Maternal inheritance of mitochondrial genomes and complex inheritance of chloroplast genomes in Actinidia Lind.: evidences from interspecific crosses

The inheritance pattern of chloroplast and mitochondria is a critical determinant in studying plant phylogenetics, biogeography and hybridization. To better understand chloroplast and mitochondrial inheritance patterns in Actinidia (traditionally called kiwifruit), we performed 11 artificial interspecific crosses and studied the ploidy levels, morphology, and sequence polymorphisms of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) of parents and progenies. Sequence analysis showed that the mtDNA haplotypes of F1 hybrids entirely matched those of the female parents, indicating strictly maternal inheritance of Actinidia mtDNA. However, the cpDNA haplotypes of F1 hybrids, which were predominantly derived from the male parent (9 crosses), could also originate from the mother (1 cross) or both parents (1 cross), demonstrating paternal, maternal, and biparental inheritance of Actinidia cpDNA. The inheritance patterns of the cpDNA in Actinidia hybrids differed according to the species and genotypes chosen to be the parents, rather than the ploidy levels of the parent selected. The multiple inheritance modes of Actinidia cpDNA contradicted the strictly paternal inheritance patterns observed in previous studies, and provided new insights into the use of cpDNA markers in studies of phylogenetics, biogeography and introgression in Actinidia and other angiosperms.     

Heterogeneity of three molecular data partition phylogenies of mints related to M. x piperita (Mentha; Lamiaceae)

Phylogenetic reconstructions with molecular tools are now widely used, thanks to advances in PCR and sequencing technologies. The choice of the molecular target still remains a problem because too few comparative data are available. This is particularly true for hybrid taxa, where differential introgression of genome parts leads to incongruity between data sets. We have studied the potential of three data partitions to reconstruct the phylogeny of mints related to M. x piperita. These included nuclear DNA (ITS), chloroplast DNA (non-coding regions trnL intron, intergenic spacers trnL-trnF, and psbA-trnH), and AFLP and ISSR, markers. The taxonomic sampling was composed of hybrids, diploid and polyploid genomes. Since the genealogy of cultivated mint hybrids is known, they represent a model group to compare the usefulness of various molecular markers for phylogeny inference. Incongruities between ITS, chloroplast DNA, and AFLP-ISSR phylogenetic trees were recorded, although DNA fingerprinting data were congruent with morphological classification. Evidence of chloroplast capture events was obtained for M. x piperita. Direct sequencing of ITS led to biased results because of the existence of pseudogenes. Sequencing of cloned ITS further failed to provide evidence of the existence of the two parental copy types for M. x piperita, a sterile hybrid that has had no opportunity for concerted evolution of ITS copies. AFLP-ISSR data clustered M. x piperita with the parent that had the largest genome. This study sheds light on differential of introgression of different genome regions in mint hybrids.     

几种猕猴桃种间杂交亲和性的探讨

以中华猕猴桃、毛花猕猴桃、美味猕猴桃及阔叶猕猴桃进行种间杂交,结果表明:各杂交组合的座果率与对照组一样,均为100％;父本花粉萌发率与对照组相近,其杂交当代果实重量、种子数量及种子千粒重也与对照组相近;相同的母本,其当代杂交果实的重量及种子数量与父本花粉萌发率的高低成正比。这不但说明了各杂交组合所采用亲本之间的亲和性,而且为生产上利用不同种生活力强的猕猴桃雄株花粉进行人工辅助授粉提供依据。     

Paternal chloroplast inheritance patterns in pine hybrids detected with trnL-trnF intergenic region polymorphism

The inheritance patterns of the chloroplast genomes of shortleaf pine (Pinus echinata Mill.), loblolly pine (Pinus taeda L.) and slash pine (Pinus elliottii Engelm.) were investigated through the trnL-trnF intergenic spacer polymorphism analysis. The DNA sequences of this spacer differ among these three closely related Pinus species. A modified 'cold' PCR-SSCP (single-strand conformation polymorphism) analysis of this spacer shows that the artificial hybrids (F1) from the shortleaf pine (seed parent) 2 loblolly pine (pollen parent) cross, exhibit the loblolly pine profile. Additionally, nine putative hybrids between shortleaf pine and loblolly pine, previously identified by the IDH (Isocitrate dehydrogenase) allozyme marker, presented the shortleaf pine profile indicating that shortleaf pine, not loblolly pine, sired all of the putative hybrids. Nondenatured polyacrylamide-gel electrophoresis of the trnL-trnF intergenic spacer demonstrated that the artificial hybrids (F1) from the cross, slash pine (seed parent) 2 shortleaf pine (pollen parent), present the shortleaf pine profile. Those results confirmed that the chloroplast genome is paternally inherited in these three species of the genus Pinus. The significance of the trnL-trnF intergenic region polymorphism and our modified 'cold' SSCP protocol for population genetic studies is discussed.     

