Identification of differentially expressed genes during flower opening by suppression subtractive hybridization and cDNA microarray analysis in Eustoma grandiflorum

The forward and reverse suppression subtractive cDNA libraries were constructed in petals of Eustoma grandiflorum at bud stage (stage 1) and anthesis (stage 7). Approximately 1000 clones were isolated from stage 1- (S1) and stage 7-specific (S7) libraries. The clones were sequenced and assembled, which yielded 98 contigs and 444 singletons. BLAST search was conducted on these assembled sequences. Generally, probes isolated from the S7 library exhibited higher expression at stage 7 by microarray analysis, as did those of the S1 library at stage 1. A clone set from the S7 library contained genes from later steps of anthocyanin biosynthesis pathway, terpene synthases, GAST (gibberellic acid-stimulated) family proteins, xyloglucan endotransglucosylase/hydrolase, glycosidases, and stress- and senescence-related proteins. In contrast, the S1 library contained genes associated with flavonol biosynthesis, phenylpropanoid metabolism, terpenoid metabolism, and floral organ development. Gene expression profiling for flavonoid biosynthesis was in accordance with preferential accumulation of flavonols at bud stages and anthocyanins at anthesis.     

鸟巢蕨转录组高通量测序及分析

采用新一代高通量测序技术Illumina HiSeq 2000对鸟巢蕨转录组（Asplenium nidus）进行测序,共获得29 254 595个序列读取片段（reads）,包含了5 908 586 517个碱基序列（bp）信息。对reads进行序列组装,共获得42 907个单基因簇（Unigene）,平均长度936 bp,序列信息达到了40.16 Mb。数据库中的序列同源性比较表明,24 993个Unigene与其他物种的已知基因具有不同程度的同源性。鸟巢蕨转录组中的Unigene根据GO功能大致可分为细胞组分、分子功能和生物学过程3大类51个分支,其中有大量的Unigene与代谢进程、结合活性、催化活性和细胞进程相关。将Unigene与COG数据库进行比对,根据其功能大致可分为24类。KEGG数据库作为参考,依据代谢途径可将Unigene定位到116个代谢途径分支。SSR位点查找发现,从42 907个Unigene中共找到6 067个SSR位点。SSR不同重复基序类型中,出现频率最高的为AG/CT,其次是AC/GT、A/T和AGG/CCT。针对这些序列,设计了20对引物进行了扩增效率和多态性检测,其中7对引物在不同蕨类材料中表现出多态性。     

新疆蟠桃中发现油桃茎痘相关病毒和亚洲李属病毒

采用高通量测序(High-throughput sequencing,HTS)技术检测新疆蟠桃感染病毒的情况。采集具有穿孔、脉间褪绿等症状的蟠桃嫩叶,提取总RNA,用于高通量测序,共获得13.36 Gb的数据,包含44563187对reads,其中比对到油桃茎痘相关病毒(nectarine stem pitting-associated virus,NSPa V,KT273409)和亚洲李属病毒(asian prunus virus,APV2,KT893294)基因组的分别有9943对和40036对,经拼接各得到长4978和9393 nt的contig,基因组覆盖率均接近100%(5′端分别差10个和7个碱基),核酸一致度分别为95.0%和92.9%,将此分离物分别命名为NSPa V-Tao和APV2-Tao4。用RT-PCR方法对23个蟠桃树样品进行了检测,结果NSPa V和APV2检出率分别为43.5%和69.6%。通过比较已知NSPa V病毒的核苷酸序列,发现NSPa V-Tao可能是其他分离物重组的结果。     

枳根cDNA全长文库的构建与分析

为了解柑橘优良砧木枳（Poncirus trifoliata）根部的基因表达信息,利用SMART技术构建了枳的根组织全长cDNA文库。检测结果显示所建枳根原始文库的容量为1.08 × 106 cfu ? mL-1,扩增后文库容量为1.30 × 109 cfu ? mL-1,重组率为95%。通过文库随机测序获得182个长度大于100 bp的ESTs序列（GenBank登录号为JK316116 ~ JK316297）,序列拼接后得到96个Unigenes,包括12个Contigs和84个singletons。其中,68个Unigenes可与GenBank中登录的基因相匹配,56个与已知的柑橘基因相匹配,36个为全长基因。对24个全长基因完成了功能注释,并进一步开展了生物信息学分析,发现有6个应激反应基因和3个根发育相关基因。     

