Breeding cold tolerance strain by chemical mutagenesis in Volvariella volvacea

Volvariella volvacea is a mushroom well-adapted to high temperature. It optimally grows at 30–35 °C. To breed cold-tolerant strains to expand cultivation region and season, the basidiospores and gills of V. volvacea were treated with chemical mutagens, ethyl methane sulfonate (EMS) and diethyl sulfate (DES), respectively. Two cold-tolerant strains, strains Em-16 and Em-18, were successfully obtained from EMS mutagenesis of gills through 0 °C screening, colony morphology screening and fruiting screening. The biological efficiencies of Em-16 and Em-18 strains were 24.55% and 23.61% in the first flush at 27 °C and their biological efficiencies were 46.1% and 40.5% higher than the control strain V41, respectively. Their fruiting bodies had a longer storage life at 16 °C, compared with the control strain V41. Amplified fragment length polymorphism analysis shows that the strains Em-16 and Em-18 are new strains.     

香蕉黑星病菌形态与生物学特性

为更好地了解香蕉黑星病和指导病害防治,对该病病原菌Macrophomamusae的形态作了系统描述并详细测定了该菌的生物学特性。生物学特性研究表明,在PDA平板上,香蕉黑星病菌在10～35℃均能生长,适宜生长温度为20～30℃,最适生长温度是28℃,PDA、PSA和V8培养基上生长较好,病原菌在pH3～10均能生长,pH4～8生长较好,pH7生长最佳,马铃薯琼脂培养基中添加不同的糖对病原菌生长有不同的影响,其中以蔗糖、果糖、葡萄糖最好,淀粉和乳糖最差,PDA培养基中添加2%的不同的氮源对病原菌生长有抑制作用,在添加氯化铵的PDA上不能生长,光照对病原菌的生长有明显促进作用,振荡培养对病原菌生长的促进作用不明显,病原菌的致死温度为55℃10min。分生孢子在蒸馏水中萌发缓慢,Ca2+有促进孢子萌发的作用,其中10mmol/LCa2+对孢子萌发的促进作用最为明显。     

香蕉WAT1基因的鉴定及表达分析

【目的】基于香蕉基因组数据筛选Walls are thin 1(WAT1)基因,分析它们的序列及表达特性。【方法】以拟南芥WAT1为参考序列,通过本地Blast筛选获得香蕉WAT1基因,分析其核苷酸、启动子及编码蛋白特性,并利用实时定量PCR技术研究其在不同组织部位、不同激素和逆境胁迫处理下的表达情况。【结果】筛选获得5个香蕉WAT1基因(命名为MaWAT1-1~5)。蛋白亚细胞定位预测结果显示,MaWAT1-1、MaWAT1-2、MaWAT1-4主要定位在液泡和细胞膜上,MaWAT1-3主要定位在细胞质和细胞膜,MaWAT1-5定位在细胞膜和叶绿体。基因结构分析和系统进化树分析均将MaWAT1s分为两组,MaWAT1-1、MaWAT1-2、MaWAT1-4聚为一组(含6个外显子和5个内含子),MaWAT1-3和MaWAT1-5归为一组(含7个外显子和6个内含子)。启动子顺式作用元件分析结果显示:MaWAT1s启动子含有大量激素和胁迫响应相关元件。实时定量PCR结果显示,MaWAT1-4在叶片中表达量最高,MaWAT1-1在根和假茎中表达量最高,其余均在根中表达量最高;大多数MaWAT1s的表达受GA3、SA、盐胁迫和干旱等显著诱导,受高温显著抑制,同时部分成员的表达受IAA、ABA、JA、低温、机械损伤和枯萎病影响显著。【结论】MaWAT1s的表达受多种激素和逆境影响显著,可能在香蕉生长发育和抗逆防御反应中发挥着重要作用。     