Inheritance patterns of ITS1, chloroplasts and mitochondria in artificial hybrids of the seaweeds Fucus serratus and F. evanescens (Phaeophyceae)

Patterns of nuclear and organelle inheritance among artificial hybrids of the seaweeds Fucus serratus and F. evanescens were detected using single-strand conformation polymorphism (SSCP). Three alleles were identified in the 231?bp rDNA-ITS1 gene (nuclear): two in F. serratus and one in F. evanescens. Alleles differed by 1–2?bp and all hybrids possessed one allele from each parent. Two haplotypes were present in the 288?bp Rubisco spacer (chloroplast), differentiated by a 33?bp indel. Two haplotypes differing by a single nucleotide were found in a 135?bp region of nad11 gene (mitochondrion). Both organelles are maternally inherited, as all hybrids contained the haplotypes of the parent contributing the egg. Although laboratory hybrids among Fucus spp. have been produced previously, this is the first time that both nuclear and cytoplasmic genetic markers have been used to document inheritance patterns. SSCPs analysed on an automated sequencer offer a rapid and powerful approach for identifying suspected hybrids from field samples, as well as a screen for intraspecific and intra-individual variation in DNA regions prior to confirmation of variations by sequencing.     

基于DNA序列的白头翁属植物自然杂交证据

对长白山区兴安白头翁(Pulsatilla dahuricaSprengel)、朝鲜白头翁[P.cernua(Thunb.)Berchtold&Presl]以及其自然杂交种(P.dahurica×P.cernua)进行ITS和psbA-trnH序列分析,结果表明,自然杂交种与兴安白头翁遗传距离为0;ITS电泳显示,在6个信息位点的峰值同时具有朝鲜白头翁和兴安白头翁信息的双重峰,进一步证实前者是朝鲜白头翁和兴安白头翁的自然杂交种.     

甘草属种间杂交种叶绿体DNA父系遗传的发现及分析

通过对甘草属乌拉尔甘草(Glycyrrhiza uralensis)、光果甘草(G.glabra)、胀果甘草(G.inflata)及其人工杂交种组合G.uralensis♀×G.glabra♂、G.glabra♀×G.uralensis♂、G.uralensis♀×G.inflata♂、G.inflata♀×G.uralensis♂共68份材料的核基因ITS序列、叶绿体rbc L、mat K、trn H-psb A基因的序列分析,探讨了甘草属叶绿体DNA遗传方式。结果表明:(1)亲本种和人工杂交种ITS序列长度均为614 bp,其中34份人工杂交种ITS序列存在4处变异位点,且人工杂交种均检测出来自父本、母本ITS序列相同位点碱基的叠加,检测率为100%。(2)亲本种与人工杂交种的叶绿体基因rbc L、mat K、trn H-psb A序列长度相同,共有4处变异位点,人工杂交种在变异位点处的碱基与其相对应的父本碱基一致率高达97.1%。以上结果说明,该研究获得34份人工杂交种为100%杂交成功的F_1子代,核基因ITS序列可用于甘草属杂交种的遗传鉴定;甘草属叶绿体rbcL、mat K、trn H-psb A基因具有父系遗传特性,推测甘草属质体的遗传方式主要表现为父系遗传,这种质体遗传方式的发现为甘草属杂交种和遗传多样性研究提供了新的认识,也为杂交种的亲本鉴定提供分子依据。     

猕猴桃观花品种‘江山娇’与中华猕猴桃回交后代的性别分离与开花性状变异

以猕猴桃种间杂种品种‘江山娇’（Actinidia chinensis Planch×A.eriantha Benth）与中华猕猴桃（A.chinensis Planch）雄株杂交得到的杂交后代群体为实验材料,于2012、2013和2016年分别对该群体的雌雄性别比及其开花性状进行了调查。结果表明,杂交群体的雌雄性别比例小于1：1,即雄株偏多。杂交后代的花瓣颜色以红色为基础,但红色的分布区域、深浅及类型出现明显分离。聚类分析显示,这些杂交后代可通过花瓣CMYK色卡取值分为4个类群,其中猩红色与紫罗兰红色2个变异类型与观察表型完全一致。杂交后代群体的始花期、开花天数、花朵大小、开花量及单花花瓣数等性状均出现广泛分离,且因不同年份而出现变化。群体中杂交个体进入始花期的平均时间跨度为14 d,群体的始花期进入高峰时个体平均比例仅占群体的25.5%。2016年群体开花时间最长,最多有47.4%的个体开放10~13 d;2013年杂交群体的开放时间最短,有55.2%的后代开花3~5 d。本研究筛选出一批花瓣数多、花朵较大或单花序花朵较多的优良单株,并在后代群体中共发现21个含不同花数的花序组合类型。     

