黑喉石斛叶绿体基因组特征及比较分析

本研究结合Illumina高通量测序和Nanopore测序技术,对黑喉石斛（Dendrobium ochreatum）的叶绿体基因组（cpDNA）进行测序,组装得到其cpDNA全长序列并绘制结构图谱。黑喉石斛cpDNA全长154 935 bp,注释共得到125个基因,其中有unigenes 103种,包含73种蛋白编码基因,26种tRNA基因和4种rRNA基因。特征分析结果表明：黑喉石斛cpDNA中共存在58个SSR （simple sequence repeat）位点,大部分为单核苷酸重复和二核苷酸重复类型,碱基组成以A/T碱基类型为主;其偏好的密码子共32个,其中有30个以A/U碱基结尾。以黑喉石斛与其他13种石斛的cpDNA构建系统发育进化树,结果显示,鼓槌石斛（D. chrysotoxum）和梳唇石斛（D. strongylanthum）与黑喉石斛具有较近的亲缘关系,IR/SC边界分析结果也映证了此结论。以黑喉石斛为参照,与3种近缘石斛的cpDNA进行全序列长对比,结果显示,4种石斛的序列变异主要发生在单拷贝区,基因间隔区的变异明显高于基因编码区。     

分子生物技术在确定物种迁移后遗传变异中的应用

植物的迁徙和定居受人类活动的影响，确定种群在原发地和定居地的遗传变异及其成因，有助于理解生物多样性的格局和育种选择的实践。介绍了应用生物技术确定北美芥菜自欧洲迁来后遗传变异的实质，欧洲苏格兰松的地理起源和高极地孑遗区的范围。分子生物方法同大化石和孢粉证据结合可能较单一的方法更能全面的反映种或基因的系统进化过程和历史。     

Analysis of chromosome, mtDNA and cpDNA patterns in five somatic hybrids between Brassica alboglabra Bailey and Brassica campestris L.

Summary Somatic hybrids between Brassica alboglabra (CC) and B. campestris (AA) were produced through protoplast fusion. Hybrids of this combination have already been produced through sexual crosses and among other traits in the offspring, a CMS of ogura type was discovered. The reason for generating somatic hybrids was to create new nuclear-cytoplasmic combinations and/or cytoplasmic variants, which could be exploited in combination with the sexual hybrids. Mitosis and meiosis were studied and all hybrids were found to be of the AACCCC genomic constitution. The fusion of somatic cells resulted in altered cytoplasmic genomes.     

Molecular Phylogeny of the ＂True Citrus Fruit Trees＂ Group （Aurantioideae,Rutaceae） as Inferred from Chloroplast DNA Sequence

The genus Citrus L.has a long controversial taxonomy history,and a well-resolved molecular phylogeny of the "true citrus fruit trees" group in the future will provide new information for advancing breeding techniques and developing better conservation strategies.In the present study,three cpDNA fragments(TrnL-TrnF,PsbH-PetB,and TrnS-TrnG)of 30 genotypes chosen from the six genera of the "true citrus fruit trees" group were analyzed.A molecular phylogenetic tree of the "true citrus fruit trees" group was reconstructed based on plastid DNA sequences.The results confirmed that the "true citrus fruit trees" group was monophyletic,and thereby the group was divided into genera as previously suggested based on morphological characters.The cpDNA data also suggested that Poncirus might be the first genus separated from the other five genera in the group.The genus Fortunella were of hybrid origin and Citrus might be as its putative paternal parent.The genera Microcitrus,Eremocitrus,and Clymenia were possibly monophyletic and their common ancestor might branch out from Citrus.Furthermore,the phylogenetic relationships within the Citrus genus were discussed.     

