DNA profiling of pineapple cultivars in Japan discriminated by SSR markers

We developed 18 polymorphic simple sequence repeat (SSR) markers in pineapple (Ananas comosus) by using genomic libraries enriched for GA and CA motifs. The markers were used to genotype 31 pineapple accessions, including seven cultivars and 11 breeding lines from Okinawa Prefecture, 12 foreign accessions and one from a related species. These SSR loci were highly polymorphic: the 31 accessions contained three to seven alleles per locus, with an average of 4.1. The values of expected heterozygosity ranged from 0.09 to 0.76, with an average of 0.52. All 31 accessions could be successfully differentiated by the 18 SSR markers, with the exception of ‘N67-10’ and ‘Hawaiian Smooth Cayenne’. A single combination of three markers TsuAC004, TsuAC010 and TsuAC041, was enough to distinguish all accessions with one exception. A phenogram based on the SSR genotypes did not show any distinct groups, but it suggested that pineapples bred in Japan are genetically diversed. We reconfirmed the parentage of 14 pineapple accessions by comparing the SSR alleles at 17 SSR loci in each accession and its reported parents. The obtained information will contribute substantially to protecting plant breeders’ rights.     

Mapping and use of QTLs controlling pod dehiscence in soybean

While the cultivated soybean, Glycine max (L.) Merr., is more recalcitrant to pod dehiscence (shattering-resistant) than wild soybean, Glycine soja Sieb. & Zucc., there is also significant genetic variation in shattering resistance among cultivated soybean cultivars. To reveal the genetic basis and develop DNA markers for pod dehiscence, several research groups have conducted quantitative trait locus (QTL) analysis using segregated populations derived from crosses between G. max accessions or between a G. max and G. soja accession. In the populations of G. max, a major QTL was repeatedly identified near SSR marker Sat_366 on linkage group J (chromosome 16). Minor QTLs were also detected in several studies, although less commonality was found for the magnitudes of effect and location. In G. max × G. soja populations, only QTLs with a relatively small effect were detected. The major QTL found in G. max was further fine-mapped, leading to the development of specific markers for the shattering resistance allele at this locus. The markers were used in a breeding program, resulting in the production of near-isogenic lines with shattering resistance and genetic backgrounds of Japanese elite cultivars. The markers and lines developed will hopefully contribute to the rapid production of a variety of shattering-resistant soybean cultivars.     

Association of molecular markers with cold tolerance and green period in zoysiagrass (Zoysia Willd.)

Cold tolerance and the green period are key traits in the breeding of zoysiagrass (Zoysia Willd.). Identification of molecular markers associated with cold tolerance and the green period of zoysiagrass will contribute to efficient selection of elite cultivars. These two traits were measured in 96 zoysiagrass accessions in 2004 and 2005–2006, respectively. The mapping population was screened with 29 pairs of simple sequence repeat (SSR) primers and 54 pairs of sequence-related amplified polymorphism (SRAP) primers. A multi-loci in silico mapping approach implemented with an empirical Bayes method was applied for association mapping of cold tolerance and green period. We detected 254 SSR polymorphic loci and 338 SRAP polymorphic loci, among which three SSR loci (Xgwm131-3B-187, Xgwm469-6D-194 and Xgwm234-5B-244) and one SRAP locus (Me11Em7-406) were significantly associated with cold tolerance with effect values of 57.83%, 38.05%, 36.92% and 37%, respectively. Three SSR loci (Xgwm132-6B-225, Xgwm111-7D-34 and Xgwm102-2D-97) and two SRAP loci (Me19Em5-359 and Me16Em8-483) were significantly associated with the green period with effect values of 79.54%, 62.59%, 99.04%, 49.01% and 82.57%. These markers will be useful for genetic improvement of the cold tolerance and green period of zoysiagrass by marker-assisted breeding.     

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.     

Genetic studies on saline and sodic tolerances in soybean

Salt-affected soils are generally classified into two main categories: saline and sodic (alkaline). Developing and using soybean (Glycine max (L.) Merr) cultivars with high salt tolerance is an effective way of maintaining sustainable production in areas where soybean growth is threatened by salt stress. Early classical genetics studies revealed that saline tolerance was conditioned by a single dominant gene. Recently, a series of studies consistently revealed a major quantitative trait locus (QTL) for saline tolerance located on linkage group N (chromosome 3) around the SSR markers Satt255 and Sat_091; other minor QTLs were also reported. In the case of sodic tolerance, most studies focused on iron deficiency caused by a high soil pH, and several QTLs associated with iron deficiency were identified. A wild soybean (Glycine soja Sieb. & Zucc.) accession with high sodic tolerance was recently identified, and a significant QTL for sodic tolerance was detected on linkage group D2 (chromosome 17). These studies demonstrated that saline and sodic tolerances were controlled by different genes in soybean. DNA markers closely associated with these QTLs can be used for marker-assisted selection to pyramid tolerance genes in soybean for both saline and sodic stresses.     

