Simple sequence repeats as useful resources to study transcribed genes of cotton

Microsatellites or Simple Sequence Repeats(SSRs) are informative molecular genetic markers in many crop species. SSRs are PCR-based, highly polymorphic, abundant, widely distributed throughout the genome and inherited in a co-dominant manner in most cases. Here we describe the presence of SSRs in cDNAs of cotton. Thirty one SSR primer pairs of 220 (∼14%) tested led to PCR amplification of discrete fragments using cotton leaf cDNA as template. Sequence analysis showed 25% of 24randomly selected cDNA clones amplified with different SSR primer pairs contained repeat motifs. We further showed that sequences from the SSR-containing cDNAs were conserved across G. barbadense and G. hirsutum, revealing the importance of the SSR markers for comparative mapping of transcribed genes. Data mining for plant SSR-ESTs from the publicly available databases identified SSRs motifs in many plant species,including cotton, in a range of 1.1 to4.8% of the submitted ESTs for a given species. This revised version was published online in August 2006 with corrections to the Cover Date.     

A preliminary genetic analysis of fibre traits and the use of new genomic SSRs for genetic diversity in jute

Jute is one of the most important fibre crops, which is second only to cotton in providing environment-friendly (biodegradable and renewable) ligno-cellulose fibre. In order to improve this largely neglected crop, we conducted a preliminary study involving the following: (i) analysis of nature and extent of the genetic variability for fibre yield and four other related traits in a set of 81 genotypes belonging to two commercially cultivated Corchorus species (45 genotypes of C. olitorius + 36 genotypes of C. capsularis), (ii) development and analysis of a set of simple sequence repeat (SSR) markers from C. olitorius, and (iii) use of a sub-set of SSRs for assessment of genetic diversity in the above set of 81 genotypes. The results suggested quantitative nature of fibre yield and other related traits, with a preponderance of dominance component in genetic variance. A sub-set of 45 SSRs derived from C. olitorius, when used for a study of DNA polymorphism and genetic diversity, showed high transferability of these C. olitorius SSRs to C. capsularis. The average number of alleles for individual SSRs was surprisingly low (3.04 for both species, 2.02 for C. capsularis and 2.51 for C. olitorius), and so was the average polymorphic information content (PIC; 0.23 and 0.24 in two species). In the dendrogram obtained using a similarity matrix, the 81 genotypes were grouped into three clusters, which largely corresponded to the two species, Cluster I belonging mainly to C. capsularis and the other two closely related clusters (clusters II and III) belonging to C. olitorius. It was also shown that a minimum of 15 SSRs could give the same information as 41 SSRs, thus making many SSRs redundant. The SSR markers developed during the present study and to be developed in future will prove useful not only for evaluation of genetic diversity, but also for molecular mapping/QTL analysis, and for comparative genome analysis of the two Corchorus species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genetic mapping of AFLP markers in Japanese bunching onion (<Emphasis Type="Italic">Allium fistulosum</Emphasis>)

Summary The first genetic linkage map of Japanese bunching onion (Allium fistulosum) based primarily on AFLP markers was constructed using reciprocally backcrossed progenies. They were 120 plants each of (P₁)BC₁ and (P₂)BC₁ populations derived from a cross between single plants of two inbred lines: D1s-15s-22 (P₁) and J1s-14s-20 (P₂). Based on the (P₂)BC₁ population, a linkage map of P₁ was constructed. It comprises 164 markers – 149 amplified fragment length polymorphisms (AFLPs), 2 cleaved amplified polymorphic sequences (CAPSs), and 12 simple sequence repeats (SSRs) from Japanese bunching onion, and 1 SSR from bulb onion (A. cepa) – on 15 linkage groups covering 947 centiMorgans (cM). The linkage map of P₂ was constructed with the (P₁)BC₁ population and composed of 120 loci – 105 AFLPs, 1 CAPS, and 13 SSRs developed from Japanese bunching onion and 1 SSR from bulb onion – on 14 linkage groups covering 775 cM. Both maps were not saturated but were considered to cover the majority of the genome. Nine linkage groups in P₂ map were connected with their counterparts in P₁ map using co-dominant anchor markers, 13 SSRs and 1 CAPS.     