Analysis of phylogenetic relationships in Mangifera by restriction site analysis of an amplified region of cpDNA

The phylogenetic relationship of 13 Mangifera species collected in Thailand were examined. Among these species, M. foetida, M. odorata, and M. sylvatica have been cultivated as fruit crops for local markets in Thailand, as well as M. indica (common mango). The other nine wild species also seem to have potential values as rootstocks and breeding materials for tolerance to critical environmental conditions. By restriction fragment length polymorphism (RFLP) analysis of a particular region of cpDNA, these species were classified into two groups. The first group consisted of M. indica and M. sylvatica, and the second group consisted of M. caloneura, M. cochinchinensis, M. collina, M. flava, M. foetida, M. gedebe, M. griffithii, M. macrocarpa, M. oblongifolia, M. odorata, and M. pentandra. The species belonging to the same group are monomorphic for 20 restriction enzymes tested. The present study indicated low diversity in the amplified region of cpDNA among Mangifera species tested, even though the species in the second group were scattered to different subgenera and sections.     

Origin of Weedy Rice Grown in Bhutan and the Force of Genetic Diversity

In Bhutan, weedy rice (Oryza sativa L.) was grown together with cultivated rice on terraced paddy fields lower than 2620 m above sea level. Seeds of cultivars and weedy strains were collected at 22 collection sites located from 1000 to 2620 m above sea level. Cultivars with round seeds were frequently found in fields higher than 2250 m, and those with slender seeds in fields lower than 1630 m. All cultivars and weedy strains were divided into indica or japonica types by isozyme (multi-locus) and morpho-physiological (multicharacter) analyses. Japonica cultivars predominated in highland; Indica cultivars predominated in lowland. Plastid type was confirmed by the length polymorphism for the ORF100 region. The japonica cultivars carried non-deletion type ORF100. The indica cultivars carried deletion type ORF100. In contrast, weedy strains showed discrepancy in the combination of the nuclear and cytoplasm types. An intermediate type was found in weedy strains for isozyme genotypes. A recombinant type, which has indica genotypes for isozyme analysis with japonica cytoplasm, and vice versa, was frequently found in weedy strains. These findings suggested that weedy strains would be generated by natural hybridization between indica and japonica. Further, they might fail to recombine their prior genotypes sets for isozyme and cytoplasm. Morphophysiological characters did not show such a tendency. Morpho-physiological analysis of highland plants, in particular, showed indica cultivars and weedy strains with japonica cytoplasm. Hypervariable nuclear microsatellite analysis was then used to compare cultivars and weedy strains. Identical alleles were shared between indica and japonica, and also between cultivars and weedy strains. This suggested that there was gene-flow resulting from natural hybridization.     

Chloroplast DNA study in sweet cherry cultivars (Prunus avium L.) using PCR-RFLP method

The chloroplast DNA of 96 sweet cherry cultivars (Prunus avium L.) and five cultivars of sour cherry (P. cerasus L.) were analysed to reveal their haplotypes using PCR-RFLP (Polymerase Chain Reaction - Restriction Fragment Length Polymorphism) method. The main advantages of the PCR-RFLP technique are: resolutive, time and cost effective and reproducible. Approximately 9.6% of the chloroplast genome was analysed, using six universal primer pairs and two restriction enzymes. All the mutations detected were insertion-deletions, ranging between 5–30 bp. The combination of all the mutations resulted in three haplotypes (H1, H2 and H3) in the 96 sweet cherry cultivars and a single haplotype (H4) in the five cultivars of sour cherry. The cpDNA polymorphism determined by PCR-RFLP markers helped to understand the maternal inheritance of chloroplast genome in sweet cherry, clearly distinguished the sour from the sweet cherry, and supported that P. avium is not the maternal species of P. cerasus.     

雷蒙德氏棉叶绿体基因组Fosmid文库构建

 采用高盐、低pH值法提取雷蒙德氏棉叶绿体DNA;通过物理剪切法获得随机断裂的DNA片段;剪切片段末端、补平修饰后与pCC1FOS载体连接;用噬菌体包装蛋白包装重组DNA,侵染大肠杆菌EPI300,构建了雷蒙德氏棉叶绿体基因组文库。对于叶绿体DNA剪切,以1 mL注射器中等速度吸打18次为最佳参数。叶绿体基因组Fosmid文库滴度为1×10⁴ cfu·mL^-1,插入片段大小平均为38 kb,最终筛选出39个克隆用于后续研究,覆盖叶绿体基因组9.2倍。以叶绿体特异标记筛选出能够覆盖雷蒙德氏棉叶绿体全基因组的6个克隆：F66,F46,F28,F8,F55和F3,为基因组结构和功能基因分析提供了良好的基础。     