Clone identification in Japanese flowering cherry (Prunus subgenus Cerasus) cultivars using nuclear SSR markers

Numerous cultivars of Japanese flowering cherry (Prunus subgenus Cerasus) are recognized, but in many cases they are difficult to distinguish morphologically. Therefore, we evaluated the clonal status of 215 designated cultivars using 17 SSR markers. More than half the cultivars were morphologically distinct and had unique genotypes. However, 22 cultivars were found to consist of multiple clones, which probably originate from the chance seedlings, suggesting that their unique characteristics have not been maintained through propagation by grafting alone. We also identified 23 groups consisting of two or more cultivars with identical genotypes. Most members of these groups were putatively synonymously related and morphologically identical. However, some of them were probably derived from bud sport mutants and had distinct morphologies. SSR marker analysis provided useful insights into the clonal status of the examined Japanese flowering cherry cultivars and proved to be a useful tool for cultivar characterization.     

Identificaiton of a clubroot resistance locus conferring resistance to a Plasmodiophora brassicae classified into pathotype group 3 in Chinese cabbage (Brassica rapa L.)

In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) genes Crr1 and Crr2 are effective against the mild Plasmodiophora brassicae isolate Ano-01 and the more virulent isolate Wakayama-01, but not against isolate No. 14, classified into pathotype group 3. ‘Akiriso’, a clubroot-resistant F₁ cultivar, showed resistance to isolate No. 14. To increase the durability of resistance, we attempted to identify the CR locus in ‘Akiriso’. CR in ‘Akiriso’ segregated as a single dominant gene and was linked to several molecular markers that were also linked to CRb, a CR locus from cultivar ‘CR Shinki’. We developed additional markers around CRb and constructed partial genetic maps of this region in ‘Akiriso’ and ‘CR Shinki’. The positions and order of markers in the genetic maps of the two cultivars were very similar. The segregation ratios for resistance to isolate No. 14 in F₂ populations derived from each of the two cultivars were also very similar. These results suggest that the CR locus in ‘Akiriso’ is CRb or a tightly linked locus. The newly developed markers in this study were more closely linked to CRb than previously reported markers and will be useful for marker-assisted selection of CRb in Chinese cabbage breeding.     

Molecular Analysis of Local Potato Cultivars from Tenerife Island Using Microsatellite Markers

We have used 19 SSR markers to fingerprint 41 local potato cultivars from 10 locations of Tenerife Island. These varieties represent relicts of the early introductions originating from South America and have been characterised previously morphologically and ecophysiologically. The SSR primers generated a varying degree of polymorphisms. A total of 67 alleles were observed, 12 of them were present in all cultivars. Several accession and group specific alleles were detected. Similarity coefficients were computed from the molecular data and cluster analyses were performed. Generally, cultivar groups with identical or related common names showed the same SSR patterns or clustered closely together. According to the molecular patterns misleading or confounded names were evident for four accessions. The dendrogram clusters were generally in good agreement with previous classifications of the accessions as Solanum tuberosum subsp. andigena, S. tuberosum subsp. tuberosum and Solanum chaucha genotypes. However in four cases the molecular patterns showed discrepancies with previous species assignments suggesting the need for a more detailed and comparative study of these accessions.     

Genetic variation in resistance to blast disease (Pyricularia oryzae Cavara) in Japanese rice (Oryza sativa L.), as determined using a differential system

A total of 324 Japanese rice accessions, including landrace, improved, and weedy types were used to 1) investigate genetic variations in blast resistance to standard differential isolates, and 2) across the genome using polymorphism data on 64 SSR markers. From the polymorphism data, the accessions were classified into two clusters. Accessions from irrigated lowland areas were included mainly in cluster I, and upland and Indica types were mainly in cluster II. The accessions were classified into three resistance subgroups, A2, B1 and B2, based on the reaction patterns to blast isolates. The accessions in A2 were postulated to have at least two resistance genes Pish and Pik-s, whereas those in B1 had various combinations of the resistance genes Pish, Pia, Pii, Pi3, Pi5(t), and Pik alleles. The B2 accessions were resistant to almost all isolates, and many accessions of cluster II were included, and had Pish, Pia, Pii, Pi3, Pi5(t), certain Pik, Piz and Pita alleles, and unknown genes. The frequencies of accessions of B1 originating in Hokkaido, and those of B2 originating in the Kanto and Tohoku regions were remarkably higher than in the other regions.     

Leaf margin phenotype-specific restriction-site-associated DNA-derived markers for pineapple (Ananas comosus L.)