鸡爪槭种下分类群的DNA条形码筛选

槭属鸡爪槭(Acer palmatum Thunb.)是北温带广泛分布的一类园艺观赏树种,由于频繁的种内杂交渐渗导致其种下分类群的形态性状特征趋同,致使传统的形态学分类难以准确鉴定,新兴的DNA条形码技术为快速、准确的鉴定鸡爪槭种下分类群提供了新的思路。本研究采用5个叶绿体DNA片段(rpl16、psbA-trnH、trnL-trnF、rbcL、matK)和核基因组ITS片段,运用PWG-distance和Tree-Building两种方法对鸡爪槭的8个分类群共32个个体进行DNA条形码分析。结果显示,单个叶绿体基因组片段(分辨率为0%~25%)或核rDNA ITS片段(12.5%)的分辨率较低,不同组合的叶绿体DNA片段(0%~62.5%)、叶绿体片段与核rDNA ITS片段(12.5%~50%)的分辨率则相对较高。其中,rpl16+psbA-trnH+trnL-trnF片段组合的分辨率最高(62.5%),较为符合DNA条形码快速、准确鉴定的要求,因此建议将其作为鸡爪槭种下分类群鉴定的DNA条形码。     

Infrageneric relationships of Korean Viola based on eight chloroplast markers

Phylogeny and infrageneric relationships were reconstructed for Korean Viola(Violaceae)using eight chloroplast sequences: the atpB-rbcL intergenic spacer,the atpF-H intergenic spacer,the matK gene,the psbA-trnH intergenic spacer,the psbK-I intergenic spacer,the rpl16 intron,the rpoC1 exon 2,and the trnL-trnF regions.The combined analyses of the eight chloroplast regions suggest that sections Dischidium and Chamaemelanium are monophyletic,whereas section Nomimium is paraphyletic; sections Dischidium and Chamaemelanium form a clade with subsections Hypocarpae and Trigonocarpae of section Nomimium,but unresolved within the clade; two subsections(subsects.Bilobatae and Vaginatae)of section Nomimium form a clade and are placed between the stem and stemless species group; and subsection Patellares of section Nomimium is monophyletic,and Viola dissecta is diverged first in the clade with high support(BS = 100,PP = 100).Our present results support the subsectional grouping based on morphological characteristics,although discordance remained at the series level.     

杯萼海桑和格氏海桑自然杂交的分子证据

Interspecific hybridization has been frequently observed in the mangrove genus Sonneratia. However, no natural hybridization has been reported between Sonneratia alba and S. griffithii to date, despite their overlapping distribution in the coast of Andaman Sea. In this study, cysteine proteinase inhibitor gene (cpi) from the nuclear genome, and two intergenic spacers (trnL-trnF and trnV-trnM) from the chloroplast genome, were sequenced to determine whether natural hybridization took place between the two species. Our results revealed two distinct types of cpi sequences from the putative hybrid matching those acquired from S. griffithii and S. alba, respectively. Sequencing of the chloroplast trnL-trnF and trnV-trnM regions showed that S. alba differed from S. griffithii by one nucleotide in each region, and the putative hybrid had the identical sequences with S. griffithii. Molecular data demonstrated clearly that there indeed existed natural hybridization between S. alba and S. griffithii, and that S. griffithii was the maternal parent in this hybridization event.     

Maternal inheritance of plastids in Encephalartos Lehm. (Zamiaceae, Cycadales).

The mode of inheritance of chloroplast DNA has been determined in Encephalartos by employing a restriction fragment length polymorphism analysis of chloroplast DNA. Artificial F1 hybrids were produced between a female specimen of E. natalensis and a male specimen of E. woodii. The hybridization patterns of all hybrids correspond, in all cases, with that of E. natalensis, and are different from that of E. woodii, thus indicating the maternal inheritance of cpDNA in cycads.     