香蕉皮脂肪酸的提取及GC-MS分析

为了给香蕉皮变废为宝加以更广泛利用提供科学依据,以提取过挥发油后的香蕉皮为材料,利用索氏提取法提取脂肪酸。并利用气相色谱质谱联用法鉴定出了21种成分及各种成分的相对含量。结果表明,香蕉皮脂肪酸中十六碳酸(Hexadecanoic acid)的含量最高,为45.90%,2种必需脂肪酸a-亚麻酸((Z,Z,Z)-9,12,15-octadecatrienoic acid)和亚油酸((Z,Z)-9,12-octadecadienoic acid)的含量分别为20.75%和19.56%。     

Identification and distribution of S haplotypes in Brassica vegetables from China

The amplification efficiency and identification diversity with gene-specific primers derived from S locus glycoprotein gene (SLG) and S locus receptor kinase gene (SRK) were compared, and the geographical distribution for S haplotypes was investigated by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 72 genotypes of 5 Brassica vegetables from China. The amplification efficiency and identification diversity with class I SRK primers were generally higher than that with class I SLG in most crops tested. Class I alleles were found in total 66 genotypes and they were classified into 16, 10, 7 and 10 groups for Chinese cabbage, purple flowering stalk, cauliflower and cabbage respectively. The number of amplification accessions and identification diversity using the primers of class II SLG and SRK were quite similar. Class II alleles were detected in 55 genotypes and further grouped into one type in mustard and three in other crops. The nucleotide sequences showed high similarity between identical S haplotypes determined by reciprocal pollination and PCR-RFLP tests. It demonstrated that the PCR-RFLP analysis was feasible for identification of S alleles, and SRK should be considered as a better marker for the identification of S haplotypes than SLG. The types of S haplotypes are highly diverse in Brassica vegetables from China. Nevertheless, they were geographically limited in some Brassica vegetables, so the exchange of germplasm resources should be enhanced for breeding.     

香蕉中一个新的S-腺苷甲硫氨酸合成酶基因的克隆及采后表达分析

利用抑制消减文库从香蕉中分离到一个cDNA片断,结合RACE技术获得该基因的全长序列。该全长cDNA共含1 285个碱基,通过Blastx同源性分析结果显示它与香蕉的一个S-adenosyl-L-methionine synthase(SAMS)基因(GenBank序列号为AF004317)具有82%的碱基同源性,编码的氨基酸顺序有93%同源性,但在cDNA的5’和3’非编码区同源性较低。设计特异引物对此基因进行RT-PCR分析表明,正常成熟的香蕉果实,采后当天表达量较高,随后略有降低,至采后12 d又达到一个相对较高值后迅速下降;高锰酸钾处理的果实,整个成熟期该基因均表现一个比较高的表达量;乙烯利处理的果实,采后表达量明显下降且处在一个比较稳定的水平。     

香蕉果实果胶裂解酶基因cDNA克隆及序列分析

利用已报道果胶裂解酶基因的保守序列设计简并引物,进行RT-PCR,得到1个大约1300bp的香蕉果胶裂解酶基因cDNA片段,命名为MA-pl。DNA序列分析表明:MA-pl片段全长1277bp,包含1个882bp的开放读码框(ORF),编码294个氨基酸;其具有所有果胶裂解酶共有的保守区域:钙协调部位(Asp72,Asp74,Asp96,andAsp100)、酶活性位点(Arg152,Pro154,Arg157)及3个重要的结构域(motifI:WVDH,motifII:DGLVDAVMGSTAITVSNNYF,motifIII:LYQRMPRCRHGYFHVVWNDY);MA-pl氨基酸序列与草莓-1、葡萄、草莓-2、拟南芥、苹果、香蕉(banana-1)的相似性分别为85.8%、74.2%、79.7%、78.6%、72.4%和71.4%。     

Twenty years of agronomic evaluation of wild cocoa trees (Theobroma cacao L.) from French Guiana

