Development and mapping of SSR markers for maize

Microsatellite or simple sequence repeat (SSR) markers have wide applicability for genetic analysis in crop plant improvement strategies. The objectives of this project were to isolate, characterize, and map a comprehensive set of SSR markers for maize (Zea mays L.). We developed 1051 novel SSR markers for maize from microsatellite-enriched libraries and by identification of microsatellite-containing sequences in public and private databases. Three mapping populations were used to derive map positions for 978 of these markers. The main mapping population was the intermated B73 × Mo17 (IBM) population. In mapping this intermated recombinant inbred line population, we have contributed to development of a new high-resolution map resource for maize. The primer sequences, original sequence sources, data on polymorphisms across 11 inbred lines, and map positions have been integrated with information on other public SSR markers and released through MaizeDB at URL:www.agron.missouri.edu. The maize research community now has the most detailed and comprehensive SSR marker set of any plant species.     

In vitro morphogenesis of Cucumis melo var. inodorus

In vitro morphogenesis of C. melo L. var. inodorus was studied by the induction of adventitious buds and somatic embryos. Organogenesis was obtained from cotyledon segments and leaf discs in culture medium supplemented with benzylaminopurine (1 mg l⁻¹) and somatic embryogenesis was induced in medium containing 2,4-dichlorophenoxyacetic acid (5 mg l⁻¹) + thidiazuron (1 mg l⁻¹). Through histological analysis it was possible to verify that in cotyledonary explants, protuberances that do not develop into well-formed shoot buds and leaf primordia are more frequently formed than complete shoot buds, resulting in a low frequency of plant recovery in the organogenic process. A high percentage of explants responded with the formation of somatic embryos; the microscopical analysis showed that the somatic embryos lacking well developed apical meristems had a low conversion rate into plants. Plant recovery was not obtained from leaf-disc explants, with high rates of contamination and formation of protuberances which did not develop into shoot buds. Histological sections showed the development of epidermis and leaf hairs, indicating those structures could be leaf primordia; however, these were not associated with a shoot apical meristem. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration and characterisation of Cucumis melo L. (+) Cucumis anguria L. var. longipes (Hook. fil.) Meeuse somatic hybrids

Cotyledon protoplasts of an albino Cucumis melo L. ‘Cantaloup Charentais’ somaclonal variant were electrofused with protoplasts of the wild species Cucumis anguria L. var. longipes (Hook. fil.) Meeuse. Selection of putative somatic hybrids was based on competence of the albino melon to grow and regenerate shoots together with the ability of the wild species to synthesise chlorophyll. The ITS region of C. anguria ribosomal DNA was sequenced to design species-specific primers, which allowed us to distinguish between parental lines and fusion products by PCR amplification. By using this method, all the organogenic lines characterised proved to be somatic hybrids. Three of sixteen selected lines produced shoots with albino and green sectors. Eleven lines remained green but shoots developed abnormally and did not produce roots in vitro. Two hybrid lines regenerated normal shoots but with a limited ability to produce roots in vitro and in vivo. Applicability of molecular characterisation to optimise the quick recovery of fusion products is discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of the Genome Sequence of Strain GiC-126 of Gloeostereum incarnatum with Genetic Linkage Map

Gloeostereum incarnatum has edible and medicinal value and was first cultivated and domesticated in China. We sequenced the G. incarnatum monokaryotic strain GiC-126 on an Illumina HiSeq X Ten system and obtained a 34.52-Mb genome assembly sequence that encoded 16,895 predicted genes. We combined the GiC-126 genome with the published genome of G. incarnatum strain CCMJ2665 to construct a genetic linkage map (GiC-126 genome) that had 10 linkage groups (LGs), and the 15 assembly sequences of CCMJ2665 were integrated into 8 LGs. We identified 1912 simple sequence repeat (SSR) loci and detected 700 genes containing 768 SSRs in the genome; 65 and 100 of them were annotated with gene ontology (GO) terms and KEGG pathways, respectively. Carbohydrate-active enzymes (CAZymes) were identified in 20 fungal genomes and annotated; among them, 144 CAZymes were annotated in the GiC-126 genome. The A mating-type locus (MAT-A) of G. incarnatum was located on scaffold885 at 38.9 cM of LG1 and was flanked by two homeodomain (HD1) genes, mip and beta-fg. Fourteen segregation distortion markers were detected in the genetic linkage map, all of which were skewed toward the parent GiC-126. They formed three segregation distortion regions (SDR1–SDR3), and 22 predictive genes were found in scaffold1920 where three segregation distortion markers were located in SDR1. In this study, we corrected and updated the genomic information of G. incarnatum. Our results will provide a theoretical basis for fine gene mapping, functional gene cloning, and genetic breeding the follow-up of G. incarnatum.     