A case of chloroplast heteroplasmy in kiwifruit (Actinidia deliciosa) that is not transmitted during sexual reproduction

We report the first case of plastid chimera within the Actinidia genus, where plastid inheritance was believed to be paternal. The heterogeneity of chloroplast DNA observed in the hexaploid Actinidia deliciosa cultivar D uno involves the presence or absence of a particular MspI restriction site in the region between the psbC gene and the tRNA-Ser(UGA) gene. The heterogeneity was first observed using restriction fragment length polymorphism and then confirmed through cloning and sequencing. The analysis of the cloned fragments revealed the presence of two haplotypes: the most frequent type was found in 123 (88.5%) out of a total of 139 colonies screened. Partial sequences of the psbC-trnS fragment from both haplotypes revealed that the polymorphism occurs within the coding region of the psbC gene and consists of a synonymous transition. A contamination-free cross involving D uno as the male parent produced only plants characterized by the most frequent haplotype, indicating either selection bias against the rare type or more likely fixation of the frequent type in tissues leading to the formation of the male gametes. The MspI restriction profiles performed on various tissues suggest that the rarer type is absent from the histogenic layer LII and that D uno is a periclinal plastid chimera.     

The effect of 2n gametes on sex ratios in Actinidia

Sex can sometimes lead to complications. In some crops, 2n gametes have been exploited by plant breeders to transfer genetic variation between taxa of different ploidy levels. However, their role and use in dioecious genera have received relatively little attention. In the dioecious genus Actinidia (kiwifruit), seedling populations usually segregate equally for females and males as sex is determined by an XX female/XY male system. While fertilization involving 2n egg cells is not expected to affect the sex ratios of progenies, fertilization involving 2n pollen is likely to produce progenies with excess males. The extent of sex ratio distortion will depend on the relative contributions of first and second division restitution, and the frequency and location of cross-overs in meiosis. In this study, seedlings recovered from crosses between females of hexaploid Actinidia deliciosa and males of two diploid species, Actinidia chinensis and Actinidia eriantha, included a proportion of pentaploid hybrids presumably derived from fertilization involving 2n pollen. Most of these pentaploids were male, and a proportion of them were likely to be carrying two Y chromosomes. If used as parents in further crosses, males with multiple Y chromosomes are likely to cause distorted sex ratios in their immediate progenies. In dioecious genera such as Actinidia, the effects on sex ratios of different mechanisms of ploidy change need to be taken into account when considering the evolution of polyploidy and the design of breeding strategies involving ploidy manipulation.     

基于叶绿体单倍型的山东丹参居群遗传结构分析

依据叶绿体psbA-trnH基因间隔区的序列多态对来自山东地区丹参的3个自然居群和1个栽培居群进行了遗传结构研究.结果显示,80个供试材料中共有10种单倍型,核苷酸多样性为0.024,叶绿体DNA单倍型进化分支分析把10种单倍型分为2大支,但每支都由4个居群的单倍型共同构成;Nei多样性指数为0.284,Shannon信息指数为0.406,多态位点百分比率为70%;4个居群总基因多样性为0.905,居群内平均基因多样性指数为0.747,遗传分化系数为0.175;不同居群间的遗传分化系数为0.017～0.199,基因流为2.013～28.912;AMOVA分析揭示居群间只有5%的遗传差异.研究结果表明,山东丹参遗传多样性水平较高,但由于彼此较高的基因交流,居群间遗传分化水平低,仅有的遗传差异主要存在于居群内;另外,序列谱系关系分析表明山东栽培丹参来源复杂且具有丰富的遗传多样性.     

Incomplete paternal inheritance of chloroplast DNA recognized in Chamaecyparis obtusa using an intraspecific polymorphism of the trnD-trnY intergenic spacer region

Using a fluorescence-based PCR-SSCP (single-strand conformation polymorphism), we verified imperfectibility in the paternal inheritance of chloroplast DNA (cpDNA) in Chamaecyparis obtusa (Cupressaceae) controlled crosses. An intraspecific sequence polymorphism of the intergenic spacer region between the trnD and trnY genes was utilized as a molecular marker. Of 361 progenies, in which the cpDNA haplotypes of their female and male parents were different, 352 (97.5%) possessed the same haplotypes as their male parents, and nine (2.5%) the same haplotypes as their female parents. The parentage of the nine progenies with female parental types was diagnosed using DNA fingerprinting based on fluorescence-based RAPD profiles. Their parentage showed convincing evidence of the low frequency of maternal inheritance. Moreover, heteroplasmy was observed in the open-pollinated seeds collected in a seed orchard. The confirmation of maternal plastid transmission in the full-sib families and the observation of heteroplasmy in seeds reveal that the paternal inheritance of cpDNA is not an exclusive phenomenon and that the mode of its inheritance is biparental in C. obtusa. Received: 15 April 2000 / Accepted: 13 July 2000     

