Development of EST-derived SSR markers using next-generation sequencing to reveal the genetic diversity of 50 chrysanthemum cultivars

Chrysanthemum plants are popular worldwide as cut flowers, potted, and in gardens. Several hundred cultivars have been commercialized, indicating that there is substantial genetic variations that can be manipulated under cultivations to produce a wide array of phenotypic variation. To study the genetic diversity of chrysanthemum cultivars in Korea, we first identified simple sequence repeats from chrysanthemum expressed sequence tags generated by FLX 454 sequencing. A total of 1109 ESTs out of 18,226 chrysanthemum ESTs were identified to carry SSRs. A total of 16 out of 46 primer pairs exhibited several polymorphisms among 50 chrysanthemum cultivars. The number of alleles per locus varied from 1 to 15, with an average of 6.25 alleles. The expected heterozygosity ranged from 0 to 0.8958, whereas polymorphism information content ranged from 0 to 0.8872. Based on polymorphisms using 16 SSR markers, a phylogenetic tree was generated revealing four groups within the 50 cultivars showing various levels of genetic diversity. The 16 polymorphic chrysanthemum SSR markers generated in this study would be useful for studies of the genetic conservation, diversity, and population structure of commercial chrysanthemum cultivars as well as closely related species.     

Identification and mapping of polymorphic SSR markers from expressed gene sequences of barley and wheat

The growing availability of EST sequences from a range of crop plantsprovides a potentially valuable source of new DNA markers. We have examined theInternational Triticeae EST Cooperative database for the presence ofdinucleotide and trinucleotide simple sequence repeats. Analysis of 24,344 ESTsidentified 388 dinucleotide repeats and 978 trinucleotide repeats in ESTs,representing 1.6% and 4.0% of the total number of ESTs, respectively. To testthe utility and cross-species transferability of EST-derived SSR markers,primers were designed to the flanking regions of 41 barley SSRs and used toscreen 11 barley and 15 wheat varieties. Sixteen of the barley SSR markers werepolymorphic in barley and five were polymorphic in wheat. This represents arelatively high level of transferability of SSR markers between barley andwheat, which has important implications for the development of new markers andcomparative mapping of barley, wheat and other cereals. An additional 56 SSRsfrom wheat ESTs were tested in the same barley and wheat varieties. Four wheatEST SSR markers were polymorphic in wheat and one in barley. Chromosomallocations in barley and wheat were determined for the majority of polymorphicmarkers.     

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.     

A survey of the response of Rhododendron to in vitro culture

The responses of 7 genotypes of Rhodendron to culture conditions and their establishment as shoot cultures are described. The genotypes represent a broad genetic diversity in the genus. After sterilization and an acclimation period of 3 to 12 months, all the selections were established as shoot cultures on Woody Plant Medium (WPM) supplemented with N⁶(-²-isopenteny) adenine (2iP). Plants with strong episodic growth cycles required the longer acclimation periods. Utilizing shoots from these cultures, the response to a cytokinin series of 0 to 32 M 2iP or BAP (6-Benzylaminopurine) was analyzed. BAP proved toxic to all but the elipidote and lepidote rhododendrons (R. mucronulatum, R. x Boule de Neige, and R. x PJM); however, even with these selections, 2iP stimulated greater shoot multiplication rates. The optimum 2iP level for shoot multiplication varied little with the genotype and levels of 4 to 16 M generally proved optimal, depending on the specific selection. Adventitious shoot production was observed in 3 selections (R. canadense, R. x Boule de Neige and R. x PJM), but only at 2iP levels above 8 M. Shoot multiplication rates of 7 to 21 times were observed, depending on the selection. Using an average utilizable shoot production rate of 40 shoots per culture per 6 week subculture period, some 75,000 shoots can be generated per square meter of culture space per year. The harvested shoots (microcuttings) rooted readily out-of-culture and the resultant plants grew like seedlings.     

Development and interspecific transferability of genic microsatellite markers in Spartina spp. with different genome size

Flow cytometry analysis showed variation of nuclear DNA content among different species of Spartina. Spartina alterniflora had the biggest genome (1763.9 Mbp) and S. cynosuroides had the smallest genome (756.35 Mbp), whereas the genomes of S. patens (969.36 Mbp) and S. spartinae (979.78 Mbp) were comparable. Mining simple sequence repeats (SSR) from 1227 expressed sequence tags (EST) generated from salt stressed S. alterniflora showed an abundance of di- and tri-nucleotide repeats. Of 100 ESSR (EST-derived SSR) loci with five or more repeats, 81 loci were successfully amplified in eight S. alterniflora genotypes and 15 (22.2%) ESSR markers were polymorphic. Eleven of the 15 polymorphic ESSRs showed amplification across six different species of Spartina while 100% cross transferability was observed with at least one species of Spartina. The average number of alleles per marker was 3.9 and 5.8 within S. alterniflora and among Spartina species, respectively. The ESSR markers discriminated different members within and between species of Spartina genus.     

