Molecular evidence for double maternal origins of the diploid hybrid Hippophae goniocarpa (Elaeagnaceae)

Homoploid hybrid plant species are rare, and the mechanisms of their speciation are largely unknown, especially for homoploid hybrid tree species. Two contrasting hypotheses have been proposed to explain the origin of Hippophae goniocarpa: (1) it is a diploid hybrid originating from H. rhamnoides ssp. sinensis x H. neurocarpa ssp. neurocarpa, and (2) it originated via marginal differentiation from H. rhamnoides ssp. sinensis. Regardless of which of these hypotheses is true (if either), previous studies have suggested that H. rhamnoides ssp. sinensis is the only maternal donor for this hybrid species. In this study, we aim to elucidate the maternal composition of H. goniocarpa and to test the two hypotheses. For this purpose, we sequenced the maternal chloroplast DNA trnL-F region of 75 individuals representing H. goniocarpa, H. rhamnoides ssp. sinensis, and H. neurocarpa ssp. neurocarpa in two co-occurring sites of the taxa. Seven haplotypes were identified from three taxonomic units, and their phylogenetic relationships were further constructed by means of maximum parsimony, maximum likelihood, and network analyses. These seven haplotypes clustered into two distinct, highly divergent lineages. Two haplotypes from one lineage were found in H. rhamnoides ssp. sinensis, and five (representing the other lineage) in H. neurocarpa ssp. neurocarpa. Hippophae goniocarpa shared four common haplotypes from both lineages, but the haplotypes detected from the two populations differed to some extent, and in each case were identical to local haplotypes of the putative parental species. Thus, both H. rhamnoides ssp. sinensis and H. neurocarpa ssp. neurocarpa appear to have together contributed to the maternal establishment of H. goniocarpa. These results clearly demonstrate that the marginal origin hypothesis should be rejected, and support the hybrid origin hypothesis. Hippophae goniocarpa exhibits a sympatric distribution with its two parent species, without occupying new niches or displaying complete ecological isolation. However, this species has effectively developed reproductive isolation from its sympatric parent species. Our preliminary results suggest that H. goniocarpa may provide a useful model system for studying diploid hybrid speciation in trees. (c) 2008 The Linnean Society of London.

高山松的二倍体杂种起源-来自三个基因组的证据

利用松科植物特殊的遗传体系（叶绿体基因组一父系遗传、线粒体基因组—母系遗传、核基因组一双亲遗传），我们对高山松及其两个亲本种进行了广泛的群体取样，通过线粒体基因nadl、叶绿体基因rbcL和trnL-F基因间区以及低拷贝核基因4CL的序列分析或PCR-RFLP分析，为高山松同倍体杂种起源假说提供了翔实的遗传学证据，同时在个体水平上探讨了高山松不同群体的遗传组成、群体遗传结构、基因交流方向、群体建立过程以及杂种基因组的进化。具体结果如下： 1．细胞质基因组分析 1)线粒体基因nudl分析 本研究对油松、高山松和云南松的19个群体、295个个体的线粒体基因nadl的一个内含子进行了序列分析或PCR-RFLP分析，共检测到3种线粒体DNA单倍型-A、B和C。油松所有的取样群体仅含单倍型A;除BX群体外，所有的云南松群体仅含单倍型B; 10个高山松群体中，5个群体固定单倍型A，4个群体固定单倍型B，1个群体(ZD)分布有A和B两种单倍型。2)叶绿体rbcL基因分析 对同一组群体的rbcL基因进行序列分析或PCR-RFLP分析，共检测到两个变异位点和三种叶绿体单倍型（TT、TC和GC）。TT和GC分别是油松和云南松种特异性叶绿体单倍型，而在高山松群体里则三种单倍型均有分布，而且TC单倍型广泛地分布在7个杂种群体中，该单倍型很可能来源于点突变或第三个已灭绝的亲本。rbcL基因检测到的高山松群体分化系数很高(Gst=0.533)。 3)叶绿体trn L-F区序列分析 叶绿体trnL-F分子标记检测到的不同单倍型的差异主要是由引物“e”下游120碱基处一个多聚T结构的长度变异所致（叶绿体SSR位点）。10个高山松群体中共检测到5种叶绿体单倍型，其中两种主要的单倍型(9T和11T)分别为油松和云南松的种特异性单倍型，其他单倍型均为非典型单倍型。群体遗传结构分析表明：杂种群体表现最高的遗传多样性，而且trnL-F分析得到的高山松群体的分化系数也很高( Gst=0.443)。 总之，对高山松、油松和云南松的同一组群体取样进行的细胞质基因组分析表明：高山松群体分布有油松和云南松种特异性的线粒体和叶绿体单倍型，该细胞质DNA单倍型的地理分布为假说“高山松为油松和云南松的的二倍体杂种”提供了翔实的遗传学证据。油松和云南松在不同的杂种群体中分别做父本和母本，即两亲本在杂交过程中发生了双向基因交流。群体遗传结构分析发现高山松群体表现最高的遗传多样性，而且群体间的分化系数很高。不同的杂种群体在遗传组成上的差异表明他们经历过不同的建立和进化历史。从线粒体和叶绿体单倍型的地理分布可以看出杂种群体的建立曾经历强烈的奠基者效应和回交。青藏高原的隆升对高山松的起源、杂种群体的适应辐射以及保持产生了重要的影响。川西南和滇西北作为青藏高原的东边边界，很可能是当初云南松和油松分布的重叠区及杂交地带，即高山松的起源地。 2．核基因4CL分析 对高山松、油松和云南松的19个群体、32个个体的低拷贝核基因4CL进行了克隆及序列分析，获得的78条序列可分为两种类型（类型A和类型B）。这两种类型明显的差别是类型A相对于类型B在内含子区有- 20bp的缺失。以华山松的3条序列为外类群，对得到的78条序列进行基因谱系分析，发现所有的序列分成明显的两支，分别对应于类型A和类型B，而且每一支均包含三个种的部分序列，表明4CL基因在这三个种分化之前就已发生重复。另一个明显的特点是某个种的一条序列与另一个种的序列比其与同种的其他序列关系更近，可能因基因交流（杂交和渐渗）、非共祖、致同进化和重组等进化事件所致。三种松树中共检测到4CL基因序列的两种类型和六个亚类型，高山松群体中没有发现杂种独特的类型或亚类型。高山松和云南松共享三种序列亚类型以及最多的序列多态性，表明这两个种之间曾存在广泛的基因交流。

