱状层，参与花粉鞘形成。第三，绒毡层细胞形成许多乌氏体，花粉发育后期，乌氏体与花粉外壁接触，将孢粉素转移到花粉外壁上。

通过体细胞杂交由大黍向水稻转移无融合生殖基因的研究

本论文以无融合生殖的大黍(Panicum maximum Jacp.)作为无融合生殖基因的供体，试图通过体细胞杂交方法向水稻(Oryza sativa L．)导入无融合生殖基因。结果如下：采用PEG融合法，诱导水稻原生质体与大黍原生质体融合，经过融合体筛选、培养，成功地获得了再生水稻植株。在融合前，水稻原生质体经过2.5 mM碘乙酰胺(IOA)在室温(22～25℃)条件下处理15分钟，大黍原生质体经过60Kr软x射线照射或不做任何处理。经双亲处理选择系统获得移栽成活的25株再生植株;经水稻单亲处理选择系统获得移栽成活再生植株3株。这两类融合再生植株（经双亲处理选择系统获得的25株和经单亲处理选择系统的3株）在花器官形态、结构及生殖特性上与对照亲本水稻植株有显著的差异，出现多花药（一朵颖花具7至11枚，甚至13枚花药）、多胚珠（一个子房内2～3个胚珠）及多胚囊（一个胚珠中2个以上胚囊）等现象;雌、雄性育性显著降低或完全消失，仅有5株能够少量结实，I-KI溶液着色的花粉从0至68%不等;胚胎学检查表明不能结实的植株雌性均不育，即不能分化出正常的胚囊结构。进一步的检查正在进行中。

水稻胚囊发育畸变的细胞学和遗传学研究

1．水稻多卵卵器的起源：被子植物的卵器中通常只有一个卵细胞。我们在水稻多胚品系胚囊中观察到二卵卵器和三卵卵器，本研究对其大孢子发生和胚囊发育进行了细胞胚胎学观察，揭示了水稻多卵卵器的起源．观察结果表明，该品系能进行正常的大孢子发生。大孢子母细胞进行正常的减数分裂形成四个大孢子靠近合点端的大孢子发育，其它三个退化。功能大孢子第一次有丝分裂后两个子核被一中央大液泡分隔在胚囊珠孔端和合点端，与此同时胚囊出现不均衡生长，珠孔端迅速膨大，合点端几乎不增大，致使二核末期的胚囊呈倒梨形．紧接着发生第二次有丝分裂，合点端核分裂时纺锤丝与胚囊纵轴平行，而珠孔端核分裂时纺锤丝与胚囊纵轴成4 5度夹角．由此产生的四核胚囊中，合点端一核向胚囊中部或中上部（胚囊珠孔端）迁移，四核胚囊再经一次有丝分裂形成两种类型的核分布偏离蓼型的八核胚囊。一种类型是珠孔端四个核，中部与合点各二个核，在胚囊细胞化过程中，珠孔端四核 分化成四细胞卵器，其中卵细胞和助细胞各二个，中部的二核分化成二极核中央细胞，合点 端的二核形成反足细胞。另一种类型是珠孔端六个核，合点端二个核，在胚囊细胞化过程中， 两端各一核向中部迁移分化成二极核中央细胞，珠孔端剩余的五核分化成五细胞卵器，其 中卵细胞三个，助细胞二个，合点端的一核迅速分裂形成反足细胞． 2．水稻同源三倍体TAR的生殖特性：TAR的单穗结实率平均可达10%，核型分析表明此三倍体产生的后代个体仍为具有36条染色体的三倍体．细胞胚胎学初步观察显示TAR为一具兼性无融合生殖特性的水稻新种质，其胚珠几乎都能进行胚囊的分化，但其中仅有33%的胚囊有较正常的结构，9%的胚囊在散粉前进行胚胎发生，58%的胚囊发育显著异常，表现为极性紊乱、多极核或缺失雌性生殖单位等。 3．水稻亚种间杂种败育的细胞学基础：对普通栽培稻不同品种类型间杂种颖花败育的细胞学基础及雌性败育的过程进行的细胞学研究表明：1）引起杂种颖花败育的原因有胚囊败育，花粉败育、开花时花药不开裂和雌雄异熟．其中胚囊败育而丧失受精能力是引起低结实率的最重要的因素，开花时花药不开裂和雌雄异熟在一定程度上形成了雌雄性细胞时间和空间的隔离屏障。2）杂种植株的所有大孢子母细胞都能进行正常的减数分裂形成四个大孢子，败育主要发生在靠近合点端的功能大孢子分化形成胚囊的早期，有的胚囊母细胞在进行第一次有丝分裂前便萎缩解体，多数能完成一次或二次有丝分裂形成二核或四核败育胚囊．败育的共同特征是无液泡的分化，细胞质少或退化，在败育胚囊残迹部位，解体的珠心细胞和萎缩的胚囊残溃混杂垛叠．已受精的杂种子房没有观察到胚及胚乳发育的异常．籼粳杂种胚囊败育频率较高． 4．籼粳杂种生殖障碍的基因定位：应用具有1 37个标记位点的籼粳杂交窄叶青8号/京系17)F1花药培养获得的127个双单倍体OH）群体构建的R FLP图谱，对控制籼粳杂种颖花败育的基因座位进行了定位研究。结果在第1、3、4、5、6、7、8、1 2染色体上检测到1 0个基因座位，其中第3、12染色体上的2个不育基因位点str3和str12与同一杂交组合F2分离群体中发现的异常分离热点处于相同的染色体区段．stj-6的基因加性效应为负值，有增加籼粳亲和性的作用;其余的不育基因座位皆有增加籼梗杂种不育性的作用． 5．籼粳杂种胚囊败育的遗传分析和基因定位：利用DH系构建的分子图谱及DH系衍生的2个回交群体定位了引起籼梗杂种胚囊败育的2个互补的主效基因esa-l（E1或e1位点）和esa-2（E2或e2位点），它们分别位于第6和第1 2染色体．在不育基因位点，籼稻基因型为EIEle2e2，粳稻基因型为elelE 2E 2，杂交后代中基因型为EIE2，Ele2、elE 2的雌配子体正常发育，携带ele2基因型的雌配子体表现败育．胚囊育性受配子体基因型控制，孢予体遗传背景影响胚囊败育基因的表达.