Validation of a set of informative simple sequence repeats markers for variety identification in Pak‐choi (Brassica rapa L. ssp. chinensis var. communis)

A core set of 21 simple sequence repeats (SSR) markers was developed for Pak‐choi (Brassica rapa ssp. chinensis var. communis) variety identification. We initially selected 74 SSR markers which exhibited high polymorphism and reproducibility in SSR detection from 2129 SSRs. Using the 74 SSR‐based dendrogram for 45 inbred lines as calibration, 21 core SSRs were selected out. The utility of this core set SSRs was firstly tested in 45 inbred lines and finally verified in 102 commercial varieties. We also constructed a molecular ladder for each core SSR as a reference standard. Diversity analysis of this core SSR panel in 102 varieties demonstrated that each marker generates 2–3 alleles (averaged 2.33), with polymorphism information content values ranging from 0.01 to 0.56 (averaged 0.31). The averaged values of Shannon information index, observed heterozygosity, expected heterozygosity and Wright's fixation index were 0.59, 0.43, 0.38 and −0.09, respectively. Furthermore, the 21 SSR‐based classifications for 102 varieties were consistent with traditional classification based on morphology. This core SSR panel represents an effective tool for genetic variation analysis in Pak‐choi.     

Characterization of novel sugarcane expressed sequence tag microsatellites and their comparison with genomic SSRs

Microsatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST–SSRs derived from sucest was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST–SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST–SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76‐314 was grouped apart from the other clones evaluated. The results here demonstrate that EST–SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.     

Development of genomic simple sequence repeat markers from an enriched genomic library of grass pea (Lathyrus sativus L.)

Simple sequence repeat (SSR) or microsatellite markers are a valuable tool for several purposes such as evaluation of genetic diversity, fingerprinting, marker‐assisted selection and breeding. In this study, a SSR genomic enriched library was developed in Lathyrus sativus (grass pea) by affinity capture of restriction fragments to biotinylated microsatellite oligonucleotides. About 400 randomly selected clones were sequenced, and SSRs were present in approximately 30% of them. Clones contained 75%, 9% and 16% of simple, interrupted and compound SSRs, respectively. Of the 10 SSRs tested, 7 primer pairs produced clearly distinguishable DNA banding patterns. Successively, SSR primer pairs were successfully tested to reveal polymorphism in a set of four different grass pea germplasm accessions. The transferability of SSR markers was high among three related species of Lathyrus, namely Lathyrus cicera, Lathyrus ochrus and Lathyrus tingitanus, and the legume crop, Pisum sativum. These results indicate that the novel SSR markers are informative and will be useful and convenient for genetic analysis in grass pea and related species.     

High‐throughput development of SSR marker candidates and their chromosomal assignment in rye (Secale cereale L.)

Shotgun survey sequences of flow‐sorted individual rye chromosomes were data mined for the presence of simple sequence repeats (SSRs). For 787,850 putative SSR loci, a total of 358,660 PCR primer pairs could be designed and 51,138 nonredundant SSR marker candidates were evaluated by in silico PCR. Of the 51,138 SSR primer candidates, 1,277 were associated with 1,125 rye gene models. A total of 2,112 of the potential SSR markers were randomly selected to represent about equal numbers for each of the rye chromosomes, and 856 SSRs were assigned to individual rye chromosomes experimentally. Potential transferability of rye SSRs to wheat and barley was of low efficiency with 4.3% (2,189) and 0.4% (223) of rye SSRs predicted to be amplified in wheat and barley, respectively. This data set of rye chromosome‐specific SSR markers will be useful for the specific detection of rye chromatin introgressed into wheat as well as for low‐cost genetic and physical mapping in rye without the need for high‐tech equipment.     

