Editor's choice: Genome-Wide SNP Genotyping to Infer the Effects on Gene Functions in Tomato

The genotype data of 7054 single nucleotide polymorphism (SNP) loci in 40 tomato lines, including inbred lines, F₁ hybrids, and wild relatives, were collected using Illumina''s Infinium and GoldenGate assay platforms, the latter of which was utilized in our previous study. The dendrogram based on the genotype data corresponded well to the breeding types of tomato and wild relatives. The SNPs were classified into six categories according to their positions in the genes predicted on the tomato genome sequence. The genes with SNPs were annotated by homology searches against the nucleotide and protein databases, as well as by domain searches, and they were classified into the functional categories defined by the NCBI''s eukaryotic orthologous groups (KOG). To infer the SNPs'' effects on the gene functions, the three-dimensional structures of the 843 proteins that were encoded by the genes with SNPs causing missense mutations were constructed by homology modelling, and 200 of these proteins were considered to carry non-synonymous amino acid substitutions in the predicted functional sites. The SNP information obtained in this study is available at the Kazusa Tomato Genomics Database (http://plant1.kazusa.or.jp/tomato/).     

Construction of a Microsatellite Linkage Map with Two Sequenced Rice Varieties

Based on the successful development of new microsatellite markers from the data of two whole-sequenced rice varieties, japonica variety Nipponbare and indica variety 9311, an F₂ population of 90 lines, which was derived from a single cross between Nipponbare and 9311, was applied to construct a genetic linkage framework map. The map covered 2 455.7 cM of total genomic length, and consisted of 152 simple sequence repeats (SSRs) loci including 46 pairs of new SSR primers developed by our research institute. The average genetic distance between two markers was 16.16 cM. In addition, markers RM345 and RM494, which have not been mapped on the Temnykh's map et al. (2001) were anchored on the sixth chromosome of this map. We compared this research with maps of Temnykh et al.(2001) and LAN et al. (2003) regarding the aspects of type and size of population, type and quantity of markers, and the marker arrangement order on chromosome, etc. Results indicated that the similarity of marker linear alignment was 93.81% between this map and T-map, Finally, the important significance of using sequenced rice varieties to construct linkage map was also discussed.     

Tomato SNP Discovery by EST Mining and Resequencing

Many economically important crop species are relatively depauparate in genetic diversity (e.g., soybean, peanut, tomato). DNA polymorphism within cultivated tomato has been estimated to be low based on molecular markers. Through mining of more than 148,000 public tomato expressed sequence tags (ESTs) and full-length cDNAs, we identified 764 EST clusters with potential single nucleotide polymorphisms (SNPs) among more than 15 tomato lines. By sequencing regions from 53 of these clusters in two to three lines, we discovered a wealth of nucleotide polymorphism (62 SNPs and 12 indels in 21 Unigenes), resulting in a verification rate of 27.2% (28 of 103 SNPs predicted in EST clusters were verified). We hypothesize that five regions with 1.6–13-fold more diversity relative to other tested regions are associated with introgressions from wild relatives. Identifying polymorphic, expressed genes in the tomato genome will be useful for both tomato improvement and germplasm conservation.     

Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.     

High-density Linkage Map of Cultivated Allotetraploid Cotton Based on SSR, TRAP, SRAP and AFLP Markers

A high-density linkage map was constructed for an F2 population derived from an Interspecific cross of cultivated allotetraploid species between Gossypium hirsutum L. and G. barbadense L. A total of 186 F2 individuals from the Interspecific cross of ＂CRI 36 × Hal 7124＂ were genotyped at I 252 polymorphic loci Including a novel marker system, target region amplification polymorphism （TRAP）. The map consists of 1 097 markers, including 697 simple se- quence repeats （SSRs）, 171 TRAPs, 129 sequence-related amplified polymorphisms, 98 amplified fragment length polymorphisms, and two morphological markers, and spanned 4 536.7 cM with an average genetic distance of 4.1 cM per marker. Using 45 duplicated SSR loci among chromosomes, 11 of the 13 pairs of homologous chromosomes were Identified In tetraploid cotton. This map will provide an essential resource for high resolution mapping of quantitative trait loci and molecular breeding in cotton.     

