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.     

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.     

Mapping of simple sequence repeat (SSR) DNA markers in diploid and tetraploid alfalfa

Cultivated alfalfa (Medicago sativa) is an autotetraploid. However, all three existing alfalfa genetic maps resulted from crosses of diploid alfalfa. The current study was undertaken to evaluate the use of Simple Sequence Repeat (SSR) DNA markers for mapping in diploid and tetraploid alfalfa. Ten SSR markers were incorporated into an existing F₂ diploid alfalfa RFLP map and also mapped in an F₂ tetraploid population. The tetraploid population had two to four alleles in each of the loci examined. The segregation of these alleles in the tetraploid mapping population generally was clear and easy to interpret. Because of the complexity of tetrasomic linkage analysis and a lack of computer software to accommodate it, linkage relationships at the tetraploid level were determined using a single-dose allele (SDA) analysis, where the presence or absence of each allele was scored independently of the other alleles at the same locus. The SDA diploid map was also constructed to compare mapping using SDA to the standard co-dominant method. Linkage groups were generally conserved among the tetraploid and the two diploid linkage maps, except for segments where severe segregation distortion was present. Segregation distortion, which was present in both tetraploid and diploid populations, probably resulted from inbreeding depression. The ease of analysis together with the abundance of SSR loci in the alfalfa genome indicated that SSR markers should be a useful tool for mapping tetraploid alfalfa. Received: 10 September 1999 / Accepted: 11 November 1999     

Genetic diversity of the endangered Chinese endemic herb Dayaoshania cotinifolia (Gesneriaceae) revealed by simple sequence repeat (SSR) markers

Dayaoshania cotinifolia W.T. Wang is a critically endangered perennial herb endemic to southern China. Simple sequence repeat (SSR) markers were employed to assess genetic diversity in two populations of D. cotinifolia. Eight primer pairs generated a total of 36 alleles, with a mean of 4.5 alleles per locus. The expected heterozygosity (H_e = 0.416) and observed heterozygosity (H_o = 0.508) indicated a moderate level of genetic diversity, though the genetic differentiation between the populations was low (F_st = 0.014), a result that was supported by a higher gene flow (N_m = 18.000). No severe bottleneck effect was detected in the two populations. Thus, the endangered status of this species is most likely due to anthropologic effects rather than a lack of genetic diversity. In situ conservation strategies should be promoted, and the sizes of the populations should be increased through artificial breeding.     

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.     

Robust simple sequence repeat markers for spruce (Picea spp.) from expressed sequence tags

Traditionally, simple sequence repeat (SSR) markers have been developed from libraries of genomic DNA. However, the large, repetitive nature of conifer genomes makes development of robust, single-copy SSR markers from genomic DNA difficult. Expressed sequence tags (ESTs), or sequences of messenger RNA, offer the opportunity to exploit single, low-copy, conserved sequence motifs for SSR development. From a 20,275-unigene spruce EST set, we identified 44 candidate EST-SSR markers. Of these, 25 amplified and were polymorphic in white, Sitka, and black spruce; 20 amplified in all 23 spruce species tested; the remaining five amplified in all except one species. In addition, 101 previously described spruce SSRs (mostly developed from genomic DNA), were tested. Of these, 17 amplified across white, Sitka, and black spruce. The 25 EST-SSRs had approximately 9% less heterozygosity than the 17 genomic-derived SSRs (mean H=0.65 vs 0.72), but appeared to have less null alleles, as evidenced by much lower apparent inbreeding (mean F=0.046 vs 0.126). These robust SSRs are of particular use in comparative studies, and as the EST-SSRs are within the expressed portion of the genome, they are more likely to be associated with a particular gene of interest, improving their utility for quantitative trait loci mapping and allowing detection of selective sweeps at specific genes.     

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.     

Development of simple sequence repeat (SSR) markers and their use in identification of Dendrobium varieties

The aim of this study was to develop simple sequence repeat (SSR) markers for Dendrobium varieties/species, many of which have medicinal and horticultural values. Two genomic DNA libraries of Dendrobium Sonia enriched with GA repeats and CA repeats were constructed. Fourteen polymorphic SSR markers were identified when screened against 42 popular commercial Dendrobium hybrids. The average allele number was 12.0 ± 1.9 and the observed heterozyosity was averaged at 0.70. All 42 hybrids tested, except for two tissue culture mutants, were uniquely identified with the markers used. Sibling hybrids were closely clustered. Hybrids were also closer to parents. These SSR markers can be used for molecular ecology research, genetic mapping and marker‐assisted breeding. They can also help protection for new Dendrobium varieties.     

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。     

Polymorphic microsatellite and cryptic simple repeat sequence markers in pineapples (Ananas comosus var. comosus)

A rapid method for isolating microsatellite loci in pineapples, based on the 5′‐anchored polymerase chain reaction technique, revealed 137 microsatellite loci (consisting of 62 dinucleotide, 24 trinucleotide, 49 tetranucleotide and 2 hexanucleotide repeats) and 16 cryptically simple repeat sequences. We report on the characterization of 19 polymorphic microsatellite loci and one cryptic simple repeat loci in pineapples. The number of alleles per locus ranged from two to four while the observed heterozygosity ranged from 0.1705 to 1. These markers are useful as tools for detecting levels of genetic variation in pineapple varieties for germplasm management and crossbreeding purposes.     