To explore genome-wide DNA polymorphisms and identify DNA markers for leaf margin phenotypes, a restriction-site-associated DNA sequencing analysis was employed to analyze three bulked DNAs of F1 progeny from a cross between a ‘piping-leaf-type’ cultivar, ‘Yugafu’, and a ‘spiny-tip-leaf-type’ variety, ‘Yonekura’. The parents were both Ananas comosus var. comosus. From the analysis, piping-leaf and spiny-tip-leaf gene-specific restriction-site-associated DNA sequencing tags were obtained and designated as PLSTs and STLSTs, respectively. The five PLSTs and two STSLTs were successfully converted to cleaved amplified polymorphic sequence (CAPS) or simple sequence repeat (SSR) markers using the sequence differences between alleles. Based on the genotyping of the F1 with two SSR and three CAPS markers, the five PLST markers were mapped in the vicinity of the P locus, with the closest marker, PLST1_SSR, being located 1.5 cM from the P locus. The two CAPS markers from STLST1 and STLST3 perfectly assessed the ‘spiny-leaf type’ as homozygotes of the recessive s allele of the S gene. The recombination value between the S locus and STLST loci was 2.4, and STLSTs were located 2.2 cM from the S locus. SSR and CAPS markers are applicable to marker-assisted selection of leaf margin phenotypes in pineapple breeding.     

DNA markers linked to Pga1, an adzuki bean gene that confers resistance to Cadophora gregata race 1

Brown stem rot (BSR) caused by Cadophora gregata f. sp. adzukicola (syn. Phialophora gregata) is a serious soilborne disease of adzuki bean (Vigna angularis) in Japan. Cultivation of resistant cultivars is the most effective disease control method, therefore the selection of resistant lines is a priority for breeders. BSR-resistant adzuki bean lines have been screened in pathogen-infected fields. However, field selection using the pathogen and artificial inoculation methods is time-consuming and labor-intensive. In the present study, we used 105 F₃ lines derived from a cross between a BSR-resistant cultivar ‘Syumari’ and a susceptible cultivar ‘Buchishoryukei-1’ for BSR inoculation tests. Amplified fragment-length polymorphism (AFLP) analyses with 1024 primer sets revealed that six fragments were polymorphic between resistance and susceptible bulked groups. Five DNA markers (Pg77, Pg118, Pg138, Pg139 and Pg126) were developed from the nucleotide sequences of polymorphic AFLP markers and their flanking regions. Pg118, which was derived from E-ACT/M-ACT-118, was tightly linked to the resistance gene Pga1 and was converted into a codominant marker for its easier use in marker-assisted selection for adzuki bean BSR resistance. Finally, the applicability of the developed markers for BSR resistance was tested on 32 adzuki bean accessions or cultivars.     

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F₁ hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC₁ obtained from B. juncea × F₁ hybrid, and this BC₁ exhibited a plant type like that of B. juncea. Most markers were deleted in BC₂ and BC₃ plants, with only two markers persisting in the BC₃. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.     

Development of cultivar-specific DNA markers based on retrotransposon-based insertional polymorphism in Japanese pear

We developed retrotransposon-based insertional polymorphism (RBIP) markers based on the long terminal repeat (LTR) sequences of copia-like retrotransposon Ppcrt4 and flanking genome sequences, which were derived from 454 sequencing data from Japanese pear (Pyrus pyrifolia) ‘Hosui’. Out of 40 sequences including both LTR and flanking genome regions, we developed 22 RBIP markers and used them for DNA profiling of 80 pear cultivars: 64 Japanese, 10 Chinese (Pyrus ussuriensis) and 6 European (Pyrus communis). Three RBIP markers were enough to differentiate ‘Hosui’ from the other Japanese pear cultivars. The 22 RBIP markers could also distinguish 61 of the 64 Japanese pear cultivars. European pears showed almost no amplification of the 22 RBIP markers, which might suggest that retrotransposons had transposed during Asian pear evolution or reflect the genetic relationship between Asian and European pears. Sixteen of the RBIP markers could be positioned on a genetic linkage map of ‘Hosui’. The RBIP loci were distributed in 10 linkage groups, and some loci were very closely located within the same linkage group. The information obtained will be applicable to developing cultivar-specific RBIP marker sets in plants.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

Genetic diversity of the black gram [Vigna mungo (L.) Hepper] gene pool as revealed by SSR markers

In this study, 520 cultivated and 14 wild accessions of black gram (Vigna mungo (L.) Hepper) were assessed for diversity using 22 SSR markers. Totally, 199 alleles were detected with a mean of 9.05 alleles per locus. Wild black gram showed higher gene diversity than cultivated black gram. Gene diversity of cultivated accessions among regions was comparable, while allelic richness of South Asia was higher than that of other regions. 78.67% of the wild gene diversity presented in cultivated accessions, indicating that the domestication bottleneck effect in black gram is relatively low. Genetic distance analysis revealed that cultivated black gram was more closely related to wild black gram from South Asia than that from Southeast Asia. STRUCTURE, principal coordinate and neighbor-joining analyses consistently revealed that 534 black gram accessions were grouped into three major subpopulations. The analyses also revealed that cultivated black gram from South Asia was genetically distinct from that from West Asia. Comparison by SSR analysis with other closely related Vigna species, including mungbean, azuki bean, and rice bean, revealed that level of gene diversity of black gram is comparable to that of mungbean and rice bean but lower than that of azuki bean.     