血皮槭叶绿体DNA非编码区差异序列筛选和分析

[目的]筛选出高变异率的叶绿体DNA序列,以进行血皮槭天然群体的谱系地理学研究。[方法]以血皮槭16个天然群体为试材,对叶绿体基因组20个非编码区序列进行测序研究,并利用筛选出的高变异率cpDNA序列初步分析了其遗传变异。[结果]序列比对和系统发育分析结果显示血皮槭cpDNA种内变异非常低,9个cpDNA序列检测到不同程度的变异位点,其中只有4个序列psbJ-petA、ndhF-rpl32、trnD-trnT和trnH-psbA表现出较高的变异水平。[结论]筛选出的4个高变异率cpDNA序列,可以在分子水平上研究血皮槭种内的系统发育、遗传变异和种群历史动态,为进一步研究濒危种血皮槭的分子谱系地理学奠定基础。     

基于ITS和cpDNA序列的梭梭和白梭梭物种分化

【目的】基于nrDNA和cpDNA 2种基因,探讨中国同域分布的梭梭和白梭梭的物种分化,分析2种基因的单片段及其片段组合对2种植物的物种鉴别力,并分析生态位分化的程度及其对物种进化的影响,为梭梭和白梭梭的系统发育和谱系地理等研究提供基础数据。【方法】基于2个nrDNA序列(ITS2、ITS1-ITS4)和3个cpDNA非编码区序列(trnS-trnG、rps4和trnV),对古尔班通古特沙漠南缘同域分布的梭梭和白梭梭共30个个体进行序列分析;利用距离法(基于Kimura 2-parameter)研究2种植物的遗传分化;基于贝叶斯方法分析个体间的分子系统关系;并利用距离法、系统发育树法、BLAST比对法和诊断特征法评价ITS和cpDNA的单序列及其组合对2种植物的物种鉴别力。在此基础上,利用生态位分析软件ENMTools V1.4定量分析梭梭与白梭梭生态位分化的程度。【结果】1)对于梭梭和白梭梭,2个ITS序列拼接比对的总长均为1 117 bp,G+C含量平均分别为34.74%和34.82%,总的信息位点数分别占片段总长的2.33%和1.43%; 3个cpDNA序列拼接比对的总长均为2 344 bp,G+C含量平均分别为59.62%和59.39%,信息位点数分别占片段总长的0.68%和0.43%。2)基于ITS和cpDNA序列组合构建的贝叶斯系统发育树都表明梭梭和白梭梭各聚为一支。3)基于ITS和cpDNA序列组合计算的2种植物的种间最小遗传距离均大于种内的最大遗传距离,并且种间种内都存在1个明显的距离间隙,表明本研究所用的ITS和cpDNA的序列组合可以作为鉴定梭梭和白梭梭的DNA条形码序列。4)基于4种方法评价的ITS和cpDNA序列对梭梭和白梭梭的物种鉴别力表明:2个ITS的单序列及其序列组合的鉴别率均为100%; cpDNA序列中,rps4的单序列及其与trnS-trnG,trnV的两两序列组合的鉴别率均为0,而trnS-trnG,trnV的单序列及其序列组合,以及trnS-trnG+trnV+rps4组合的鉴别率均为100%。5)生态位参数D值和I值的观察值和一致性检验的模拟值都集中在0.65和0.90左右,表明梭梭和白梭梭的生态位分化不明显。【结论】基于ITS和cpDNA序列组合,梭梭和白梭梭物种间的遗传分化明显,分子系统关系清楚; ITS和cpDNA序列组合都可以作为鉴定梭梭和白梭梭的DNA条形码序列;梭梭和白梭梭的生态位分化不明显,说明生态因素很可能对2种植物的分化与进化没有起到明显的作用,2种植物很可能也具有相近的进化历史。本研究的结论可以为梭梭和白梭梭的系统发育、遗传和进化研究提供重要的理论依据和数据支撑。