Development of ten new EST-derived microsatellites in Atlantic cod (Gadus morhua L.)

Ten polymorphic microsatellite markers were developed from approximately 1,300 expressed sequence tags (ESTs) of Atlantic cod (Gadus morhua L.). Thirty two primer pairs were designed for EST sequences containing perfect di- tri- tetra- and pentanucleotide motifs and characterised in 96 unrelated fish. Ten markers were successfully amplified with number of alleles from 2 to 13 per locus and observed and expected heterozygosity ranging from 0.03 to 0.69 and 0.03 to 0.74, respectively. Loci Gmo-C131, C132 and C136 deviated from Hardy-Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C131 and Gmo-C132 and C128 and Gmo-C133. The gene identity was determined at five of the loci, confirming the associated microsatellites as Type I markers. The new microsatellites reported in this work can be used for conservation and enhancement of wild stocks for commercial harvesting. Jon-Ivar Westgaard and Tekle Tafese have contributed equally to the work.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

10种杜鹃亚属植物种间杂交的可育性研究

为探索杜鹃花亚属内异种杂交的可育性及其规律,对杜鹃亚属有鳞大花亚组(subsect. Maddenia)、三花杜鹃亚组(subsect. Triflora)、亮鳞杜鹃亚组(subsect. Heliolepida)及腋花杜鹃亚组(subsect. Scabrifolia)等4亚组10个杜鹃花种类的22个杂交组合(其中18个数据完整组合)的可育性进行了研究。结果表明:(1)所涉及的杜鹃亚属不同亚组间及三花杜鹃亚组内杂交均较困难,在18个数据完整组合中高可育与可育组合比率明显偏低,不育比率高(55.6%)。(2)在10个不可育或败育组合中,不能坐果(Cab型)、不能结实(Sab型)和可结实而种子不能发芽(Sng型)的数量分布为6∶1∶3,其不亲和或败育发生的阶段可能涵盖了从前合子期到后合子期的整个阶段。(3)亲本一方为多倍体组合的可育率(41.6%),尤其是母本为多倍体时,比二倍体组合的可育率(50.0%)低且无高可育组合出现,部分印证了倍性是导致该亚属植物不同种类杂交不亲和、不育与育性下降的重要原因,但不是唯一原因,亚组间杂交的可育率(16.7%)明显低于亚组内(三花杜鹃亚组内,58.3%)。(4)与相应的母本自然授粉结果相比,杂交明显导致多数可育组合绿苗率比率和单位可育种子数量比率的大幅度下降,这是双亲遗传差异及多倍体亲本介入后所导致的杂交衰退hybrid weakness现象。(5)在多倍体作母本的情况下,杂交单向不育或非对称遗传渗透现象明显。     

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization, development and exploitation of EST-derived microsatellites in Porphyra haitanensis Chang et Zheng (Bangiales, Rhodophyta)

The frequency, type and distribution of simple sequence repeats (SSRs) in Porphyra haitanensis genomes was investigated using expressed sequence tag (EST) data deposited in public databases. A total of 3,489 non-redundant P. haitanensis ESTs were screened for SSRs using SSRhunter software. From those, 224 SSRs in 210 ESTs were identified; trinucleotides were the most common type of SSR (64.29%), followed by dinucleotides (33.48%). Tetranucleotides, pentanucleotides, and hexanucleotides were not common. Among all identified motif types, CGG/CCG had the highest frequency (33.9%), followed by TC/AG (24.6%). From these EST-SSRs, 37 SSR primer-pairs were designed and tested using common SSR reaction conditions with 15 P. haitanensis DNAs as templates. The results showed that 28 SSR primer-pairs gave good amplification patterns. These were used to conduct SSR analyses of genetic variations of the 15 germplasm strains of P. haitanensis. A total of 224 alleles were detected, with the number of alleles ranging from 4 to 15. The effective number of alleles, expected heterozygosity, and polymorphism information content of the 15 germplasm strains of P. haitanensis were 2.81, 0.64, and 0.57, respectively. All of these parameters indicate that the 15 germplasm strains of P. haitanensis harbor rich genetic variation.     