Effect of two UV-absorbing greenhouse-covering films on growth and yield of an eggplant soilless crop

The use of UV absorbing films as greenhouse cover material is spreading out in protected cultivation. Although their effects on pest and disease management have received much attention, few studies focus on their effects on the crop. This study aims at assessing the consequences of UV absorbing film on the behaviour and production of an eggplant crop by comparing two different UV absorbing films (0 and 3% UV transmittance) to a standard polyethylene film (5% UV transmittance). Results show that the eggplants grown in the greenhouse with 0% transmission to UV light are about 21% taller and have about 17% higher leaf product (leaf length × width) than the plants grown in the greenhouse with 5% transmission to UV light. Finally, given that the production was slightly increased in quantity (20%) and quality (bigger fruits) in the greenhouse with absence of UV light compared to that with 5% transmission coefficient, it can be concluded that growing soilless eggplant under UV-absorbing material can be achieved with the same or better results as under standard covering material. Any other enhancement that the UV-absorbing film will bring (lower pest and disease impact on the crop, lower pesticide load and costs) will therefore be to the benefit of the grower.     

Identification of differentially expressed genes in fragrant rose Jinyindao with suppressive subtraction hybridization

To investigate the floral fragrance new genes, scent mutant of rose was used here. The suppressive subtraction hybridization (SSH) technique and micoarray analysis of the clones were used to isolate the cDNA fragments, which showed differential expression between the rose scent mutant ‘Wangriqinghuai’ and wild type ‘Jinyindao’ (Rosa × hybrida), and RT-PCR was used to identify up-regulated expressed genes. 16 positive contigs of JSSH were obtained. Some ESTs such as RcOMT1, RcOMT2, RhMYB92 and RhGP1 were known to regulate scent metabolism, and 5 ESTs with no homology in NCBI may represent new genes involved in rose flower fragrance metabolism. SSH technique combined with cDNA micoarray would be useful for analysis and isolation of the genes related to rose floral scent.     

Molecular characterization and analysis of somaclonal variation in chrysanthemum cultivars using RAPD markers

Molecular characterization using RAPD analysis was carried out in eight cut flowers and two pot plant cultivars of chrysanthemum. Three of them (‘Refocus’, ‘Red Reagan’, and ‘Sheena Select’) were established in vitro and the occurrence of somaclonal variation was studied using the same molecular technique. Two induction media (MS + 0.1 mg l⁻¹ NAA + 0.1 mg l⁻¹ BA, and MS + 2.0 mg l⁻¹ IAA + 0.5 mg l⁻¹ Kinetin), and two proliferation media (MS + 0.1 mg l⁻¹ NAA + 0.2 mg l⁻¹ BA, and MS + 4.0 mg l⁻¹ IAA + 2.0 mg l⁻¹ Kinetin) were employed in order to evaluate the effect of the medium composition in the shoots’ stability. Likewise, the effect of the culture age was considered in assessing genetic stability. Monthly subcultures were carried out, identifying the origin and history of the shoots, throughout a nine-month proliferation period followed by acclimatization. Molecular markers were obtained in every subculture cycle and from the acclimatized plants. Only one shoot from the 7th subculture of the cultivar ‘Refocus’ showed a different band pattern. The use of RAPD for chrysanthemum cultivar characterization and somaclonal variation detection is discussed.     

牡丹EST序列中的微卫星分析

NCBI的dbEST数据库中2 204条牡丹EST序列经过预处理获得2 048个高质量的序列,经拼接,获得318个Contigs和636个Singlets.其中142条拼接序列共检测出167个微卫星(简单序列重复,SSR),平均1.18个SSR/Contig或Singlet.这些微卫星从二至四核苷酸重复都有出现,二、三核苷酸重复类型占多数(97.61％),其中二核苷酸重复为46.71％,三核苷酸重复为50.90％.二核苷酸重复类型中出现最多的是AG/TC和GA/CT,分别占SSR总数的20.36％和16.77％,三核苷酸重复类型以AGA/TCT、GAT/ATC最多,分别占总SSR的5.40％.最后对含有微卫星的Contigs和Singlets进行基因注释、功能分类.     