高山松的群体历史及其适应性进化研究

物种形成一直以来是进化生物学的一个中心议题。因为杂交能够快速创造出新的遗传变异和提高基因组重组率，加速物种形成和适应性进化，所以杂交物种形成是其重要的组成部分。但是直到最近几年，人们才逐渐意识到同倍体杂种形成的重要性和普遍性。此外，人工杂交种群结果证实了杂种的适应性起源于亲本基因的超亲分离现象，这些基因之间通过加和效应或上位效应相互连锁，并在杂种后代中逐渐固定，因此，探讨维持这种适应机制的杂种的连锁不平衡模式就显得尤为重要。到目前为止，关于自然杂种的核苷酸多态性和连锁不平衡结构的报道非常稀见，对杂种的进化历史知之更少。高山松能生长于亲本种不能正常繁殖生长的青藏高原上，这种极端环境为杂种的适应性进化和与亲本的生态隔离造就了绝佳的条件，其生态和生殖的稳定性极大地方便了我们研究植物物种形成和适应性进化的遗传机理，对高山松开展的生态学、遗传学的系列分析已使高山松成为同倍体杂种形成的经典范例。 该论文通过随机挑选7个核基因位点对5个高山松群体、3个油松群体和3个云南松群体的164个大配子体单倍体基因组进行了核苷酸多态性分析。研究发现：所有基因座位的单倍型组成和基因谱系的拓扑结构都支持高山松的多次杂交起源。高山松的平均核苷酸多态性与油松相当，θW达到0.0107;比云南松高出一倍;高于已报道的裸子植物类群遗传变异水平。高山松如此高的遗传多态性与其杂交特性和有效群体大有关。我们运用分子钟和共祖模拟分析，推测出高山松的有效群体为7.32 x 105。等位基因共祖时间模拟分析发现，杂交过程早于青藏高原的隆升，也即是说，在杂种稳定成种以前，两个亲本种之间存在广泛的渐渗杂交。此外，高山松杂种的群体间分化严重，并且在不同的群体中，我们找到了多个偏离中性的基因座位。由此说明，高山松复杂的进化历史和适应的策略区域化。 在显著偏离中性的基因座位上，选取群体历史清晰的高山松群体，对其全基因序列多态性和连锁不平衡结构进行了深入调查，以期找到选择作用位点或区域。Ara-like和Dhn1两个基因在高山松与亲本之间都没有发现固定变异。在高山松中，Ara-like基因和Dhn1基因核苷酸多态性式样和连锁不平衡结构存在明显差异，这说明在两个基因上选择作用的程度和方向不尽相同。 我们对Dhn1基因的多态性分布、LD结构和中性偏离水平进行了分析，结果表明，该基因可能在PdNX和PdLZ群体中受到平衡选择的作用。从Dhn1基因谱系结构可以看出有两种来源的等位基因造成这种平衡多态性，一种属于祖先类型，另一种是从云南松继承衍生而来。这两种等位基因之间的分化很大，极有可能造成编码蛋白的亚功能化。事实上，Dhn1编码的脱水蛋白在植物对环境的抗逆过程中发挥着重要作用。由此我们推测高山松在高海拔极端环境中的适应性进化与Dhn1基因的平衡作用有关。 对Ara-like和Dhn1基因进行HKA检测，结果表明，与Dhn1基因相比，在Ara-like基因上，双维管束亚属与单维管束亚属之间存在显著分化，沉默突变位点的分化Ksil达到了0.1392，远远高于平均水平0.0508;在双维管束亚属共发现了43个固定突变位点，其中有6个能导致氨基酸突变，它们有可能导致了Ara-like基因功能的分化。结合裸子植物近缘物种间共享多态性的普遍性，我们推测Ara-like基因可能在单维管束亚属中的进化速率加快，暗示其在单维管束亚属中的适应性进化。 最后，基因内连锁不平衡分析结果显示，随机筛选的基因座位之间不存在连锁不平衡。高山松平均的基因内LD程度非常低，仅有18%的信息位点之间显著连锁。平均基因内LD在油松和高山松中的衰减速率很快，尤其在杂种中下降最快，在不到200bp以内就降到0.1以下。 LD的结果印证了高山松的有效群体大和多次起源特性。另外，我们也怀疑杂交物种形成过程中染色体组的重组和重排频繁发生，也是造成自然杂种现有群体的LD水平低的一个主要原因。 通过LD衰减曲线估计高山松的平均单位重组率,比亲本油松高17倍，比云南松高45倍。这个结果表明，杂种共适应的基因间要维持超亲分离，需要强烈的自然选择压力，才能保持它们之间的连锁不平衡。