Characterization and compilation of polymorphic simple sequence repeat (SSR) markers of peanut from public database

ABSTRACT: BACKGROUND: There are several reports describing thousands of SSR markers in the peanut (Arachis hypogaea L.) genome. There is a need to integrate various research reports of peanut DNA polymorphism into a single platform. Further, because of lack of uniformity in the labeling of these markers across the publications, there is some confusion on the identities of many markers. We describe below an effort to develop a central comprehensive database of polymorphic SSR markers in peanut. FINDINGS: We compiled 1,343 SSR markers as detecting polymorphism (14.5%) within a total of 9,274 markers. Amongst all polymorphic SSRs examined, we found that AG motif (36.5%) was the most abundant followed by AAG (12.1%), AAT (10.9%), and AT (10.3%).The mean length of SSR repeats in dinucleotide SSRs was significantly longer than that in trinucleotide SSRs. Dinucleotide SSRs showed higher polymorphism frequency for genomic SSRs when compared to trinucleotide SSRs, while for EST-SSRs, the frequency of polymorphic SSRs was higher in trinucleotide SSRs than in dinucleotide SSRs. The correlation of the length of SSR and the frequency of polymorphism revealed that the frequency of polymorphism was decreased as motif repeat number increased. CONCLUSIONS: The assembled polymorphic SSRs would enhance the density of the existing genetic maps of peanut, which could also be a useful source of DNA markers suitable for high-throughput QTL mapping and marker-assisted selection in peanut improvement and thus would be of value to breeders. KEYWORDS: SSR, motif, polymorphism, cultivated peanut.     

Development and characterization of EST-derived simple sequence repeat (SSR) markers for pasture grass endophytes.

Fungal endophytes of the genus Neotyphodium are common in temperate pasture grass species and confer both beneficial and deleterious agronomic characteristics to their hosts. The aim of this study was to develop molecular markers based on simple sequence repeat (SSR) loci for the identification and assessment of genetic diversity among Neotyphodium endophytes in grasses. Expressed sequence tags (ESTs) from both Neptyphodium coenophialum and Neotyphodium lolii were examined, and unique SSR loci were identified in 9.7% of the N. coenophialum sequences and 6.3% of the N. lolii sequences. A variety of SSRs were present, although perfect trinucleotide repeat arrays were the most common. Primers were designed to 50 SSR loci from N. coenophialum and 57 SSR loci from N. lolii and were evaluated using 20 Neotyphodium and Epichlo? isolates. A high proportion of the N. coenophialum and N. lolii primers produced amplification products from the majority of isolates and most of these primers detected genetic variation. SSR markers from both N. coenophialum and N. lolii detected high levels of polymorphism between Neotyphodium and Epichlo? species, and low levels of polymorphism within N. coenophialum and N. lolii. SSR markers may be used in appropriate combinations to discriminate between species. Comparison with amplified fragment length polymorphism (AFLP) data demonstrated that the SSR markers were informative for the assessment of genetic variation within and between endophyte species. These markers may be used to identify endophyte taxa and to evaluate intraspecific population diversity, which may be correlated with variation for endophyte-derived agronomic traits.     

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     

Assessment of genetic diversity among sorghum landraces and their wild/weedy relatives in western Kenya using simple sequence repeat (SSR) markers

Understanding the extent of gene exchange between cultivated sorghum and its wild/weedy relatives and the evolutionary processes (including farmers’ practices) that act to shape the structure of genetic diversity within and between them is an important aspect for germplasm conservation strategies, biosafety risk assessment, and crop improvement programs. In this study, molecular characterization and genetic diversity analyses were conducted on wild, weedy and cultivated sorghums collected at a local-scale in a traditional farming system in the Lambwe Valley of western Kenya. Nine simple sequence repeat (SSR) markers were used to genotype 294 cultivated sorghum and 200 wild sorghum individuals. The nine SSR markers were highly polymorphic with a number of alleles that varied from 2 to 19. Overall, wild sorghums had higher genetic diversity, observed heterozygosity, total number of alleles, polymorphic information content and more genotypes per locus than the cultivated types. A Mantel test demonstrated that there was significant isolation-by-distance for wilds and cultivated materials. STRUCTURE, cluster and principal coordinate analyses consistently assigned wild and cultivated individuals to different groups but failed to place hybrids/weedy types as a single separate group from wilds. Our results provide strong evidence of significant genetic diversity retained within wilds, larger divergence between wild and cultivated materials and reduced gene flow than those previously reported in Kenya. These results demonstrate the value of the Lambwe Valley region as a genetic reservoir and the importance to conduct genetic diversity studies at the local scale to design and execute appropriate in situ conservation programs and policies.     

Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera)

Simple sequence repeat (SSR) markers were developed in the water lotus (Nelumbo nucifera Gaertn.) from an SSR-enriched genomic library. Of the SSR markers tested, 11 primer pairs produced clearly distinguishable DNA banding patterns. Forty-three alleles were detected with the 11 markers. The allele number per locus ranged from 2 to 5 with an average of 3.9. Polymorphism values ranged from 0.11 to 0.66 with an average of 0.51. These primers were also applicable to another Nelumbo species, Nelumbo lutea (Willd.) Pers. (American lotus) and hybrids between N. nucifera and N. lutea. These results indicate that the SSR markers developed in this study are informative and will be useful for genetic analysis in Nelumbo species.     

Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs), can greatly accelerate breeding programs. The aim of this study was to develop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative trait loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA) _n repeats yielded 793 clones containing SSR loci. The majority of SSRs consisted of perfect dinucleotide repeats, only 7% being trinucleotide repeats. After exclusion of redundant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR loci and PCR products in the expected size range were amplified from 101 loci. These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four primer pairs were tested in an F₂ population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amplification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provide valuable tools for molecular breeding of white clover but may also have applications in related taxa. Received: 3 April 2000 / Accepted: 12 May 2000     

Development and characterisation of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.)

Enrichment methods were optimised in order to isolate large numbers of simple sequence repeat (SSR) markers for perennial ryegrass (Lolium perenne L.), with the aim of developing a comprehensive set of loci for trait mapping and cultivar identification. Two libraries were constructed showing greater than 50% enrichment for a variety of SSR-motif types. Sequence characterisation of 1853 clones identified 859 SSR-containing clones, of which 718 were unique. Truncation of flanking sequences limited potential primer design to 366 clones. One-hundred selected SSR primer pairs were evaluated for amplification and genetic polymorphism across a panel of diverse genotypes. The efficiency of amplification was 81%. A relatively high level of SSR polymorphism was detected (67%), with a range of 2–7 alleles per locus. Mendelian segregation of alleles detected by selected SSR-locus primer pairs was demonstrated in the F₁ progeny of a pair cross. Cross-species amplification was detected in a number of related pasture and turfgrass species, with high levels of transfer to other Lolium species and members of the related genus Festuca. The identity of putative SSR ortholoci in these related species was confirmed by DNA sequence analysis. These loci constitute a valuable resource of ideal markers for the molecular breeding of ryegrasses and fescues. Received: 8 May 2000 / Accepted: 13 June 2000     

Identification and characterization of simple sequence repeat (SSR) markers from Fragaria×ananassa expressed sequences

The availability of expressed sequence data derived from gene discovery programmes enables mining for simple sequence repeats (SSRs), providing useful genetic markers for crop improvement. These markers are inexpensive, require minimal labour to produce and can frequently be associated with functionally annotated genes. This study reports on the development and characterization of expressed sequence tag (EST)–SSR markers in the cultivated strawberry, Fragaria×ananassa. Fourteen primer pairs were assessed for polymorphism in 13 F.×ananassa genotypes. The markers show reliable amplification and considerable polymorphism, demonstrating the utility of EST–SSRs for genetic analysis of commercial strawberry germplasm.     

Development of simple sequence repeat (SSR) markers from a genome survey of Chinese Bayberry (Myrica rubra)

ABSTRACT: BACKGROUND: Chinese bayberry (Myrica rubra Sieb. et Zucc.) is a subtropical evergreen tree originating in China. It has been cultivated in southern China for several thousand years, and annual production has reached 1.1 million tons. The taste and high level of health promoting characters identified in the fruit in recent years has stimulated its extension in China and introduction to Australia. A limited number of co-dominant markers have been developed and applied in genetic diversity and identity studies. Here we report, for the first time, a survey of whole genome shotgun data to develop a large number of simple sequence repeat (SSR) markers to analyse the genetic diversity of the common cultivated Chinese bayberry and the relationship with three other Myrica species. RESULTS: The whole genome shotgun survey of Chinese bayberry produced 9.01Gb of sequence data, about 26x coverage of the estimated genome size of 323 Mb. The genome sequences were highly heterozygous, but with little duplication. From the initial assembled scaffold covering 255 Mb sequence data, 28,602 SSRs ([greater than or equal to]5 repeats) were identified. Dinucleotide was the most common repeat motif with a frequency of 84.73%, followed by 13.78% trinucleotide, 1.34% tetranucleotide, 0.12% pentanucleotide and 0.04% hexanucleotide. From 600 primer pairs, 186 polymorphic SSRs were developed. Of these, 158 were used to screen 29 Chinese bayberry accessions and three other Myrica species: 91.14%, 89.87% and 46.84% SSRs could be used in Myrica adenophora, Myrica nana and Myrica cerifera, respectively. The UPGMA dendrogram tree showed that cultivated Myrica rubra is closely related to Myrica adenophora and Myrica nana, originating in southwest China, and very distantly related to Myrica cerifera, originating in America. These markers can be used in the construction of a linkage map and for genetic diversity studies in Myrica species. CONCLUSION: Myrica rubra has a small genome of about 323 Mb with a high level of heterozygosity. A large number of SSRs were identified, and 158 polymorphic SSR markers developed, 91% of which can be transferred to other Myrica species.