Genome skimming-based simple sequence repeat (SSR) marker discovery and characterization in Grevillea robusta

Proteaceae, a largely southern hemisphere family consisting of 80 genera distributed in Australia and southern Africa as its centres of greatest diversity, also extends well in northern and southern America. Under this family, Grevillea robusta is a fast-growing species got popularity in farm and avenue plantations. Despite the ecological and economic importance, the species has not yet been investigated for its genetic improvement and genome-based studies. Only a few molecular markers are available for the species or its close relatives, which hinders  genomic and population genetics studies. Genetic markers have been intensively applied for the main strategies in breeding programs, especially for the economically important traits. Hence, it is of utmost priority to develop genomic database resources and species-specific markers for studying quantitative genetics in G. robusta. Given this, the present study aimed to develop de novo genome sequencing, robust microsatellites markers, sequence annotation and their validation in different stands of G. robusta in northern India. Library preparation and sequencing were carried out using Illumina paired-end sequencing technology. Approximately, ten gigabases (Gb) sequence data with 70.87 million raw reads assembled into 425,923 contigs (read mapped to 76.48%) comprising 455 Mb genome size (23 × coverage) generated through genome skimming approach. In total, 9421 simple sequence repeat (SSR) primer pairs were successfully designed from 13,335 microsatellite repeats. Afterward, a subset of 161 primer pairs was randomly selected, synthesized and validated. All the tested primers showed successful amplification but only 13 showed polymorphisms. The polymorphic SSRs were further used to estimate the measures of genetic diversity in 12 genotypes each from the states of Punjab, Haryana, Himachal Pradesh and Uttarakhand. Importantly, the average number of alleles (N_a), observed heterozygosity (H_o), expected heterozygosity (H_e), and the polymorphism information content (PIC) were recorded as 2.69, 0.356, 0.557 and 0.388, respectively. The availability of sequence information and newly developed SSR markers could potentially be used in various genetic analyses and improvements through molecular breeding strategies for G. robusta.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01035-w.     

Analysis of genetic relationship in mutant silkworm strains of Bombyx mori using inter simple sequence repeat （ISSR） markers

Amplified inter simple sequence repeats (ISSR) markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat (SSR) motifs were used in this study. A total of 113 markers were produced among 20 mutant swains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was (1.7080±0.4567), effective alleles (1.5194±0.3950) and genetic diversity (Ht) (0.2901±0.0415). The dendrogram produced using the unweighted pair group method with arithmetic means (UPGMA) and cluster analysis made using Nei's genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm swains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm swains maintained at the germplasm center.     

Assessment of genetic diversity and relationship among a collection of US sweet sorghum germplasm by SSR markers

Sweet sorghum (Sorghum bicolor L.) is a type of cultivated sorghums and has been recognized widely as potential alternative source of bio-fuel because of its high fermentable sugar content in the stalk. A substantial variation of sugar content and related traits is known to exist in US sweet sorghum. The objectives of the study were to assess the genetic diversity and relationship among the US sweet sorghum cultivars and lines using SSR markers and to examine the genetic variability within sweet sorghum accessions for sugar content. Sixty-eight sweet sorghum and four grain sorghum cultivars and lines were genotyped with 41 SSR markers that generated 132 alleles with an average of 3.22 alleles per locus. Polymorphism information content (PIC) value, a measure of gene diversity, was 0.40 with a range of 0.03–0.87. The genetic similarity co-efficient was estimated based on the segregation of the 132 SSR alleles. Clustering analysis based on the genetic similarity (GS) grouped the 72 sorghum accessions into 10 distinct clusters. Grouping based on clustering analysis was in good agreement with available pedigree and genetic background information. The study has revealed the genetic relationship of cultivars with unknown parentage to those with known parentage. A number of diverse pairs of sweet sorghum accessions were identified which were polymorphic at many SSR loci and significantly different for sugar content as well. Information generated from this study can be used to select parents for hybrid development to maximize the sugar content and total biomass, and development of segregating populations to map genes controlling sugar content in sweet sorghum.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow and genetic diversity in pigeonpea (Cajanus cajan)

Pigeonpea (Cajanus cajan) is an important subsistence crop in India where traditional landraces and improved hybrids are grown alongside each other. Gene flow may result in genetic erosion of these landraces and their wild relatives, whilst transgene escape from future genetically engineered varieties is another potential hazard. To assess the impact of these factors gene flow needs to be measured. A set of 10 simple sequence repeat markers have been developed, which exhibit polymorphism across a range of pigeonpea varieties. Use of these markers also offers an efficient system for the assessment of genetic diversity within populations of pigeonpea.     