An efficient selection and regeneration protocol for Agrobacterium-mediated transformation of oriental melon (Cucumis melo L. var. makuwa)

An efficient selection and plant regeneration protocol for Agrobacterium-mediated transformation using cotyledon explants of oriental melon (Cucumis melo L. var. makuwa) has been developed. All six oriental melon cultivars evaluated in the study showed a >90?% shoot regeneration frequency and produced 1.8?C3.6 shoots per cotyledon explant when cultured on Murashige and Skoog (MS) medium supplemented with 1.0?mg?L^?1 benzyladenine and 0.01?mg?L^?1 indoleacetic acid. Kanamycin (Km) and geneticin (Gt) in the shoot induction medium (SIM) were compared both qualitatively and quantitatively for their efficiency as a selection agent for the selection and regeneration of transgenic plants after Agrobacterium-mediated transformation. Shoot formation was completely inhibited at 50?mg?L^?1 Km and 10?mg?L^?1 Gt. Relatively high concentrations of both Gt and Km (>100?mg?L^?1 Km and >25?mg?L^?1 Gt) were necessary because large numbers of non-transgenic shoots survived during the selection process. The incorporation of a selectable marker (neomycin phosphotransferase II) into the genome of transgenic plants was confirmed using ??-glucuronidase (GUS), PCR and Southern blot analysis. Shoot regeneration frequencies were 41.2?% at 100?mg?L^?1 Km and 15.2?% at 30?mg?L^?1 Gt 8?weeks after transformation, whereas the transformation frequencies based on the PCR were 2.9 and 7.1?%, respectively, 16?weeks after transformation. These results demonstrate that a large portion of the regenerated shoots on SIM supplemented with 100?mg?L^?1 Km consisted of non-transformed or escaped shoots, indicating that 30?mg?L^?1 Gt is the more suitable for the selection and regeneration of transgenic plants in oriental melon.     

国外甜瓜种质资源形态性状遗传多样性分析

对250份国外甜瓜(Cucumis melo)种质资源的19个形态性状多样性进行研究。结果表明, 国外甜瓜种质资源具有丰富的形态多样性, 平均遗传多样性指数为1.378。9个质量性状(果实形状、果皮底色、覆纹颜色、覆纹形状、网纹密度、网纹粗度、果肉颜色、果肉质地和种子颜色)和4个数量性状(单果鲜重、果肉厚度、可溶性固形物含量和种子千粒重)变异明显, 其Shannon’s指数分别大于1.0和1.9。不同生态区间种质资源遗传多样性差异明显, 多样性指数由高到低依次为: 南亚(1.512)、东北欧(1.404)、西欧(1.372)、北美(1.340)和东亚(1.281)。通过聚类分析将所有甜瓜种质划分为四大组群, 即南亚组群、东北欧组群、西欧北美组群和东亚北美组群。以印度为代表的南亚甜瓜种质形态多样性水平较高, 支持了印度次生大陆为甜瓜起源中心的观点。     

新疆甜瓜地方种质资源遗传多样性的SRAP分析

为研究我国新疆甜瓜地方种质资源亲缘关系及其分类,充分高效的利用种质资源,利用SRAP(sequence-related amplified polymorphism technique)标记对117份中国新疆甜瓜地方品种和28份国内外对照材料进行亲缘关系和遗传多样性分析。结果表明,20对SRAP引物共扩增出224个带,其中多态性谱带216个,多态性比率达96%,平均每对引物扩增的带数和多态性带数分别为11.2个和10.8个,每对引物的多态性信息含量PIC值为0.73~0.94,平均为0.85;不同生态区域供试材料的Nei's基因多样性指数(H)和Shannon's信息指数(I)分别为0.1075~0.2560和0.1569~0.4061,中国新疆的南疆、东疆和北疆均高于其他生态区域供试材料,且以南疆最高,具有非常丰富的遗传多样性;不同生态区域甜瓜种质资源的遗传一致度和遗传距离分别为0.6384~0.9919和0.0081~0.4488,其中南疆、东疆和北疆两两之间的遗传一致度均在0.95以上,遗传距离均在0.04以下,三者之间遗传分化较小;中国新疆甜瓜与印度、西亚、西班牙的甜瓜种质资源亲缘关系较近,与韩国、日本、美国和前苏联的甜瓜种质资源亲缘关系较远。聚类分析结果表明,以遗传相似系数0.548为阈值,145份种质材料可分为3大类群;厚皮甜瓜与薄皮甜瓜间在分子水平上没有严格的界限,两者之间亲缘关系的远近在不同的种质材料间差异很大;117份中国新疆甜瓜地方种质资源可分为A(Ⅰ-1)、B(Ⅰ-2、Ⅰ-3、Ⅰ-5)、C(Ⅰ-6)、D(Ⅱ)等4大类6个亚类群,与传统4个变种10个品种群分类结果不同,但在每个大类或亚类群中属于同一变种或品种群的材料倾向于聚在一起。     

Characterization of highly polymorphic nuclear microsatellite loci in Juniperus communis L.