Construction of Rice RAPD Molecular Linkage Map

A rice (Oryza sativa L. ) molecular linkage map has been constructed form over 52 random amplified polymorphic DNA (RAPD) markers with the double haploid (DH) population. It covered a total genetic distance of over 898.4 cM (centimorgan) with 17.3 cM between markers and was complemental with RFLP linkage map.     

SNP genotyping reveals genetic diversity between cultivated landraces and contemporary varieties of tomato

Background

The tomato (Solanum lycopersium L.) is the most widely grown vegetable in the world. It was domesticated in Latin America and Italy and Spain are considered secondary centers of diversification. This food crop has experienced severe genetic bottlenecks and modern breeding activities have been characterized by trait introgression from wild species and divergence in different market classes.

Results

With the aim to examine patterns of polymorphism, characterize population structure and identify putative loci under positive selection, we genotyped 214 tomato accessions (which include cultivated landraces, commercial varieties and wild relatives) using a custom-made Illumina SNP-panel. Most of the 175 successfully scored SNP loci were found to be polymorphic. Population structure analysis and estimates of genetic differentiation indicated that landraces constitute distinct sub-populations. Furthermore, contemporary varieties could be separated in groups (processing, fresh and cherry) that are consistent with the recent breeding aimed at market-class specialization. In addition, at the 95% confidence level, we identified 30, 34 and 37 loci under positive selection between landraces and each of the groups of commercial variety (cherry, processing and fresh market, respectively). Their number and genomic locations imply the presence of some extended regions with high genetic variation between landraces and contemporary varieties.

Conclusions

Our work provides knowledge concerning the level and distribution of genetic variation within cultivated tomato landraces and increases our understanding of the genetic subdivision of contemporary varieties. The data indicate that adaptation and selection have led to a genomic signature in cultivated landraces and that the subpopulation structure of contemporary varieties is shaped by directed breeding and largely of recent origin. The genomic characterization presented here is an essential step towards a future exploitation of the available tomato genetic resources in research and breeding programs.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-14-835) contains supplementary material, which is available to authorized users.     

Construction of Molecular Linkage Map in Masson Pine Using RAPD Markers and Megaga metophytes from a Single Tree

A set of 300 random ohgonucleotide primers was screened for random amplified polymorphic DNA (RAPD) fragments within a sample of 79 megagametophyte DNAs of a single masson pine ( Pinus massoniana Lamb. ) to generate RAPD markers and construct the molecular linkage map for masson pine. 64 repeatable RAPD markers segregated in a 1 present to 1 absent ratio. Through multipoint linkage analysis, 47 markers were assigned to 13 different linkage groups(pairs). It covered a tdtal genetic distance of over 692.5 cM (centimorgan). The average distance between markers was 14.7 cM. It supplied a linkage framework for a more saturated linkage map of masson pine.     

A Simple Sequence Repeat- and Single-Nucleotide Polymorphism-Based Genetic Linkage Map of the Brown Planthopper,Nilaparvata lugens

In this study, we developed the first genetic linkage map for the major rice insect pest, the brown planthopper (BPH, Nilaparvata lugens). The linkage map was constructed by integrating linkage data from two backcross populations derived from three inbred BPH strains. The consensus map consists of 474 simple sequence repeats, 43 single-nucleotide polymorphisms, and 1 sequence-tagged site, for a total of 518 markers at 472 unique positions in 17 linkage groups. The linkage groups cover 1093.9 cM, with an average distance of 2.3 cM between loci. The average number of marker loci per linkage group was 27.8. The sex-linkage group was identified by exploiting X-linked and Y-specific markers. Our linkage map and the newly developed markers used to create it constitute an essential resource and a useful framework for future genetic analyses in BPH.     

RAPD标记构建家蚕分子连锁图

以大造、Ｃ１０８及其Ｆ２群体构建了１个家蚕的ＲＡＰＤ连锁框架图,该图含ＲＡＰＤ标记位点１８２个,来自大造的１０３个标记分属前２３个连锁群,来自Ｃ１０８的７９个标记分属后１６个连锁群,覆盖基因组的总长度为１１４８．３ｃＭ（ｃｅｎｔｉｍｏｒｇａｎ）,它能与本室用同一群体构建的ＳＡＤＦ图谱相整合,亦可与相应的ＲＦＬＰ图谱相互补充。     

High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.)