Molecular characterization of Jatropha genetic resources through inter-simple sequence repeat (ISSR) markers

The genetic diversity among eight Jatropha species and three Jatropha curcas accessions were analyzed using ISSR-PCR. Nine ISSR primers generated reproducible amplification banding pattern of 61 polymorphic bands out of 64 scored accounting for 98.14% polymorphism across the species. The ISSR primers viz., I1, I2, I3, I4, I5, I6, I7 and I10 generated 100% polymorphic patterns. Jaccard’s coefficient of similarity varied from 0.346 to 0.807, indicative of high level of genetic variation among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L (TNMJ1, TNMJ 22 and TNMJ 23), while second included four species viz., J. tanjorensis J. L. Ellis et Saroja., J. gossypifolia L., J. podagrica Hook and J. maheshwarii Subrum and M.P. Nayer and the third cluster included another four species viz., J. villosa Wight J. multifida L., J. integerrima Jacq and J. glandulifera Roxb. The overall grouping pattern of clustering corresponds well with principal component analysis (PCA) confirming patterns of genetic diversity observed among the species. So far, there are no reports on the molecular diversity of the Jatropha species through ISSR marker. This study provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources and also for further breeding programme towards biodiesel production.     

Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza

Inter simple sequence repeat (ISSR) polymorphism was used to determine genetic diversity and phylogenetic relationships in Oryza. Forty two genotypes including 17 wild species, representing AA,BB,CC,EE,FF,GG,BBCC,CCDD, and HHJJgenomes, two cultivated species, Oryza sativa (AA) and Oryza glaberrima (AA), and three related genera, Porteresia coarctata, Leersia and Rhynchoryza subulata, were used in ISSR analysis. A total of 30 ISSR primers were screened representing di-, tri-, tetra- and penta-nucleotide repeats, of which 11 polymorphic and informative patterns were selected to determine the genetic diversity. The consensus tree constructed using binary data from banding patterns generated by ISSR-PCR clustered 42 genotypes according to their respective genomes. ISSR analysis suggests that the genus Oryza may have evolved following a polyphyletic pathway; Oryza brachyantha (FF genome) is the most divergent species in Oryza and Oryza australiensis (EE genome) does not fall under the Officinalis complex. DNA profiles based on ISSR markers have revealed potential diagnostic fingerprints for various species and genomes, and also for individual accessions/cultivars. Additionally ISSR revealed 87 putative genome/species-specific molecular markers for eight of the nine genomes of Oryza. The ISSR markers are thus useful in the fingerprinting of cultivated and wild species germplasm, and in understanding the evolutionary relationships of Oryza. Received: 23 August 1999 / Accepted: 10 November 1999     

油菜简单重复序列SSR(simple sequence repeat)研究进展

简单重复序列(simple sequence repeat,SSR)是重复单元少于6个核苷酸重复序列,广泛分布于动植物基因组中,呈孟德尔遗传,已被作为一种理想的共显性标记应用于动植物遗传研究中。本文重点介绍了SSR分类、特点,及近几年来油菜SSR标记的开发和SSR技术在油菜基因定位、品种鉴定中的应用,并对SSR标记在油菜中的应用进行了探讨。     

Identification of polymorphic,conserved simple sequence repeats (SSRs) in cultivated Brassica species