同源四倍体水稻雌配子体发育特性及变异胚囊研究

通过秋水仙素诱导获得同源四倍体水稻10个株系，包括6个恢复系、3个保持系和1个不育系，这些株系具有加倍的染色体组。田间观察表明10个株系具有特殊的农艺性状：茎杆变粗壮、植株颜色加深、叶片变厚、叶宽适度增加、分蘖数减少、有效分蘖的比率下降等。根尖有丝分裂鉴定表明，同源四倍体水稻10个株系具有正常的有丝分裂，观察细胞的染色体数目皆为2n=48。花粉母细胞减数分裂鉴定表明10个株系具有比较理想的减数分裂行为，后期I染色体滞后、末期I微核生成和末期II异常小孢子比率较低，能较好的完成减数分裂过程，其中后期I染色体滞后比率约为10%-20%，末期I微核生成比率约为1%-6%，末期II异常小孢子比率约为1%-8%。这提示，染色体联合和分离不规则导致三价体、单价体 和落后染色体等产生，并进一步导致在后期和末期不均横分离产生异常小孢子，这可能是同源四倍体株系结实率不高的原因之一。 同源四倍体水稻正常胚囊为蓼型，变异胚囊具有多种类型，其比率显著高于二倍体对照，变化范围为39.62%－69.85%。按变异胚囊的结构特点和形成方式，分为四种类型：退化型，结构变异型，无融合生殖型和反足细胞增殖型。退化型胚囊的平均比率为29.17%，包括小胚囊（15.04%）和完全退化胚囊（14.13%），前者仍有较小胚囊腔而后者胚囊腔缺失。结构变异胚囊包括结构缺失、结构重复和位置异常，反映了蓼型胚囊八核七细胞结构的变异，其在各株系的平均比率为18.96%。无融合生殖胚囊发生比率极低，平均比率为1.77%，类型包括反足胚和卵细胞胚。反足细胞增殖胚囊是反足细胞团频繁增殖形成，伴随上述三种变异发生使异常胚囊的多样性进一步增加，其在各株系的平均比率为10.62%。相关分析表明，同源四倍体水稻结实可能主要来自三部分：正常胚囊、正常型小胚囊和反足细胞增殖型胚囊。这三种胚囊具有相对完整的蓼型结构，可能具有较好的育性，其对结实率的贡献程度估计值分别为72.44%、15.12%、12.44%。此外，完全退化型胚囊和位置异常型胚囊对结实率分别表现出显著（-0.66）和极显著（-0.92）的负相关，这表明二者可能是结实性的抑制因素。 Ten autotetraploid strains, which include six restoring lines, three maintaining lines and a sterile line, are derived from artificial induction by colchicine treatments. Variations of agronomical traits are observed which include large organs, sturdy plants, long panicle length and deep leaf color and so on. It has been confirmed that autotetraploid strains exhibit normal chromosome behaviors in mitosis and the chromosome numbers are all 48. Moreover, abnormal chromosome behaviors are investigated in meiosis including univalent, trivalent, quatrivalent, chromosome lagging and microkernel and so on. It evaluates that the percentage of chromosome lagging in anaphase I is about 10%-20%, the percentage of microkernel in telophase I is about 1%-6% and the percentage of abnormal microspore in telophase II is about 1%-8%. In all, abnormal behaviors of chromosomes could induce univalent, trivalent and et al. and subsequently induce infertile microspore. That may be one of the causes of low seed sets in autotetraploid strains. Embryo sacs of autotetraploid strains are formed according to the Polygonum type. However, these strains exhibit variations of abnormal embryo sacs at high frequencies (39.62% - 69.85%). The variations are frequently involved in the spikelets of the main panicles and the first tillers, leading to obvious decreases of the percentages of normal embryo sacs among the strains. Four types of abnormal embryo sacs are classified basing on their different structures and origins: degenerated embryo sac (DES), structure variation (SV), apomixis (Apo) and antipodal cell proliferation (ACP). Embryo sacs of DES (29.17%) exhibit small embryo sacs (15.04%) or no embryo sac (14.13%), the former showing embryo sacs with decreased size and the latter showing no sac. Embryo sacs of AS (18.96%) include three subtypes: structure deletion, structure duplication and location variation, which suggests abnormalities