We developed five nuclear microsatellite markers in Juniperus communis L. using an enriched library method. Samples from 28 juniper individuals were collected in Spain, Germany and Slovakia and were analysed at the five loci. A high level of allelic diversity with values ranging from nine to 23 alleles was found. These highly polymorphic markers will be used in ongoing population genetic studies to evaluate the genetic resources and to contribute to the maintenance of genetic diversity of juniper in Middle Europe.     

Mixed linear model association mapping for low chloride accumulation rate in oriental-type tobacco (Nicotiana tabaccum L.) germplasm

Chloride is an essential micronutrient in tobacco (Nicotiana tabaccum L.) cultivation. However, large amounts of it have many adverse effects on burning quality of tobacco leaves. The objective of this study was to evaluate the genetic variability among 70 oriental-type tobacco genotypes and determine the genomic regions associated with chloride accumulation rate using mixed linear model (MLM) procedure. A total number of 66 alleles were detected by 26 simple sequence repeat (SSR) loci with an average of 2.53 alleles per locus. A model-based Bayesian approach subdivided 70 tobacco genotypes into the three subgroups. Almost 5.85% of the 325 marker pairs showed a significant level of linkage disequilibrium (P ≤ 0.01). Using MLM procedure, 1 SSR locus (pt30027) from linkage group 13 was identified to be associated with the gene(s) controlling low chloride accumulation in oriental tobacco genotypes. Identified markers could be of great interest in marker-assisted selection in tobacco breeding programs.     

Constructing a high-density linkage map for Gossypium hirsutum × Gossypium barbadense and identifying QTLs for lint percentage

To introgress the good fiber quality and yield from Gossypium barbadense into a commercial Upland cotton variety, a high-density simple sequence repeat(SSR) genetic linkage map was developed from a BC1F1 population of Gossypium hirsutum×Gossypium barbadense. The map comprised 2,292 loci and covered 5115.16 centi Morgan(c M) of the cotton AD genome, with an average marker interval of 2.23 c M. Of the marker order for 1,577 common loci on this new map, 90.36% agrees well with the marker order on the D genome sequence genetic map. Compared with five published high-density SSR genetic maps, 53.14% of marker loci were newly discovered in this map. Twenty-six quantitative trait loci(QTLs) for lint percentage(LP) were identified on nine chromosomes. Nine stable or common QTLs could be used for marker-assisted selection. Fifty percent of the QTLs were from G. barbadense and increased LP by 1.07%–2.41%. These results indicated that the map could be used for screening chromosome substitution segments from G. barbadense in the Upland cotton background, identifying QTLs or genes from G. barbadense, and further developing the gene pyramiding effect for improving fiber yield and quality.     

Association of tomato leaf curl Palampur virus with yellow mosaic disease of Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) in India

Natural occurrence of yellow mosaic disease was observed on Armenian cucumber (Cucumis melo var. flexuoses) and wild melon (C. callosus var. agrestis) with disease incidences of ~36 and ~27%, respectively. Association of tomato leaf curl Palampur virus (ToLCPV) with the disease was investigated by Polymersae chain reaction (PCR) using begomovirus-specific primers. Full-length genome was amplified by rolling circle amplification (RCA) method from representative samples of C. melo and C. callosus. RCA products obtained were cloned and sequenced. Analyses of sequence data revealed the presence of full-length begomoviral genome of 2756 nucleotides with the gene arrangement of a typical begomovirus: HQ848383 (C. melo) and GU253914 (C. callosus). Both the isolates shared 99% sequence identity together and high 97–99% identities and the closest phylogenetic relationships with ToLCPV strains reported worldwide, hence identified as two new members of ToLCPV. Natural occurrence of ToLCPV on C. melo and C. callosus is the first report.     

甜瓜诱变材料的同工酶电泳聚类分析

本文应用PAGE技术,分析了经秋水仙碱诱变的甜瓜的四个品种、一个新品系的13份材料和3个对照的POD、EST、MDH、ADH、GDH、SOD的同工酶．根据16份材料的谱带特征二态数字矩阵,用MEGA程序计算了它们间的Numberofdifference,Proportionofdifference和Kimura-2-parameterdistance遗传距离,分别用UPGMA和Neighbor-joining进行聚类分析．结果表明,甜瓜材料经秋水仙碱诱变均产生了一定的变异,品种内的遗传分化程度与形态性状和细胞学检测相符合．同工酶遗传变异的大小可以反映诱变材料变异大小,在一定程度上可为有效选择育种材料提供依据．     

Genic microsatellite markers derived from EST sequences of switchgrass (Panicum virgatum L.)