The application of simple sequence repeat (SSR) genotyping for the characterization of genetic variation in crop plants has been hindered by ready access to useful primer pairs and potentially limited conservation of the repeat sequences among related species. In this phase of work, we report on the identification and characterization of SSRs that are conserved in Brassica napus L. (rapeseed) and its putative progenitors, B. oleracea L. (cabbage, and related vegetable types) and B. rapa (vegetable and oil types). Approximately 140 clones from a size-fractionated genomic library of B. napus were sequenced, and primer pairs were designed for 21 dinucleotide SSRs. Seventeen primer pairs amplified products in the three species and, among these, 13 detected variation between and within species. Unlike findings on SSR information content in human, no relationship could be established between the number of tandem repeats within the target sequence and heterozygosity. All primer pairs have been designed to work under identical amplification conditions; therefore, single-reaction, multiplex polymerase chain reaction (PCR) with these SSRs is possible. Once moderate numbers of primer pairs are accessible to the user community, SSR genotyping may provide a useful method for the characterization, conservation, and utilization of agricultural crop diversity.     

肉质色不同萝卜遗传多样性的SSR分子标记分析

利用微卫星(SSR)标记技术,从600对SSR引物中筛选86对扩增条带清晰的引物,检测了来自我国不同地区37个肉质不同颜色萝卜品种的遗传多样性。86对引物共扩增到976个条带,每对引物扩增出2～17个条带,平均为10.7个,其中多态性条带892个,多态性条带比例为91.39%;共检测出753个基因型,每对引物检测2～20个基因型,平均8.7个,其中有效基因型443.99,有效基因型比例为58.96%;Shannon多态性指数变幅为0.44～2.77,平均1.76。当相似系数为0.81时,可将供试萝卜分成3类,第I类包括6份白色肉质萝卜,第II类包括3份红色肉质萝卜和6份白色肉质萝卜,第III类包括22份红色肉质萝卜。各红色肉质萝卜品种间的遗传相似系数有97%大于0.80,而各白色肉质萝卜品种间的遗传相似系数有91%大于0.80。红色肉质萝卜遗传多样性略低于白色肉质萝卜,红色肉质萝卜与白色肉质萝卜间平均相似系数为0.83,说明不同肉色的萝卜间亲缘关系较密切,在分类上红色肉质萝卜可能是白色萝卜的一个变种。     

Assessment of genetic diversity in Salvadora persica L. based on inter simple sequence repeat (ISSR) genetic marker

Studies on the genetic variation in marginal populations and differentiation between them are essential for assessment of best gene conservation strategies and sampling schemes. In this study, ISSR markers were used to establish the level of genetic relationships and polymorphism 50 genotypes of Salvadora persica collected from 6 different regions of Hormozgan province. The ISSR analysis with 9 anchored primers also generated 105 scorable loci, of which 85 were polymorphic (80.95%). Parameters of genetic diversity and its partitioning were calculated. The genetic analysis demonstrated that S. persica maintain relatively high genetic diversity (PIC was 0.63, Na was 1.27 and Ho and He were 0.15 and 0.17 respectively). The coefficient of genetic differentiation among populations based on FST equaled 0.20. Genetic identities between population's pairs were high (mean I?=?0.88). These values are high as compared with other widespread congener species. Cluster analysis based on the Unweighted Pair Group Method with Arithmetic Averages (UPGMA) revealed 3 main clusters for the ISSR data. The levels of genetic diversity maintained within populations of S. persica indicate that an appropriate sampling design for ex situ safeguarding should capture the majority of genetic diversity found within these taxa to help ensure the long term viability of this species. Furthermore, it could be inferred that ISSR markers are suitable tools for the evaluation of genetic diversity and relationships within the Salvadora persica.     

Detection of haplotypic variation and natural hybridization in halepensis-complex pine species using chloroplast simple sequence repeat (SSR) markers

In this investigation, nine chloroplast, paternally inherited simple-sequence repeat (cpSSR) markers were used to describe genetic variation of three closely related species belonging to the halepensis complex ( Pinus halepensis Ait., P. brutia Mill. and P. eldarica Medwed.). Both the infinite allele model (IAM) and stepwise-mutation model (SMM) have been applied to the analysis of the genetic structure of natural populations and the geographical distribution of haplotypic variation. SMM-based estimators performed better than IAM-based estimators for large values of within-population diversity and divergence between population pairs. Overall, large haplotypic variation and high genetic divergence were detected for both P. halepensis and P. brutia . The genetic structures of the three species are discussed with consideration to the evolutionary and ecological characteristics of these species. Three highly informative markers showing size variants distinguishing P. halepensis from the other two species were used to provide more information on the occurrence of natural hybridization in a Turkish sympatric population of P. halepensis and P. brutia . Strong evidence of introgression of ' halepensis ' chloroplast haplotypes into P. brutia seeds (but not vice versa) was detected. According to previous evidence from controlled crossings, matings between the above species seem to be successful only when P. halepensis is the pollen donor and P. brutia is the female parent (but not reciprocally). The existence of unidirectional gene flow in sympatric populations confirms previous evidence about partial reproductive barriers between P. halepensis and P. brutia . Implications of the above evidence for the evolutionary history of these species are discussed.     