SSR analysis of chromosome 8 regions associated with aroma and cooked kernel elongation in Basmati rice

Aroma and cooked kernel elongation (CKE) are the two most important quality traits, which differentiate the highly valued Basmati rice from other rice types. Previous studies on genetic analysis have shown that genes/QTLs for these two traits are linked and present on chromosome number 8. We have evaluated the genetic diversity in 33 rice genotypes representative of the traditional Basmati (TB), cross-bred Basmati derived from indica × Basmati rice crosses and non-Basmati (indica and japonica) rice varieties for chromosome number 8 using 26 SSR markers including a specific marker (SCU-SSR1) for RG28 locus; the results have been compared with whole genome based SSR allelic data. The 26 SSR markers (24 polymorphic and 2 monomorphic) amplified a total of 106 alleles; 21 of these alleles were detected to be unique, present in only one genotype. The number and size of the alleles, and polymorphism information content (PIC) values ranged between 1–8, 87–312 and 0–0.736 bp, respectively. SCU-SSR1 marker amplified a total of three alleles (128, 129 and 130 bp). All the TB varieties except Basmati 217 (129 bp) and 7/13 cross-bred Basmati varieties had the 130 bp allele. Alleles of 129 and 128 bp were present in majority of the indica and japonica varieties, respectively. The average pair-wise Jaccard similarity coefficients for TB, indica and japonica varieties were 0.512, 0.483 and 0.251, respectively. Average similarity coefficient between TB and japonica was higher (0.236) compared to that between TB and indicas (0.150). Genetic relationships as determined by Principal Component Analysis (PCA, NTSYS-pc), PowerMarker tree, and Structure analyses, clearly showed high-level differentiation between TB and indica rice varieties, which formed two distinct clusters. The cross-bred Basmati and japonica rice genotypes were placed between these two clusters. Basmati 217 and Ranbir Basmati were quite divergent from rest of the TB varieties. Some of cross-bred Basmati varieties including Super, CSR30 and kernel were closer to TB. Indica rice varieties, CSR10 (salt tolerant variety) and Pokkali (salt tolerant landrace) formed a separate distinct cluster. The Pritchard structure analysis divided the rice genotypes in four major sub-populations of TB, cross-bred Basmati, indica and japonica (including Ranbir Basmati and Basmati 217) rice varieties. Chromosome 8 data-set showed a positive correlation (Mantel test, r = 0.739) with the allelic data-set for 30 SSR markers well-distributed on 12 rice chromosomes indicating a higher level of similarity between the two. The study demonstrates the distinctness of TB from other rice types (indica and japonica) and also provides several novel markers for differentiation between TB rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.     

Mapping Co, a gene controlling the columnar phenotype of apple, with molecular markers

The columnar phenotype is a very valuable genetic resource for apple breeding because of its compact growth form determined by the dominant gene Co. Using bulked segregant analysis combined with several DNA molecular marker techniques to screen the F₁ progeny of Spur Fuji × Telamon (heterozygous for Co), 9 new DNA markers (6 RAPD, 1 AFLP and 2 SSRs) linked to the Co gene were identified. A total of 500 10-mer random primers, 56 pairs of selective AFLP primers and 8 SSR primer pairs were screened. One RAPD marker S1142₆₈₂, and the AFLP marker, E-ACT/M-CTA₃₄₆, were converted into SCAR markers designated SCAR₆₈₂ and SCAR₂₁₆, respectively. These markers will enable early selection in progenies where Co is difficult to identify. The Co gene was located between the SSR markers CH03d11 and COL on linkage group 10 of the apple genetic linkage map. Finally, a local genetic map of the region around the Co gene was constructed by linkage analysis of the nine new markers and three markers developed earlier.     

A genetic linkage map of <Emphasis Type="Italic">Populus adenopoda</Emphasis> Maxim. × <Emphasis Type="Italic">P. alba</Emphasis> L. hybrid based on SSR and SRAP markers

Populus adenopoda Maxim. and P. alba L. [section Populus (aspen), genus Populus] are two tree species of ecological and economic value. To date, no high-density genetic maps are available for these two species. In this study, 1100 interspecific hybrids were obtained by controlled crossing and embryo culture. Simple sequence repeat (SSR) and sequence-related amplified polymorphisms (SRAP) were used to genotype 189 F₁ individuals. The genetic linkage map of P. adenopoda × P. alba generated from this study includes 212 markers (192 SSRs and 50 SRAPs) and consists of 26 linkage groups spanning 2178.5 cM, with an average distance of 11.7 cM between markers. This is the first SSR- and SRAP-containing genetic linkage map for aspen. The SSRs on the map will serve both as bridges for comparison with the poplar maps published to date and as a direct link to the Populus genomic sequence. Future studies focusing on the data presented here should enhance the density and precision of the map for identifying and localizing quantitative trait loci and promote genomic research on the genus.     