The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina''s GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.     

Control of Root-knot Nematodes on Tomato in Stone Wool Substrate with Biological Nematicides

The efficacy of four biological nematicides on root-galling, root-knot nematode (Meloidogyne incognita) reproduction, and shoot weight of tomato (Solanum lycopersicum) grown in stone wool substrate or in pots with sandy soil was compared to an oxamyl treatment and a non-treated control. In stone wool grown tomato, Avid® (a.i. abamectin) was highly effective when applied as a drench at time of nematode inoculation. It strongly reduced root-galling and nematode reproduction, and prevented a reduction in tomato shoot weight. However, applying the product one week before, or two weeks after nematode inoculation was largely ineffective. This shows that Avid® has short-lived, non-systemic activity. The effects of Avid® on nematode symptoms and reproduction on soil-grown tomato were only very minor, probably due to the known strong adsorption of the active ingredient abamectin to soil particles. The neem derived product Ornazin® strongly reduced tomato root-galling and nematode reproduction only in stone wool and only when applied as a drench one week prior to nematode inoculation, suggesting a local systemic activity or modification of the root system, rendering them less suitable host for the nematodes. This application however also had some phytotoxic effect, reducing tomato shoot weights. The other two products, Nema-Q™ and DiTera®, did not result in strong or consistent effects on nematode symptoms or reproduction.     

The Potential of Five Winter-grown Crops to Reduce Root-knot Nematode Damage and Increase Yield of Tomato

Broccoli (Brassica oleracea), carrot (Daucus carota), marigold (Tagetes patula), nematode-resistant tomato (Solanum lycopersicum), and strawberry (Fragaria ananassa) were grown for three years during the winter in a root-knot nematode (Meloidogyne incognita) infested field in Southern California. Each year in the spring, the tops of all crops were shredded and incorporated in the soil. Amendment with poultry litter was included as a sub-treatment. The soil was then covered with clear plastic for six weeks and M. incognita-susceptible tomato was grown during the summer season. Plastic tarping raised the average soil temperature at 13 cm depth by 7°C.The different winter-grown crops or the poultry litter did not affect M. incognita soil population levels. However, root galling on summer tomato was reduced by 36%, and tomato yields increased by 19% after incorporating broccoli compared to the fallow control. This crop also produced the highest amount of biomass of the five winter-grown crops. Over the three-year trial period, poultry litter increased tomato yields, but did not affect root galling caused by M. incognita. We conclude that cultivation followed by soil incorporation of broccoli reduced M. incognita damage to tomato. This effect is possibly due to delaying or preventing a portion of the nematodes to reach the host roots. We also observed that M. incognita populations did not increase under a host crop during the cool season when soil temperatures remained low (< 18°C).     

重要花卉植物高密度遗传连锁图谱构建研究进展

遗传连锁图谱是以遗传标记间重组频率为基础的染色体或基因组内位点相对位置的线性排列图,高密度遗传图谱构建可实现物理图谱和遗传图谱的整合,对促进基因图位克隆具有重要作用。利用遗传图谱可有效地提高育种效率和改良品种。重要花卉植物高遗传图谱精密度尚无法满足精细定位研究的要求,百合、紫薇、郁金香、向日葵等重要花卉高密度遗传图谱构建研究较少,制约了花卉植物分子育种研究进程。概述了高密度遗传图谱构建流程及作图方法,综述了牡丹、梅花、月季、菊花、兰花、荷花、桂花等重要花卉植物遗传图谱构建研究进展,讨论了重要花卉植物高密度遗传图谱构建存在的主要问题,对今后重要花卉植物遗传图谱构建研究的发展方向及其在育种中的应用前景进行了展望,以期为花卉植物基因定位、辅助基因组组装、比较基因组学、基因克隆、分子标记辅助育种等提供参考。     

Chloroplast to chromoplast transition in tomato fruit: spectral confocal microscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue

Background and Aims

There are several studies suggesting that tomato (Solanum lycopersicum) chromoplasts arise from chloroplasts, but there is still no report showing the fluorescence of both chlorophylls and carotenoids in an intermediate plastid, and no video showing this transition phase.