Assessing the genetic diversity of tobacco germplasm using intersimple sequence repeat and inter-retrotransposon amplification polymorphism markers

The genetic diversity of 118 tobacco accessions, including flue‐cured tobacco, sun‐/air‐cured tobacco, burley tobacco, oriental tobacco and wild tobacco, was characterised using intersimple sequence repeat (ISSR) and inter‐retrotransposon amplification polymorphism (IRAP) markers. ISSR and IRAP banding patterns and genetic distance (GD) values showed the low level of genetic diversity within and among cultivated tobacco types. There was higher GD and average heterozygosity among wild tobacco types than those among cultivated tobacco. Genetic diversity of tobacco germplasm was low, with a high level of genetic identity (>0.77) between the different types. However, neighbour‐joining cluster analysis of marker‐based GDs showed that the accessions from the same tobacco type, as classified by manufacturing quality traits, were nearly clustered into the same group. These results will help in the formulation of appropriate strategies for variety improvement in tobacco, and ISSR and IRAP markers of the genetic diversity will contribute to further study and improvement of tobacco.     

Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soybean

 Simple Sequence Repeat (SSR) allele sizing provides a useful tool for genotype identification, pedigree analysis, and for estimating genetic distance between organisms. Soybean [Glycine max (L.) Merr.] cultivars are identified for Plant Variety Protection (PVP) purposes by standard pigmentation and morphological traits. However, many commercial soybeans arise from a limited number of elite lines and are often indistinguishable based on these traits. A system based on SSR markers would provide unique DNA profiles of cultivars. Fluorescent labeling of alleles combined with automated sizing with internal size standards in each gel lane was used as an alternative to standard [³²P] labeling to assess genetic variability in soybean. Allelic frequencies at 20 SSR loci were determined in 35 soybean genotypes that account for greater than 95% of the alleles in North American soybean cultivars based upon pedigree analysis. An average of 10.1 alleles per locus (range: 5–17), with a mean gene diversity of 0.80 (range: 0.50 to 0.87) were observed at the 20 SSR loci. The 20 loci successfully distinguished modern soybean cultivars that are identical for morphological and pigmentation traits, as well as 7 soybean genotypes reported to be indistinguishable using 17 RFLP probes. Pedigrees of 7 cultivars were studied to estimate stability of SSRs in soybean across generations. Of the 7 pedigrees 6 had one locus in the progeny with an allele(s) that was not present in either parent. These new alleles are most likely the result of mutation. The mutation rate of SSR alleles in soybean was similar to that reported in humans. To avoid difficulty associated with mutation, DNA fingerprint data should be determined from the bulk of 30-50 plants of a cultivar. Received: 24 March 1997 / Accepted: 4 April 1997     

Development and use of simple sequence repeat SSR markers in Rubus species

The isolation of polymorphic codominant microsatellite markers in Rubus and in particular red raspberry will provide a tool to investigate gene flow between cultivated and wild raspberries. Microsatellite loci were isolated by screening a PstI size selected genomic library with AC₍₁₃₎ and AG₍₁₃₎. Positive clones were sequenced and primer pairs designed to the sequences flanking identified SSRs. One primer of each pair was fluorescently labelled to facilitate polymerase chain reaction (PCR) product identification on an automated DNA sequencer. We describe 10 polymorphic microsatellite loci developed and demonstrate their usefulness in different Rubus species.     