Almost 500 clones of wild cocoa trees (Theobroma cacao L.) grown from pods collected in 1987 from wild mother-trees in the Camopi and Tanpok river basins (southeastern French Guiana) have been distributed in around fifteen cocoa producing countries since 1988. The name of those clones always bears the GU prefix (for “Guyane”, i.e. French Guiana). All the germplasm of the same geographical origin present in the CIRAD collection at Paracou-Combi (Sinnamary, French Guiana), i.e. more than 1600 trees, has been abundantly studied for its morphological characterization, its agronomic assessment or its genetic diversity. Other assessment work, primarily on resistance to certain diseases, has been carried out by CIRAD in Montpellier, or in various countries by other organizations.     

香蕉延长因子1A（MaEF1A）基因的分离及特征分析

采用筛选香蕉果实采后cDNA文库的方法,首次获得了一条长为1 341 bp的cDNA序列,命名为MaEF1A。生物信息学分析结果表明,它与麝香百合延长因子1A 3的mRNA有84%的同源性,与烟草延长因子1A的mRNA有83%的同源性,推测它可能为编码香蕉延长因子1A（elongation factor-1 alpha...     

Application of thermal sums concept to estimate the time to harvest new banana hybrids for export

Application of the thermal sum concept was developed to determine the optimal harvesting stage of new banana hybrids to be grown for export. It was tested on two triploid hybrid bananas, FlhorBan 916 (F916) and FlhorBan 918 (F918), created by CIRAD's banana breeding programme, using two different approaches. The first approach was used with F916 and involved calculating the base temperature of bunches sampled at two sites at the ripening stage, and then determining the thermal sum at which the stage of maturity would be identical to that of the control Cavendish export banana. The second approach was used to assess the harvest stage of F918 and involved calculating the two thermal parameters directly, but using more plants and a longer period. Using the linear regression model, the estimated thermal parameters were a thermal sum of 680 degree-days (dd) at a base temperature of 17.0 °C for cv. F916, and 970 dd at 13.9 °C for cv. F918. This easy-to-use method provides quick and reliable calculations of the two thermal parameters required at a specific harvesting stage for a given banana variety in tropical climate conditions. Determining these two values is an essential step for gaining insight into the agronomic features of a new variety and its potential for export.     

Nutritional quality of greenhouse lettuce at harvest and after storage in relation to N application and cultivation season

The effect of five levels of nitrogen fertilization on the growth and nutritional quality of Cos lettuce (Lactuca sativa L. cv. Parris Island) at harvest and after storage was studied during autumn and winter in South-West Greece. Plants were cultivated hydroponically in a greenhouse and the nitrate, chlorophyll and ascorbic acid (vitamin C) concentrations within the plant tissues were measured at harvest and following storage at 5 or 10 °C for 10 days. Nitrate accumulated in the leaves with increasing amounts of N within the nutrient solution and was higher in the winter than in the autumn. At the lowest N level (20 mg L⁻¹), the inner leaves accumulated more nitrate than the outer leaves, whereas at higher N levels (140, 200 or 260 mg L⁻¹) nitrate accumulation was higher in the outer leaves. Overall, the highest nitrate concentrations were detected in the petiole and the proximal end of the leaf, but at the lowest N application rate (20 mg L⁻¹) nitrate accumulated in the distal region of the leaf too. Although the nitrate concentrations within the leaves did not change significantly during 10 days storage at 5 or 10 °C, the chlorophyll and vitamin C concentrations decreased. Chlorophyll loss was higher in lettuce that was grown under low N levels and was higher at 10 °C than at 5 °C, but was reduced by enclosure of the lettuce in polyethylene film. It is concluded that the optimum N application rate for Cos lettuce grown hydroponically under cover during autumn and winter in South-West Greece, and in other areas with a similar climate, is 200 mg N L⁻¹ because at this N rate yield is satisfactory and leaf nitrate concentrations are below the maximum acceptable level for human consumption. Nutritional value (vitamin C concentration) and market quality (chlorophyll content) are highest at harvest and decrease during storage, but quality in terms of nitrate concentration does not change.     