Stoloniferous shoot protoplast,an efficient cell system in potato for somatic cell genetic manipulations

The present study reports that protoplasts isolated from stoloniferous shoots (SS) of potato represent an efficient system for somatic cell genetic manipulations. SS were established from single-node cuttings on MS medium supplemented with either 0.1 or 0.2 M sucrose (Suc), and protoplasts were isolated and cultured within the alginate strip, following an improved method. SS induced by 0.1 M Suc yielded 8–22 × 10⁵ protoplasts g⁻¹ fresh mass, with a high morphogenic competence. However, 0.2 M Suc-induced SS yielded protoplasts that contained large amounts of starch grains, resulting in their high degree of fragility, delayed cell division and poor morphogenic competence. For symmetric somatic hybridization (electrofusion) between Solanum tuberosum Gp. Tuberosum androgenic (di)haploid (2n = 2x = 24) ‘C-13’ and diploid (2n = 2x = 24) wild species S. pinnatisectum, protoplasts isolated from 0.1 M Suc-induced SS were also found to be most responsive. Out of several putative somatic hybrids, there were two tetraploids and five diploids, with 48 and 24 chromosomes, respectively at all the three shoot layers (L₁–L₃). This precluded the occurrence of mixoploidy vis-à-vis chimaerism in regenerants, as common in somatic fusion involving mesophyll protoplasts of S. pinnatisectum. Nuclear microsatellite analyses based on the two single-locus nSSR loci (STM0037 and STM2030) confirmed that one of the tetraploids was a true nuclear hybrid (heterokaryon), while the other a homokaryon of the Tuberosum parent ‘C-13’. The use of 0.2 M Suc-induced SS protoplasts for fundamental studies on tissue- and/or cell type-specific transient gene expression underlying tuberization has been discussed.     

Comparative analysis of genetic diversity in Indian bitter gourd (Momordica charantia L.) using RAPD and ISSR markers for developing crop improvement strategies

A genetic analysis of 38 diverse Indian bitter gourd (Momordicacharantia var. charantia, and var. muricata) accessions was performed using 29 RAPD and 15 ISSR markers. RAPD primers yielded 208 amplicons of which 76 (36.5%) were polymorphic providing an average of 2.6 amplicons per primer. RAPD amplicons per primer ranged from 3 (OPE-19, OPW-09) to 15 (OPW-05), and varied in size from 200 bp to 3000 bp. Fifteen ISSR primers provided a total of 125 bands of which 94 (74.7%) were polymorphic. Polymorphic ISSR markers ranged from 0 (UBC-841) to 12 (UBC-890) providing a mean of 6.3 amplicons per primer that ranged in size from 150 bp to 2700 bp. Nevertheless, the concordance among bitter gourd accession groupings after cluster analysis was relatively high (r = 0.77), indicating that RAPD- and ISSR-based diversity assessments in this germplasm array were generally consistent. The M.charantia var. charantia (domesticated) and var. muricata (wild, free-living) accessions examined were genetically distinct, and these differences provided for the development of strategies for genetic analyses and crop improvement in this species.     

High frequency shoot regeneration from cotyledon explants of cucumber via organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from cotyledon explants of cucumber. About 86.2% of cotyledon explants derived from 5-day-old in vitro raised seedlings produced green, compact nodular organogenic callus in MS medium containing NAA (2.69 μM) and BA (4.44 μM) after two successive transfers at 20 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MS medium supplemented with NAA (1.34 μM), BA (8.88 μM), zeatin (0.91 μM) and l-glutamine (136.85 μM) with shoot induction frequency of 75.6%. Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 20 days. Shoots (1.0 cm length) were excised from callus and were elongated in MS medium fortified with GA₃ (1.44 μM) and BA (4.44 μM). The elongated shoots were rooted in MS medium supplemented with IBA (3.42 μM) and BA (4.44 μM). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 80%. This protocol yielded an average of 35 shoots per cotyledon explant in a culture duration of 120–140 days.     