Assessment of genetic diversity within and among Basmati and non-Basmati rice varieties using AFLP,ISSR and SSR markers

Molecular markers provide novel tools to differentiate between the various grades of Basmati rice, maintain fair-trade practices and to determine its relationship with other rice groups in Oryza sativa. We have evaluated the genetic diversity and patterns of relationships among the 18 rice genotypes representative of the traditional Basmati, cross-bred Basmati and non-Basmati (indica and japonica) rice varieties using AFLP, ISSR and SSR markers. All the three marker systems generated higher levels of polymorphism and could distinguish between all the 18 rice cultivars. The minimum number of assay-units per system needed to distinguish between all the cultivars was one for AFLP, two for ISSR and five for SSR. A total of 171 (110 polymorphic), 240 (188 polymorphic) and 160 (159 polymorphic) bands were detected using five primer combinations of AFLP, 25 UBC ISSR primers and 30 well distributed, mapped SSR markers, respectively. The salient features of AFLP, ISSR and SSR marker data analyzed using clustering algorithms, principal component analysis, Mantel test and AMOVA analysis are as given below: (i) the two traditional Basmati rice varieties were genetically distinct from indica and japonica rice varieties and invariably formed a separate cluster, (ii) the six Basmati varieties developed from various indica × Basmati rice crosses and backcrosses were grouped variably depending upon the marker system employed; CSR30 and Super being more closer to traditional Basmati followed by HKR228, Kasturi, Pusa Basmati 1 and Sabarmati, (iii) AFLP, ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.42–0.50), and (iv) the partitioning of the variance among and within rice groups (traditional Basmati, cross-bred Basmati, indica and japonica) using AMOVA showed greater variation among than within groups using SSR data-set, while reverse was true for both ISSR and AFLP data-sets. The study emphasizes the need for using a combination of different marker systems for a comprehensive genetic analysis of Basmati rice germplasm. The high-level polymorphism generated by SSR, ISSR and AFLP assays described in this study shall provide novel markers to differentiate between traditional Basmati rice supplies from cheaper cross-bred Basmati and long-grain non-Basmati varieties at commercial level.The first two authors have equal contribution     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

Genetic diversity in soybean genotypes from north‐eastern China and identification of candidate markers associated with maturity rating

Random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to estimate the genetic relationships among 101 soybean cultivars developed in north‐eastern China. Fifty‐three fragments of the 100 RAPD markers and 35 SSR markers tested were polymorphic across the 101 soybean cultivars. Similarity values among these soybean cultivars ranged from 45.2% to 100% for RAPD data, and ranged from 36.1% to 100% for SSR data. The similarity matrices for SSR data and RAPD data were moderately correlated (r = 0.31, P < 0.05). Cluster analyses indicated that the cultivars released from the same seed company were mostly grouped together. A principal component analysis, based on the combined RAPD and SSR data, yielded a good separation of soybean varieties with different maturity ratings [represented by soybean Heat Unit (HU)]. The varieties with HU < 2200 were well separated from those with HU > 2200. Four RAPD markers and eight SSR markers were significantly associated with the maturity ratings of soybean.     

Mapping of the leaf rust resistance gene <Emphasis Type="Italic">Lr38</Emphasis> on wheat chromosome arm 6DL using SSR markers

Leaf rust caused by the fungus Puccinia triticina is one of the most important diseases of wheat (Triticum aestivum) worldwide. The use of resistant wheat cultivars is considered the most economical and environment-friendly approach in controlling the disease. The Lr38 gene, introgressed from Agropyron intermedium, confers a stable seedling and adult plant resistance against multiple isolates tested in Europe. In the present study, 94 F₂ plants resulting from a cross made between the resistant Thatcher-derived near-isogenic line (NIL) RL6097, and the susceptible Ethiopian wheat cultivar Kubsa were used to map the Thatcher Lr38 locus in wheat using simple sequence repeat (SSR) markers. Out of 54 markers tested, 15 SSRs were polymorphic between the two parents and subsequently genotyped in the population. The P. triticina isolate DZ7-24 (race FGJTJ), discriminating Lr38 resistant and susceptible plants, was used to inoculate seedlings of the two parents and the segregating population. The SSR markers Xwmc773 and Xbarc273 flanked the Lr38 locus at a distance of 6.1 and 7.9 cM, respectively, to the proximal end of wheat chromosome arm 6DL. The SSR markers Xcfd5 and Xcfd60 both flanked the locus at a distance of 22.1 cM to the distal end of 6DL. In future, these SSR markers can be used by wheat breeders and pathologists for marker assisted selection (MAS) of Lr38-mediated leaf rust resistance in wheat.     