32种杜鹃花属植物在迁地保育条件下的自交研究

迁地保育条件下的杜鹃花属植物自交可育性一直缺乏系统的比较研究,该文对5亚属13亚组32种杜鹃花属植物的自交进行了数据采集与分析,初步揭示了迁地保育条件下杜鹃花属植物自交育性特征。结果表明:(1)自交可育与不育是杜鹃花属植物有性生殖中的两个并存现象,自交能育型种类或多于不育型。在受试的32种杜鹃花中,自交不育型10种、弱可育型5种、可育型7种、高可育型10种,其中27种的自交育性为首次报道,涵盖包括杜鹃亚属杜鹃组(sect.Rhododendron)和马银花亚属(subgen.Azaleastrum)及常绿杜鹃亚属(subgen.Hymenanthes)中银叶杜鹃亚组(subsect.Argyrophylla)等5个亚组在内的未被研究的类群。(2)通过与自然授粉有关育性指标的比较,发现不同种类的自交可育性指标有大幅度降低及增高这两类截然不同的现象,从而提出了自交可能是部分杜鹃花属植物的适应策略,或者对不利环境及其媒介条件的主动响应。(3)在云锦杜鹃亚组(subsect.Fortunea)这个被认为最原始的杜鹃花类群中,具备从自交不育到高可育的所有类型,并可能由此奠定整个杜鹃花属的遗传基础,而类群与种类分布的不同区域气候环境长期直接的或通过影响传粉媒介间接的作用,则可能是最终塑造该属植物自交育性多样化的外部动力。(4)该文还依据有关自交的研究结果,对杜鹃花属植物的迁地保育、育种、相关学科发展进行了讨论,并认为后合子期败育的理论不能完美地解释自交不能坐果的现象,而多倍体似不会导致自交不育。     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Development of 14 EST-SSRs for Betula maximowicziana and their applicability to related species

Simple sequence repeats (SSRs) markers were developed for Betula maximowicziana using 2698 expressed sequence tags (ESTs) from the NCBI database. Out of 112 designed primer pairs, 54 showed clear PCR amplification and 14 of these revealed polymorphism in eight individuals sampled across the species’ range. The number of alleles detected and the expected heterozygosity ranged from 1 to 3 and 0.000 to 0.570, respectively, when these 14 loci were examined in 49 individuals from a single population. In the cross species transferability test, eight of the 14 loci were also polymorphic in all four of the diploid, tetraploid and hexaploid Betula species examined. These results showed high transferability of the developed EST-SSRs and that these markers are likely to be useful in studies of the population genetics of species in the genus Betula.     

Identification and characterization of EST-SSRs and cpSSRs in endangered <Emphasis Type="Italic">Cycas hainanensis</Emphasis>

We report on the identification and characterization of six EST-linked simple sequence repeats (EST-SSRs) and chloroplast SSRs (cpSSRs) in endangered Cycas hainanensis. The number of alleles ranged from two to eight for EST-SSRs, two to three for cpSSRs. Observed and expected heterozygosities ranged from 0.042 to 0.417 and 0.042 to 0.811 for EST-SSRs, respectively. Expected heterozygosities ranged from 0.156 to 0.457 for cpSSRs. All these markers gave successful cross-species amplification in C. fairylakea. These markers will allow analyses of the baseline genetic variability and population structure of C. hainanensis to provide strategies for effective conservation and management. The experiments were carried out in South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, P.R. China.     

Transferable EST-SSR markers for the study of polymorphism and genetic diversity in bread wheat

Nearly 900 SSRs (simple sequence repeats) were identified among 15,000 ESTs (expressed sequence tags) belonging to bread wheat ( Triticum aestivum L.). The SSRs were defined by their minimum length, which ranged from 14 to 21 bp. The maximum length ranged from 24 to 87 bp depending upon the length of the repeat unit itself (1–7 bp). The average density of SSRs was one SSR per 9.2 kb of EST sequence screened. The trinucleotide repeats were the most abundant SSRs detected. As a representative sample, 78 primer pairs were designed, which were also used to screen the dbEST entries for Hordeum vulgare and Triticum tauschii (donor of the D-genome of cultivated wheat) using a cut-off E (expectation) value of 0.01. On the basis of in silico analysis, up to 55.12% of the primer pairs exhibited transferability from Triticum to Hordeum, indicating that the sequences flanking the SSRs are not only conserved within a single genus but also between related genera in Poaceae. Primer pairs for the 78 SSRs were synthesized and used successfully for the study of (1) their transferability to 18 related wild species and five cereal species (barley, oat, rye, rice and maize); and (2) polymorphism between the parents of four mapping populations available with us. A subset of 20 EST-SSR primers was also used to assess genetic diversity in a collection of 52 elite exotic wheat genotypes. This was done with a view to compare their utility relative to other molecular markers (gSSRs, AFLPs, and SAMPL) previously used by us for the same purpose with the same set of 52 bread wheat genotypes. Although only a low level of polymorphism was detected, relative to that observed with genomic SSRs, the study suggested that EST-SSRs can be successfully used for a variety of purposes, and may actually prove superior to SSR markers extracted from genomic libraries for diversity estimation and transferability.Communicated by R. Hagemann