Chromosome observation method at metaphase and pro-metaphase stages in diploid and octoploid strawberries

Chromosome observation is necessary to elucidate the structure, function, organization, and evolution of octoploid strawberry plants’ genes and genomes. However, distinguishing strawberries’ chromosomes from one another using light microscopy is extremely difficult, not only because of their small size and large number, but also because current chromosome observation methods are insufficient. Chromosome preparation and staining using maceration enzymes, acetic acid, and DAPI (4′,6-diamidino-2-phenylindole) were improved for this study to obtain clear images of somatic chromosomes in Fragaria vesca (2n = 14) and Fragaria×ananassa (2n = 56). Collected root tips of octoploid plants were placed in 0.002 M 8-hydroxyquinoline solution for 1 h and stored at 4 °C for 16 h. Subsequently, they were fixed using 3:1 absolute alcohol:glacial acetic acid for 40 min, hydrolyzed in the 1N HCl solution at room temperature for 2 h, macerated using an enzyme solution for 25 min at 42 °C, and stained in 1.5% lacto-propionic orcein solution. On the other hand, in case of DAPI staining, the macerated root tips of octoploid plants were soaked in 60% acetic acid for 5 min before staining. Clear digital images of F. vesca and F.×ananassa were obtained using light and fluorescent microscopy. Their respective 14 and 56 chromosomes were counted. Fluorescent microscopy yielded clear chromosome images at the pro-metaphase in F. vesca and F.×ananassa. This chromosome observation method alleviates the difficulties that have heretofore hindered chromosome analyses of strawberry plants.     

Rapid in vitro propagation of Dendrobium hybrids through direct shoot formation from foliar explants,and protocorm-like bodies

In vitro propagation protocol for Dendrobium hybrids Sonia 17 and 28, two highly priced commercial cut flower cultivars through direct organogenesis from in vitro derived foliar explants was established. Rapid clonal propagation was achieved by subsequent induction of protocorm-like bodies (PLBs) and its conversion to shoots. No significant differences were observed in the induction of direct shoots, shoot multiplication, PLBs formation and subsequent shoot development and rooting of shoots between the two cultivars. Leaf explants from flower stalk node derived shoots cultured on half-strength Murashige and Skoog (MS) medium supplemented with 44.4 μM N⁶-benzyladenine (BA) developed more than seven shoots per explant. The isolated shoots transferred onto the same medium induced more than eight PLBs from the base within 60 days, which upon transferral to fresh medium having the same level of BA facilitated rapid proliferation. More than 200 PLBs were yielded from fifth subculture. Half-strength MS medium containing 6.97 μM kinetin (Kn) facilitated conversion of more than 90% PLBs to shoots. PLBs exhibited proliferation without decline up to the 15th subculture. Half-strength MS medium with 2 g l⁻¹ activated charcoal was the best for in vitro rooting. Plantlets of the hybrids exhibited more than 80% ex vitro establishment.     

Analysis of factors governing water flow traits in fruiting plants twigs

We compared hydraulic traits of 18 tropical/subtropical fruit-producing species plants and a further 18 from temperate zone. Plants were classified into four categories by height: tall tree (>10 m), small tree (4–9 m), shrub (1–4 m) and vine. We measured ratios [(cross-section area of xylem)/(cross-section area of twig)], and the diameters and numbers of xylem vessels in microscopic images. We calculated the water flow index (WFI: Σr⁴ S⁻¹ × xylem ratio, where, r is the vessel radius, and S is the xylem cross-section area) according to Hagen–Poiseuille's law. Vine had thick vessels and remarkably higher WFI than free-standing trees in both temperate and tropical fruit species. Vessel diameter increased as trees being taller in both in latitudinal groups. Xylem vessel number decreased with height in temperate fruit trees but not in tropical species. WFI increased with tree height of both latitudinal groups. There were no significant effects of latitude on WFI.     