Molecular mapping of powdery mildew resistance genes in wheat: A review

Powdery mildew, caused by Blumeria graminis f. sp. tritici (syn. Erysiphe graminis f. sp. tritici), is one of the most important diseases of common wheat (Triticum aestivum L.) worldwide. Molecular mapping and cloning of genes for resistance to powdery mildew in hexaploid wheat will facilitate the study of molecular mechanisms underlying resistance to powdery mildew diseases and help understand the structure and function of powdery mildew resistance genes, and permit marker-assisted selection in breeding programs. So far, 48 genes/alleles for resistance to powdery mildew at 32 loci have been identified and located on 16 different chromosomes, of which 21 resistance genes/alleles have been tagged by restriction fragment length polymorphisms (RFLPs), random-amplified polymorphic DNAs (RAPDs), amplified fragment length polymorphisms (AFLPs), sequence characterized amplified regions (SCARs), sequence-tagged sites (STS) or simple sequence repeats (SSRs). Several quantitative trait loci (QTLs) for adult plant resistance (APR) to powdery mildew have been associated with molecular markers. The detailed information on chromosomal location and molecular mapping of these genes has been reviewed. Isolation of powdery mildew resistance genes and development of valid molecular markers for pyramiding resistance genes in breeding programs is also discussed.     

Genetic relationships of Brassica vegetables determined using database derived simple sequence repeats

Sequence databases were screened to identify simple sequence repeats (SSRs) in Brassica oleracea sequences. A total of 512 B. oleracea DNA sequences were screened and 43 potential SSRs were identified. Thirty-six primer pairs were designed to amplify target sequences. Of the 36 primer pairs, six failed to amplify fragments of expected sizes, and 17 primer pairs failed to generate polymorphisms. Thirteen SSRs were used to assess genetic similarity between 54 B. oleracea cultivars, belonging to 3 variteal groups (cabbage, cauliflower, and broccoli). Pairwise genetic similarities were calculated for cultivars, and a dendrogram of relationships was produced. All cabbage cultivars were distinguished from each other and clustered in two separate groups. Five cauliflower cultivars could not be distinguished with SSR markers used in the study. Three broccoli cultivars clustered with cauliflower cultivars, and two cauliflower cultivars grouped with broccoli cultivars. The varietal group with the narrowest genetic variation in the study was cauliflower (B. oleracea var. botrytis) followed by broccoli (B. oleracea var. italica) and cabbage (B. oleracea var. capitata) groups. Polymorphism information content (PIC) values and number of alleles produced per marker ranged between 0.25 to 0.86 and 1 to 8, respectively, for database derived SSR markers.     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

Development of molecular markers to assess the level of introgression of Populus tremula into P. alba natural populations

Morphological traits traditionally adopted to discriminate between Populus alba L. and P. tremula L. have frequently led to misclassifi‐cation of their spontaneous hybrid P. × canescens Sm. Moreover, they may not be of any help in cases of spontaneous backcross phenomena. These limitations can be overcome by molecular markers, which are not environmentally influenced nor subjectively assessed. In this study, the effectiveness of amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers in species and hybrid discrimination was evaluated by analysing a set of reference samples of P. alba, P. tremula and P. × canescens. Species‐specific and species‐indicative AFLPs, as well as diagnostic SSR alleles, were recorded in both P. alba and P. tremula reference samples. The results allowed a clear distinction between the two poplar species and their hybrid. Using these diagnostic markers, a natural population of P. alba trees sampled along the Ticino river basin in northern Italy was analysed, and P. × canescens individuals, intermingled with P. alba trees, were detected.