Phylogeography of the widespread plant Ailanthus altissima (Simaroubaceae) in China indicated by three chloroplast DNA regions

Ailanthus altissima (Mill.) Swingle, a temperate tree species, has a wide distribution in China. To infer its refugia and patterns of migration during past climatic changes in China, genetic variations among different populations were studied. Gene sequences of three chloroplast DNA spacer regions, psbA-trnH, trnL-trnF, andtrnD-trnT, were obtained from 440 individuals of 44 populations. The distribution of haplotype and the relationships among them were investigated by haplotype network. In addition, the genetic diversity of the sampled regions was inferred, and the biogeographic history was also reconstructed. Twelve haplotypes were identified, among which, five were unique. The phylogenetic analysis and geographical distribution of haplotypes indicate that multiple glacial refugia existed in mainland China during the Quaternary oscillations. Due to the combined effects of contiguous range expansion and allopatric fragmentation, significant genetic structure was not found in this study. Based on biogeographic and demographic analysis, three main dispersal routes were identified for the major haplotypes, whereas others were more likely localized demographic expansion.     

A one-step organelle capture: gynogenetic kiwifruits with paternal chloroplasts

Androgenesis, the development of a haploid embryo from a male nucleus, has been shown to result in the instantaneous uncoupling of the transmission of the organelle and nuclear genomes (with the nuclear genome originating from the male parent only and the organelle genomes from the female parent). We report, for the first time, uncoupling resulting from gynogenesis, in Actinidia deliciosa (kiwifruit), a plant species known for its paternal mode of chloroplast inheritance. After pollen irradiation, transmission of nuclear genes from the pollen parent to the progeny was inhibited, but transmission of the chloroplast genome was not. This demonstrates that plastids can be discharged from the pollen tube into the egg with little or no concomitant transmission of paternal nuclear genes. Such events of opposite inheritance of the organelle and nuclear genomes must be very rare in nature and are unlikely to endanger the long-term stability of the association between the different genomes of the cell. However, they could lead to incongruences between organelle gene trees and species trees and may constitute an alternative to the hybridization/introgression scenario commonly invoked to account for such incongruences.     

Phylogeographic study reveals the origin and evolutionary history of a Rhododendron species complex in Taiwan

This study infers a single origin and a once-widespread distribution of the Rhododendron pseudochrysanthum species complex in Taiwan based on chloroplast DNA (cpDNA) variation. In total, 124 individuals from five endemic Rhododendron species were used for amplifications of two chloroplast intergenic spacers: trnL-trnF and atpB-rbcL. The haplotype and nucleotide diversities were much lower for the R. pseudochrysanthum complex, comprised of the species R. pseudochrysanthum, R. morii, Rhododendron rubropunctatum, and Rhododendron hyperythrum, than for Rhododendron formosanum. Two measures of pairwise population differentiation, N(ST) and F(ST), consistently revealed mostly non-significant levels of genetic divergence between populations of the R. pseudochrysanthum complex. No genetic difference was found among the four species of the R. pseudochrysanthum complex by analysis of molecular variance (AMOVA), which is concordant with the parsimonious topology of cpDNA haplotypes for the complex. Nested clade analysis (NCA) of the cpDNA haplotypes indicated that restricted gene flow with isolation-by-distance characterized the recolonization pattern of the R. pseudochrysanthum complex. In contrast, the NCA analysis indicated a contiguous range expansion for cpDNA haplotypes of R. formosanum. This research suggests a once-widespread distribution of the R. pseudochrysanthum complex probably via north-to-south colonization of mid-elevations during low-temperature periods of the Pleistocene. Population fragmentation followed the warmer climate which began in the Holocene and resulted in the present-day range contraction into high elevations.     

猕猴桃属种间体细胞杂种

利用PEG融合方法,分别进行了中华猕猴桃(Actinidia chinensis var.chinensis)(2n=2x=58)子叶愈伤组织来源的原生质体与美味猕猴桃(A.deliciosa var.deiciosa)(2n=6x=174)子叶愈伤组织原生质体、以及狗枣猕猴桃(A.kolomikta)(2n=2x=58)叶肉原生质体种间原生质体融合。结果表明:中华猕猴桃与美味猕猴桃融合的1个克隆和中华猕猴桃与狗枣猕猴桃融合的4个克隆的RAPD谱带分别具有双亲特异的DNA谱带;经流式细胞仪分析,前者细胞核倍性推测为8倍体,后者细胞核为3倍体、4倍体和5倍体。初步鉴定这5个克隆是猕猴桃属种间体细胞杂种。