紫背天葵高通量转录组测序分析

为探讨紫背天葵(Gynura bicolor)花青素等物质合成的遗传基础,采用高通量测序技术(Illumina HiSeq 2500)对其嫩叶进行转录组测序,共获得21 387 624个序列读取片段(reads),将测序数据进行序列组装后,获得33 314个单基因簇(Unigene),其中超过1 kb的7 792个Unigene中共检测到2 387个SSR位点。对所得Unigene进行不同数据库注释,22 048和14 417个Unigene分别在Nr和Swiss Prot数据库有同源比对信息,并发现有29个Unigene与花青素合成相关;Pfam功能注释到13 909个Unigene分为5 198类相关蛋白功能区域,有12个Unigene涉及到花青素合成;GO注释到11 613个Unigene分为细胞组分、分子功能及生物学过程等3大类51个功能组,有24个Unigene与花青素合成有关;COG注释到的6 589个Unigene功能系统分为24类,而KOG注释到的13 498个Unigene功能系统分为25类;以KEGG数据库为参考,将4 466个Unigene定位到108个代谢途径分支,其中有47个Unigene与类黄酮生物合成相关。     

Auxin-dependent development and habituation of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures

The influence of IAA (1.0 mg dm⁻³), and IBA (1.16 mg dm⁻³), on the development of highbush blueberry (Vaccinium × covilleanum But. et Pl.) ‘Herbert’ in vitro shoot cultures was examined. Depending on the kind of auxin and 2iP concentration in vitro cultures consisted of various number of axillary (AX) and adventitious (AD) shoots. Three different categories of AD shoots were found: leaf shoots (AD-L), node-adjoin shoots (AD-P), and base-adjoin shoots (AD-M, madshoots). The AX shoots were the least habituated (towards auxins, cytokinins and vitamins) whereas the AD-M shoots (madshoots) the most. In comparison to IAA, IBA caused dying or callusing higher number of initial explants. However, IBA generally suppressed development of AD shoots, especially madshoots whereas slightly weakened multiplication of AX shoots. IBA significantly enhanced elongation of AX shoots also. Axillary shoots obtained on IBA-media had relative long internodes and rigid, well-developed leaves. The adventitious shoots, especially base-adjoin (AD-M) ones, were easily distinguishable as were more thin and fragile, more or less vitrified, and had short internodes and smaller, sometimes unfolded leaves. Development of blueberry in vitro cultures on auxin-free and IAA-supplemented media was similar. AX shoots grown on such media resembled AD shoots. 2iP applied in higher doses along with IAA promoted much proliferation of AD than AX shoots. In contrast, 2iP applied in higher doses together with IBA stimulated significantly only growth of AX shoots whereas in general, development of adventitious shoots was not affected. Micropropagation carried out through routine method based on subculturing of shoot explants or shoot clumps on the medium supplemented with IAA (4 mg dm⁻³) and 2iP (10–15 mg dm⁻³) as well as stimulation of shoot elongation on the blank medium causes in fact the propagation of highbush blueberry through highly habituated adventitious madshoots. Replacement of IAA by IBA facilitates micropropagation of highbush blueberry cv. Herbert through axillary shoots.     

Effect of reciprocal transfers of onion sets between inductive and non-inductive temperatures on the incidence of bolting and bulbing and seed yield

The experiment was conducted at the experimental grounds of the School of Plant Sciences, University of Reading during 1996. Onion sets (22.5 mm diameter) of two cultivars (Hygro and Delta) were transferred from a low (10 °C) to a high (30 °C) temperature and vice versa on six occasions at 15 days intervals. Two control treatments where sets remained at 10 and 30 °C throughout were also included in the experiment for comparison. In both cultivars (Hygro and Delta), plants did not flower when sets were maintained at 30 °C throughout or when given 15 days at 10 °C followed by 30 °C for 75 days. Highest percentage of bolting was observed when sets were maintained for the longest period at 10 °C followed by the shortest period at 30 °C or when given 10 °C throughout. Highest number of florets and seed yield per umbel was recorded in treatments where sets were maintained at 10 °C for 90 days. Mean bulb weight increased where sets remained for longer period at higher temperature (30 °C) either before or after transfer. Bulb yield m⁻² increased when sets were initially stored at 10 °C for a short period followed by 30 °C throughout.