Identification of seven haplotype-specific SFBs in European pear (Pyrus communis) and their use as molecular markers

The European pear (Pyrus communis) carries the S-RNase-mediated gametophytic self-incompatibility (GSI) system. The S-haplotype is conferred by an S-locus, which contains the style-specific expressed S-RNase, and the pollen-specific expressed F-box genes (SFB). Both the S-RNase and the SFB genes are multi-allelic and each is characteristic of one of the S-haplotypes. Therefore, they are ideal markers for molecular S-genotyping. In this work, for the first time, seven haplotype-specific SFBs were isolated from European pears. Particular primers for each of these SFBs were generated, thus providing an additional tool for S-genotyping of European pear cultivars.     

Screening of cold-resistant seedlings of a Chinese wild grape (Vitis piasezkii Maxim var. pagnucii) native to loess plateau of eastern Gansu province,China, as rootstocks

Seedlings of a Chinese wild grape (Vitis piasezkii Maxim var. pagnucii) native to loess plateau of Eastern Gansu province, China, were evaluated to screen cold-resistant rootstocks in Lanzhou area. After 14-year investigation two selections of LDP-191and LDP-294 were screened as rootstocks for two table grape cultivars, ‘Fujiminori’ and ‘Red Globe’, respectively. The two graft unions demonstrated very high cold-resistance as well as good graft compatibility. Furthermore, they could survive through low temperatures in winter without soil coverage together with good fruit quality of the cultivars grafted.     

香蕉trihelix转录因子MaGTL1a的分离及特性

【目的】研究香蕉MaGTL1a转录因子的特性及其基因表达规律,探讨MaGTL1a转录因子在香蕉果实成熟过程中的作用。【方法】以香蕉果肉c DNA为模板,采用RT-PCR获得MaGTL1a序列;利用烟草瞬时表达法和酵母系统分析MaGTL1a亚细胞定位和转录调控活性;通过实时荧光定量PCR分析MaGTL1a在香蕉果实成熟过程中的表达;运用烟草BY2悬浮细胞瞬时表达法研究MaGTL1a启动子活性。【结果】MaGTL1a c DNA序列含有1个2 283 bp的开放阅读框,编码761个氨基酸,属于trihelix转录因子的GT-2亚家族;亚细胞定位和转录活性分析显示,MaGTL1a定位于细胞核,并在酵母和植物体内具有转录激活活性;实时荧光定量PCR和启动子活性试验表明,MaGTL1a转录水平和启动子活性均受乙烯诱导,并且MaGTL1a转录水平随着香蕉果实的成熟进程而明显增强。【结论】MaGTL1a是一个受乙烯诱导和核定位的转录激活子,可能参与了香蕉果实成熟的调控。     

Application method and rate of Trichoderma species as a biological control against Pythium aphanidermatum (Edson) Fitzp. in the production of microgreen table beets (Beta vulgaris L.)