Methods

Pigment fluorescence within individual plastids, isolated from tomato fruit using sucrose gradients, was observed at different ripening stages, and an in situ real-time recording of pigment fluorescence was performed on live tomato fruit slices.

Key results

At the mature green and red stages, homogenous fractions of chloroplasts and chromoplasts were obtained, respectively. At the breaker stage, spectral confocal microscopy showed that intermediate plastids contained both chlorophylls and carotenoids. Furthermore, an in situ real-time recording (a) showed that the chloroplast to chromoplast transition was synchronous for all plastids of a single cell; and (b) confirmed that all chromoplasts derived from pre-existing chloroplasts.

Conclusions

These results give details of the early steps of tomato chromoplast biogenesis from chloroplasts, with the formation of intermediate plastids containing both carotenoids and chlorophylls. They provide information at the sub-cellular level on the synchronism of plastid transition and pigment changes.     

单核苷酸多态性基因分型技术原理与进展

在基因组规模了解遗传变异与生物功能之间的关系可望为生物学带来全新的深入认识。本从等位基因分型机理、反应形式和检测方法等三个方面讨论SNP分型方法的现状,并简要介绍了目前应用的一些分型方法。     

不同番茄种质叶表次生代谢物质

多毛番茄(Solanum habrochaites)为重要的番茄种质资源, 其叶表存在大量次生代谢物质, 对多种虫害具有趋避或/和毒害作用。利用气相色谱-质谱联用(GC-MS)技术分析栽培番茄(S. lycopersicum ) 9706与3份多毛番茄(LA2329、LA1777和PI134417)材料叶表次生代谢物质。结果表明, 3份多毛番茄叶表可检测到的次生代谢物质种类和总含量均高于普通番茄, 同时多毛番茄亚种间次生代谢物质的种类和含量也存在差异。普通番茄叶表次生代谢物质为3种单萜和3种倍半萜类物质, 其中单萜和倍半萜类物质分别占次生代谢物质总量的60.3%和39.7%。多毛番茄LA2329和LA1777叶表倍半萜类物质的种类和含量较高, 有些萜类物质具有物种特异性。如LA2329中含量最高的α-姜烯, 其含量为2 409.1 μg·g^–1; LA1777中含量较高的γ-榄香烯和E-β-法尼烯, 含量分别为573.3 μg·g^–1和289.9 μg·g^–1, 在其它番茄材料中未检测到这3种倍半萜类物质。PI134417中含量最高的是月桂酸乙酯, 其含量为5 312.8 μg·g^–1, 在普通番茄中这一物质未见报道。PI134417中甲基酮类物质含量也较高, 其中2-十一烷酮和2-十三烷酮的含量分别为689.8 μg·g^–1和1 459.7 μg·g^–1。研究结果可为番茄种质资源利用和次生代谢物质开发提供理论依据。     

The Effects of Root-knot Nematode Infection and Mi-mediated Nematode Resistance in Tomato on Plant Fitness

The Mi-1.2 resistance gene in tomato (Solanum lycopersicum) confers resistance against several species of root-knot nematodes (Meloidogyne spp.). This study examined the impact of M. javanica on the reproductive fitness of near-isogenic tomato cultivars with and without Mi-1.2 under field and greenhouse conditions. Surprisingly, neither nematode inoculation or host plant resistance impacted the yield of mature fruits in field microplots (inoculum=8,000 eggs/plant), or fruit or seed production in a follow-up greenhouse bioassay conducted with a higher inoculum level (20,000 eggs/plant). However, under heavy nematode pressure (200,000 eggs/plant), greenhouse-grown plants carrying Mi-1.2 had more than ten-fold greater fruit production than susceptible plants and nearly forty-fold greater estimated lifetime seed production, confirming prior reports of the benefits of Mi-1.2. In all cases Mi-mediated resistance significantly reduced nematode reproduction. These results indicated that tomato can utilize tolerance mechanisms to compensate for moderate levels of nematode infection, but that the Mi-1.2 resistance gene confers a dramatic fitness benefit under heavy nematode pressure. No significant cost of resistance was detected in the absence of nematode infection.