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

Caracterização citogenética das espécies matrinxã (Brycon amazonicus), piraputanga (Brycon hilarii) e sua geração filial, utilizados na piscicultura brasileira

Pós-graduação em Ciências Biológicas (Zoologia) - IBB

Stable hybrid containing haploid genomes of two obligate diploid Candida species

Candida albicans and Candida dubliniensis are diploid, predominantly asexual human-pathogenic yeasts. In this study, we constructed tetraploid (4n) strains of C. albicans of the same or different lineages by spheroplast fusion. Induction of chromosome loss in the tetraploid C. albicans generated diploid or near-diploid progeny strains but did not produce any haploid progeny. We also constructed stable heterotetraploid somatic hybrid strains (2n + 2n) of C. albicans and C. dubliniensis by spheroplast fusion. Heterodiploid (n + n) progeny hybrids were obtained after inducing chromosome loss in a stable heterotetraploid hybrid. To identify a subset of hybrid heterodiploid progeny strains carrying at least one copy of all chromosomes of both species, unique centromere sequences of various chromosomes of each species were used as markers in PCR analysis. The reduction of chromosome content was confirmed by a comparative genome hybridization (CGH) assay. The hybrid strains were found to be stably propagated. Chromatin immunoprecipitation (ChIP) assays with antibodies against centromere-specific histones (C. albicans Cse4/C. dubliniensis Cse4) revealed that the centromere identity of chromosomes of each species is maintained in the hybrid genomes of the heterotetraploid and heterodiploid strains. Thus, our results suggest that the diploid genome content is not obligatory for the survival of either C. albicans or C. dubliniensis. In keeping with the recent discovery of the existence of haploid C. albicans strains, the heterodiploid strains of our study can be excellent tools for further species-specific genome elimination, yielding true haploid progeny of C. albicans or C. dubliniensis in future.

Cytogenetic markers as diagnoses in the identification of the hybrid between Piauçu (Leporinus macrocephalus) and Piapara (Leporinus elongatus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model "diploid embryogenic callus protoplast + diploid mesophyll-derived protoplast". Protoplasts were isolated from embryogenic calli of 'Pera' and 'Westin' sweet orange cultivars (Citrus sinensis) and from young leaves of 'Fremont', Nules', and 'Thomas' mandarins (C. reticulata), and 'Nova' tangelo [C. reticulata x (C. paradisi x C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when 'Pera' sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of 'Pera' sweet orange + 'Fremont' mandarin, 3 'Pera' sweet orange + 'Nules' mandarin, and 2 'Pera' sweet orange + 'Nova' tangelo, and all the diploid regenerated plants showed the 'Pera' sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96 h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.

Cytogenetic characterization of the Passiflora edulis Sims x Passiflora cincinnata Mast. interspecific hybrid and its parents.

The genus Passiflora L. consists of approximately 530 widely distributed species, including Passiflora edulis, which has drawn interest because of its commercial and agronomic value. Passiflora cincinnata is another important species owing to its long flowering period and resistance or tolerance to diseases and pests. In the present study, the meiotic segregation and pollen viability of an interspecific hybrid (P. edulis x P. cincinnata) and its parents were analyzed. The genomic contents were characterized using chromomycin A3 (CMA3)/40-60-diamidino-2-phenylindole (DAPI) staining, fluorescent in situ hybridization with 5S/45S ribosomal DNA (rDNA), genomic in situ hybridization (GISH), and inter-simple sequence repeat (ISSR) markers. The results indicated the diploid chromosome number for the parents and interspecific hybrid was 2n = 18. We also observed regular meiosis, one pair of S rDNA sites, and two pairs of 45S rDNA sites that colocalized with two pairs of CMA3 /DAPI- bands. The GISH data revealed three distinct chromosomal groups in the hybrid. The genetic origins of the interspecific hybrid, and its relationship with its parents were also confirmed using ISSR markers.

Determination of preventive maintenance lead time using hybrid analysis

The time for conducting Preventive Maintenance (PM) on an asset is often determined using a predefined alarm limit based on trends of a hazard function. In this paper, the authors propose using both hazard and reliability functions to improve the accuracy of the prediction particularly when the failure characteristic of the asset whole life is modelled using different failure distributions for the different stages of the life of the asset. The proposed method is validated using simulations and case studies.