Seed balls of ‘Early Wonder Tall Top’ table beet (Beta vulgaris L.) were incubated for 2 days at 21 °C in moist exfoliated grade 3 vermiculite (3 g seed balls [168 seed balls] and 1.1 g vermiculite) containing equal weights of Trichoderma harzianum Rifai strain KRL-AG2 G41 and T. virens strain G-41 (ThTv) at 0, 0.25, 0.50, 0.75 or 1.00 mg ThTv per seed ball. ThTv also was applied to the peat-lite growth medium 14 days before planting, at the same rates per seed ball as the seed ball treatments. Four days before planting, the peat-lite was inoculated with Pythium aphanidermatum (Edson) Fitzp. (Pa) at 0, 0.5 and 1.0× the rate that resulted in 96% damping-off when non-ThTv-treated dry seed balls were sown in peat-lite containing 1.0 Pa. Increasing ThTv level per seed ball decreased percentage damping-off, with 1.0 Pa giving greater percentage damping-off than 0.5 Pa. At 1 mg ThTv per seed ball, damping-off was 5% and 19% at 0.5 and 1.0 Pa, respectively. Including Agro-Lig UF (mostly humic acids) in the incubating seed-ball-ThTv mixture further decreased damping-off by an average 13 and 10 percentage points in 0.5 and 1.0 Pa, respectively. Increasing ThTv per seed ball in growth media decreased percentage damping-off, but not to the extent achieved with seed ball treatment, with 1 mg ThTv per seed ball giving 20% and 55% damping-off in 0.5 and 1.0 Pa, respectively. Decreasing incidence of damping-off with increasing ThTv application to seed balls or growth media was associated with increasing shoot fresh weight m⁻² at 14 days after planting, a response attributable to increased percentage plant survival and not to a ThTv growth-promoting effect.     

Yield,dry matter and polysaccharides content of the mushroom Agaricus blazei produced on asparagus straw substrate

The aim of this work is to evaluate the potential of asparagus (Asparagus officinalis L.) straw as a raw material for cultivating Agaricus blazei Murrill (ABM). On non-supplemented asparagus straw substrate, the yield and biological efficiency (BE) of ABM were respectively 6.7 kg/m² and 30.2%. Addition of appropriate amounts of cottonseed hull or cow manure to the substrate increased the mushroom yield significantly. The mushroom yield on asparagus straw + cottonseed hull substrate was higher than that on asparagus straw + cow manure substrate. Maximum mushroom yield (9.8 kg/m²) and BE (44.1%) were obtained on the substrate consisting of asparagus straw (600 kg) and cottonseed hull (300 kg). No significant differences were found in either the dry matter contents or the polysaccharides contents of fruit bodies among the treatments.     

Identification of S-alleles in pear (Pyrus communis L.) cv. ‘Rocha’ and other European cultivars

In this work we report the cloning and identification of S-RNase alleles responsible for gametophytic self-incompatibility (GSI) of ‘Rocha’ pear and of 13 other European pear cultivars that might be used as its pollinators. Partial sequences of S-RNase alleles were amplified by PCR with specific primers hybridising in conserved regions of previously identified S-RNase alleles of Pyrus communis, cloned and sequenced and the S-genotype of eight pear cultivars was fully determined. Three cultivars (‘General Léclerc’ (SqSl), ‘Tosca’ (SbSl) and ‘Alexandrine Douillard’ (SbSk)) shared no S-alleles with ‘Rocha’ (SaSe) and shall be totally compatible with this cultivar. None of the cultivars analysed showed an identical amplification pattern to the one observed in ‘Rocha’, so the other cultivars shall be at least semi-compatible. One new allele was identified in P. communis cv. ‘Beurré d’Avril’ (designated as St). The determination of both S-RNase alleles of cvs ‘Rocha’, ‘Beurré Precoce Morettini’ (SeSk) and ‘Tosca’ and the identification of one S-RNase allele in cvs ‘Carapinheira’ (Sb), ‘Amêndoa’ (Se), ‘Pérola’ (Sk) and ‘Beurré d’Avril’ (St) are important contributions for the effort recently developed worldwide to establish groups of sexual compatibility among European pears.     

Genetic diversity and relationships of wild and cultivated olives at regional level in Spain