Study of simple sequence repeat (SSR) markers from wheat expressed sequence tags (ESTs)

The increasing availability of expressed sequence tags (ESTs) in wheat (Triticum aestivum) and related cereals provides a valuable resource of non-anonymous DNA molecular markers. We examined 170,746 wheat ESTs from the public (International Triticeae EST Cooperative) and Génoplante databases, previously clustered in contigs, for the presence of di- to hexanucleotide simple sequence repeats (SSRs). Analysis of 46,510 contigs identified 3,530 SSRs, which represented 7.5% of the total number of contigs. Only 74% of the sequences allowed primer pairs to be designed, 70% led to an amplification product, mainly of a high quality (68%), and 53% exhibited polymorphism for at least one cultivar among the eight tested. Even though dinucleotide SSRs were less represented than trinucleotide SSRs (15.5% versus 66.5%, respectively), the former showed a much higher polymorphism level (83% versus 46%). The effect of the number and type of repeats is also discussed. The development of new EST-SSRs markers will have important implications for the genetic analysis and exploitation of the genetic resources of wheat and related species and will provide a more direct estimate of functional diversity.     

Simple Sequence Repeat Analysis of Genetic Diversity in Primary Core Collection of Peach （Prunus persica）

In this study, the genetic diversity of 51 cultivars in the primary core collection of peach （Prunus persica （L.） Batsch） was evaluated by using simple sequence repeats （SSRs）. The phylogenetic relationships and the evolutionary history among different cultivars were determined on the basis of SSR data. Twenty-two polymorphic SSR primer pairs were selected, and a total of 111 alleles were identified in the 51 cultivars, with an average of 5 alleles per locus. According to traditional Chinese classification of peach cultivars, the 51 cultivars in the peach primary core collection belong to six variety groups. The SSR analysis revealed that the levels of the genetic diversity within each variety group were ranked as Sweet peach 〉 Crisp peach 〉 Flat peach 〉 Nectarine 〉 Honey Peach 〉 Yellow fleshed peach. The genetic diversity among the Chinese cultivars was higher than that among the introduced cultivars. Cluster analysis by the unweighted pair group method with arithmetic averaging （UPGMA） placed the 51 cultivars into five linkage clusters. Cultivar members from the same variety group were distributed in different UPGMA clusters and some members from different variety groups were placed under the same cluster. Different variety groups could not be differentiated in accordance with SSR markers. The SSR analysis revealed rich genetic diversity in the peach primary core collection, representative of genetic resources of peach.     

Genetic diversity of the endangered and endemic species Psathyrostachys huashanica natural populations using simple sequence repeats (SSRs) markers

Psathyrostachys huashanica Keng., a species endemic to China, is only distributed in Huashan Mountain in Shaanxi Province. It has been listed as “national first-class protected rare species.” In this study, the microsatellites of barley were used to analyze genetic diversity of P. huashanica populations sampled from three valleys (Huangpu, Xian and Huashan Valleys) in Mt. Huashan. A total of 33 alleles of 11 loci were detected from 266 individuals. The observed average number of alleles (A) is 2.75; the effective number of alleles (Ae) is 1.67. The percentage of polymorphic loci (PPB) is 58.33% in Huangpu Valley, 75% in Xian Valley, 83.33% in Huashan Valley, and the total PPB is 83.33%. About 77.6% of (F_ST = 0.324) genetic diversity was observed within the subpopulations. Genetic differentiation within each subpopulations was higher than that among the subpopulations. Mean genetic distance is 0.17 (range: 0.010–0.401). Correlation analysis detected significant correlation between genetic distance and vertical distance of altitude in Huashan Valley. Differentiation mainly occurred between the higher altitude subpopulations and the lower altitude subpopulations, suggesting that altitude might be the major factor that restricted the gene flow between different altitude subpopulations and resulted in differentiation of subpopulations.     

Application of simple sequence repeat (SSR) markers for molecular diversity and heterozygosity analysis in maize inbred lines

There is an important role of understanding the genetic diversity among and within inbred lines at the molecular level for maize improvement in different breeding programs. The present study was devoted to estimate the level of genetic diversity among the inbred lines of maize using the simple sequence repeat analysis (SSR). The application of six different SSR markers successfully provided the information on similarity or diversity as well as the heterozygosity of the allelic loci for all the eight inbred line of maize.