of the eight nuclei, seven celled pattern of the Polygonum type. Embryo sacs of Apo (only 1.77%) include two origins of apomictic embryos: antipodal embryo and egg embryo. Embryo sacs of ACP are observed frequently (10.62%) in autotetraploid strains which subsequently increase the variations of abnormal embryo sacs. It evaluates by the Pearson’s correlation analysis that seed set is probably contributed by three origins of embryo sacs: normal embryo sacs, small embryo sacs (normal pattern) and embryo sacs of ACP. These three origins exhibit comparatively good structure of the Polygonum type and could account for seed set at a percentage of 72.44%, 15.12%, 12.44%, respectively. Moreover, the subtype of no embryo sac (NES) negatively related to seed set at the P>0.01 level (-0.92) and the subtype of location variation (LV) negatively related to seed set at the P>0.05 level (-0.66). Which suggest the two subtypes may have strong stress on seed set.

Responses of vegetative gametophytes of Undaria pinnatifida to high irradiance in the process of gametogenesis

The population of Undaria pinnatifida in its ecologic niche sustains itself in high temperature summer in the form of vegetative gametophytes, the haploid stage in its heteromorphic life cycle. Gametogenesis initiates when seawater temperature drops below the threshold levels in autumn in the northern hemisphere. Given that the temperature may fall into the appropriate range for gametogenesis, the level of irradiance determines the final destiny of a gametophytic cell, either undergoing vegetative cell division or initiating gametogenesis. In elucidating how vegetatively propagated gametophytes cope with changes of irradiance in gametogenesis, we carried out a series of culture experiments and found that a direct exposure to irradiance as high as 270 mu mol photons m(-2) s(-1) was lethal to dim-light (7-10 mu mol photons m(-2) s(-1)) adapted male and female gametophytes. This lethal effect was linearly corelated with the exposure time. However, dim-light adapted vegetative gametophytes were shown to be able tolerate as high as 420 mu mol photons m(-2) s(-1) if the irradiance was steadily increased from dim light levels (7-10 mu mol photons m(-2) s(-1)) to 90, 180 and finally 420 mu mol photons m(-2) s(-1), respectively, at a minimum of 1-3 h intervals. Percentage of female gametophytic cells that turned into oogonia and were eventually fertilized was significantly higher if cultured at higher but not lethal irradiances. Findings of this investigation help to understand the dynamic changes of population size of sporophytic plants under different light climates at different site-specific ecologic niches. It may help to establish specific technical details of manipulation of light during mass production of seedlings by use of vegetatively propagated gametophytes.