Genetic diversity and relationships between local cultivars and wild olive trees from three important Spanish olive-growing regions, Andalusia (South), Catalonia and Valencia (from Eastern Mediterranean Coastal area), were studied by means of eight SSR loci. Distinct allelic composition and heterozygosity levels were found in wild olive populations and cultivars. The observed patterns of genetic variation revealed: a) the independent clustering of Andalusian wild olives in a separate gene pool, b) the belonging of wild populations and most cultivars from Catalonia to another gene pool, c) the joined clustering of Andalusian and a set of Valencian cultivars in a third gene pool, and d) clustering of wild individuals from Valencia to the three different gene pools. These results suggest that wild populations of Andalusia may represent true oleasters, the ones from Catalonia may be feral forms derived from cultivar seed spreading, while the population of Valencia seems to be the most admixed one. The significant differentiation between Andalusian and most Catalonian cultivars is indicating an independent selection of olive cultivars in the two regions. The detection of a certain wild genetic background in some Catalonian and Valencian cultivars and the similarity found between wild and cultivated forms may suggest the use of local wild trees in olive domestication. The proposed scenario for the development of olive cultivars in Andalusia includes an empirical selection of outstanding local wild genotypes followed by various generations of crosses and various replanting campaigns, as well as possible introductions of ancestral cultivars. Therefore, our findings would lead us to support the hypothesis that the current diversity found in Spanish olive cultivars may be regionally differentiated and due to both, autochthonous and allochthonous origin. The information obtained in this work gives insights into the genetic resources of the main olive producing country, demonstrating that wild olive populations and local cultivars both represent potential sources of useful variability for olive breeding programs.     

Development of specific primers for cpSSR analysis in caper,olive and grapevine using consensus chloroplast primer pairs

Chloroplast SSR (cpSSR) markers have demonstrated utility in studying genetic relationships. DNA sequence information of the chloroplast genome is necessary for the development of cpSSR primer pairs. To overcome this limitation, “consensus” primers have been developed to amplify the homologous regions in plants where chloroplast sequences are not available. However, 80% Pinus thunbergi and Nicotiana tabacum developed “consensus” primers tested with grapevine, olive and caper showed multi-locus patterns. The presence of multi-locus patterns requires the use of agarose gel electrophoresis followed from isolation and sequencing of the bands. Herein, a PCR-strategy is proposed to construct specific cpSSR primer pairs without genomic sequence information, giving single-band amplifications that can be directly sequenced. Twelve new specific cpSSR primer pairs were developed for Capparis spinosa L., Olea europea L. and Vitis vinifera L. PCR products were sequenced to confirm the presence of microsatellite sequences, and their transportability was tested on six V. vinifera cultivars. Both single-nucleotide polymorphisms and polymorphic cpSSR were observed in the six grapevine cultivars using the specific cpSSR primers.     

Genetic characterization of asparagus doubled haploids collection and wild relatives

The genus Asparagus is very large consisting of around 150 species found as herbaceous perennials, tender woody shrubs and vines. The cultivated species (Asparagus officinalis L., diploid) is a highly prized vegetable, grown in different environments ranging from cool temperate zones to deserts, Mediterranean climates and tropical areas. As a consequence, Asparagus breeders have developed different cultivars that differ for their morpho-agronomic traits, habit and ploidic status (few triploid and tetraploid cultivars are used). Several breeding methods are used for developing cultivars, among which a well developed in vitro anther culture technique produces homozygous clones useful for F₁ hybrids constitution. A fluorescent based AFLP (amplified fragment length polymorphism) technique were applied with the aim to assess genetic diversity among a collection of 173 doubled haploid (DH) androgenetic clones, five Asparagus wild species and interspecific hybrids obtained among the cultivated species and two wild relatives. The average number of AFLP fragments generated per primer set was 105, varying in size from 50 to 550 bp. A total of 1054 AFLP fragments were detected, 20% of which were polymorphic. Genetic similarity based on DNA polymorphisms, showed that a few number of AFLP primer combinations are able to distinguish the cultivated DH clones from the wild species. Indeed, from one DH clone for each anther donors and the wild species were used to construct a dendrogram using Dice's coefficient and the unweighted pair group method with the arithmetic mean (UPGMA). Genetic distances among all DH clones were calculated using the C.S. Chord distance; and a neighbour-joining (NJ) consensus tree was constructed in order to support the breeder for parental genotype choice for asparagus hybrid constitution.