Switchgrass is a large, North American, perennial grass that is being evaluated as a potential energy crop. There is a need to assess genetic diversity in stored accessions and in remaining native stands to assist breeding and conservation efforts. Marker development will also be necessary for genetic linkage mapping. Toward this end, 32 switchgrass genic di‐, tri‐ and tetranucleotide repeat microsatellites were identified from expressed sequence tags (ESTs). These microsatellites were used to screen individuals from two different named cultivars. The markers displayed a high level of polymorphism consistent with the tetraploid, allogamous behaviour of the cultivars tested.     

‘弥河银瓜’高效植株再生体系的建立

建立了以薄皮甜瓜品种‘弥河银瓜’子叶为外植体的高效再生体系。取生长5d的甜瓜子叶为外植体,置于MS+2.0mg·L-16-BA+0.5mg·L-1IBA+3.5%蔗糖+0.7%琼脂(pH5.8)的诱导培养基上培养,可获得100%芽分化频率。在不定芽伸长过程中,生长调节剂最佳浓度为0.5mg·L-16-BA+0.1mg·L-1IAA。潮霉素是较合适的筛选抗生素。     

金瓜和白瓜花叶病毒病病原的初步鉴定

崇明是上海出口蔬菜生产基地之一,也是国家级的绿色食品园区之一,具有生产绿色蔬菜商品的生态环境和资源优势。特色出口蔬菜金瓜(Cucurbita pepo L.var.ovifera)、白瓜(Cucumis melo L.var.conomon,又名盐渍菜瓜)1997～1999统计病毒病发病率为23％,被感染的金瓜、白瓜田     

Isolation of polymorphic microsatellite markers for British Euphrasia L.

Euphrasia species in Britain attract a large amount of conservation attention due to the recognition of numerous endemic taxa in what is essentially a species‐poor flora. To develop a set of research tools to investigate the evolutionary processes underlying this diversification, a membrane enrichment procedure has been used to isolate five polymorphic microsatellite loci from Euphrasia nemorosa (Pers.) Wallr. These loci amplify polymorphic products in several other British Euphrasia species.     

粳稻SRAP分子标记遗传群的构建与分析

用超级稻品种‘沈农606’和普通粳稻‘丽江新团黑谷’为亲本杂交获得的102份F_2代单株,通过SRAP分子标记遗传分析,构建了包含14个连锁群,由129个多态性位点组成的水稻连锁图谱,此图谱覆盖基因组长度1671.5 cM,平均图距13.0 cM。连锁群上有17.2%的多态性位点表现偏分离,偏分离标记在连锁群上存在热点区域。     

黑龙江省主栽大豆品种遗传多样性的SSR分析

使用合丰25等来自13个育种单位的13份大豆（Glycinemax）品种对大豆20个连锁群上的100对SSR标记进行筛选,最终保留了扩增稳定且多态性较高的43对SSR标记,分析了黑龙江省83个主栽大豆品种的遗传多样性。结果表明,在所有供试材料中共鉴定出等位变异157个,每个位点2—7个,平均为3．65个。品种间遗传相似系数为0．216—0．937,平均为0．6384,表明黑龙江省大豆品种的遗传相似性较大,故拓宽黑龙江省大豆品种的遗传基础具有重要意义。     

山地虎耳草和棒腺虎耳草转录组SSR和SNP分析

利用Illumina HiSeqTM2000对山地虎耳草和棒腺虎耳草进行转录组测序,分析和比较其SSR和SNP特征。结果表明:山地虎耳草63 763条Unigene序列中含有4 622个SSR,发生频率为7.25%,有110种重复基元,平均每10.00kb出现一个SSR位点;棒腺虎耳草60 972条Unigene序列中含有4 542个SSR,发生频率为7.45%,有85种重复基元,平均每10.40kb出现一个SSR位点,略低于山地虎耳草。山地虎耳草和棒腺虎耳草转录组序列的SSR优势基元均为三核苷酸重复。2个物种的转录组SSR以5~10次的较低重复次数为主,长度主要集中于12~30bp,具有较高的多态性。山地虎耳草和棒腺虎耳草中分别获得118 424个和112 006个SNP位点,编码区的SNP位点分别占30.40%和28.59%,且在编码SNP中同义突变所占比例(30.27%、28.48%)远高于非同义突变(0.13%、0.11%)。比较发现,2个物种的各项检索结果基本一致,推测与选取的组织部位、组织的发育阶段以及物种的亲缘关系有关。