Programmed cell death in Laminaria japonica (Phaeophyta) tissues infected with alginic acid decomposing bacterium

TdT-mediated dUTP-biotin nick end labeling (TUNEL) is a sensitive and valid method for detecting DNA cleavage in programmed cell death (PCD). Using this method, DNA cleavage was observed in Laminaria japonica sporophytic tissues, which were infected with alginic acid decomposing bacterium. It was found that DNA cleavage occurred 5 min after the infection, the fragments with 3'-OH groups of cleaved nuclear DNA increased with time of infection and spread from the infection site. Although no typical DNA ladder (200 bp/ 180 bp) was detected by routine agarose gel electrophoresis, the cleavage of nuclear DNA fragments of 97 similar to 48.5 kb could be detected by pulsed field gel electrophoresis (PFGE). By using CaspGLOW(TM) fluorescein active caspase-3 staining method, caspase-3 activity has been detected in response to the infection of alginic acid decomposing bacterium. Our results are similar to the observations in hypersensitive response (HR) of higher plant, suggesting that the rapid cell death of L. japonica infected by alginic acid decomposing bacterium might be involved in PCD, and indicating that the occurrence of PCD is an active defense process against the pathogen's infection.

Effect of temperature and irradiance on the growth and reproduction of Enteromorpha prolifera J. Ag. (Chlorophycophyta, Chlorophyceae)

Effect of temperature and irradiance on growth and reproduction of Enteromorpha prolifera that bloomed offshore along the Qingdao coast in summer 2008, was studied. It was showed that E. prolifera propagated mainly asexually with specific growth rate (SGR) of 10.47 at 25A degrees C/40 mu mol m(-2)s(-1). Under this condition, gametes with two flagellate formed and released in 5 days. At the beginning of the development, the unicell gamete divided into two cells with heteropolarity, and then the apical cell developed into thalli primordial cells, whereas the basal cell developed into rhizoid primordial cells. In 8-day culture, the monoplast gamete developed into juvenile germling of 240 mu m in length. Unreleased gametes can develop directly within the alga body. E. prolifera could either reproduce through lateral branching or fragmenting except apomixis revealed by Microscopic observation. On aged tissue of E. prolifera, although the degraded pigments partially remained in faded algal filaments, numerous vegetative cells could still divide actively in the algal tissues.

Characterization of the life history of Bangia fuscopurpurea (Bangiaceae, Rhodophyta) in connection with its cultivation in China

Bangia fuscopurpurea (Rhodophyta) was cultivated in Putian (Fujian province, China). The characteristics of the life history concerned with cultivation were investigated and the cultivation procedure was presented. The gametophytic phase (thallus) and the sporophytic phase (conchocelis) occurred alternately in the life history of B. fuscopurpurea. Young thalli produced archeospores, and the number depended on the environmental factors. Temperature affected the number of archeospore release and percent of germination, and photo flux density (PFD) mainly affected the time of spore release and germination. Thalli matured from December to February and developed into the conchocelis phase through sexual reproduction. The conchocelis grown in shells had three developmental stages: vegetative conchocelis, conchosporangiall formation and conchospore formation. Pit-connections were present in the first 2 stages but absent after conchospore formation. Vegetative conchocelis and conchosporangial. branches can transform into each other. However, conchospores only developed into the gametophytic phase. Cultivation of B. fuscopurpurea was based on characterization of the life history, consisting of 3 steps: zygotospores collection, indoor cultivation of conchocelis and outdoor cultivation of thalli. Young thalli that developed from conchospores produced numerous archeospores before December. Over 90% of the crop was from the development of archeospores. The results indicated that conchosporelings were a good source of archeospores, and the development of the large quantity of archeospores acted as a more prevailing means to increase the population size. (C) 2008 Elsevier B.V. All rights reserved.