Genome-wide indel markers shared by diverse Asian rice cultivars compared to Japanese rice cultivar ‘Koshihikari’

Insertion-deletion (indel) polymorphisms, such as simple sequence repeats, have been widely used as DNA markers to identify QTLs and genes and to facilitate rice breeding. Recently, next-generation sequencing has produced deep sequences that allow genome-wide detection of indels. These polymorphisms can potentially be used to develop high-accuracy polymerase chain reaction (PCR)-based markers. Here, re-sequencing of 5 indica, 2 aus, and 3 tropical japonica cultivars and Japanese elite cultivar ‘Koshihikari’ was performed to extract regions containing large indels (10–51 bp) shared by diverse cultivars. To design indel markers for the discrimination of genomic regions between ‘Koshihikari’ and other diverse cultivars, we subtracted the indel regions detected in ‘Koshihikari’ from those shared in other cultivars. Two sets of indel markers, KNJ8-indel (shared in eight or more cultivars, including ‘Khao Nam Jen’ as a representative tropical japonica cultivar) and C5-indel (shared in five to eight cultivars), were established, with 915 and 9,899 indel regions, respectively. Validation of the two marker sets by using 23 diverse cultivars showed a high PCR success rate (≥95%) for 83.3% of the KNJ8-indel markers and 73.9% of the C5-indel markers. The marker sets will therefore be useful for the effective breeding of Japanese rice cultivars.     

Fine mapping of the gene for susceptibility to black spot disease in Japanese pear (Pyrus pyrifolia Nakai)

Black spot disease, which is caused by the Japanese pear pathotype of the filamentous fungus Alternaria alternata (Fries) Keissler, is one of the most harmful diseases in Japanese pear cultivation. We mapped a gene for susceptibility to black spot disease in the Japanese pear (Pyrus pyrifolia Nakai) cultivar ‘Kinchaku’ (Aki gene) at the top of linkage group 11, similar to the positions of the susceptibility genes Ani in ‘Osa Nijisseiki’ and Ana in ‘Nansui’. Using synteny-based marker enrichment, we developed novel apple SSR markers in the target region. We constructed a fine map of linkage group 11 of ‘Kinchaku’ and localized the Aki locus within a 1.5-cM genome region between SSR markers Mdo.chr11.28 and Mdo.chr11.34. Marker Mdo.chr11.30 co-segregated with Aki in all 621 F₁ plantlets of a ‘Housui’ × ‘Kinchaku’ cross. The physical size of the Aki region, which includes three markers (Mdo.chr11.28, Mdo.chr11.30, and Mdo.chr11.34), was estimated to be 250 Kb in the ‘Golden Delicious’ apple genome and 107 Kb in the ‘Dangshansuli’ Chinese pear genome. Our results will help to identify the candidate gene for susceptibility to black spot disease in Japanese pear.     

Expressed sequence tags from organ-specific cDNA libraries of tea (Camellia sinensis) and polymorphisms and transferability of EST-SSRs across Camellia species

Tea is one of the most popular beverages in the world and the tea plant, Camellia sinensis (L.) O. Kuntze, is an important crop in many countries. To increase the amount of genomic information available for C. sinensis, we constructed seven cDNA libraries from various organs and used these to generate expressed sequence tags (ESTs). A total of 17,458 ESTs were generated and assembled into 5,262 unigenes. About 50% of the unigenes were assigned annotations by Gene Ontology. Some were homologous to genes involved in important biological processes, such as nitrogen assimilation, aluminum response, and biosynthesis of caffeine and catechins. Digital northern analysis showed that 67 unigenes were expressed differentially among the seven organs. Simple sequence repeat (SSR) motif searches among the unigenes identified 1,835 unigenes (34.9%) harboring SSR motifs of more than six repeat units. A subset of 100 EST-SSR primer sets was tested for amplification and polymorphism in 16 tea accessions. Seventy-one primer sets successfully amplified EST-SSRs and 70 EST-SSR loci were polymorphic. Furthermore, these 70 EST-SSR markers were transferable to 14 other Camellia species. The ESTs and EST-SSR markers will enhance the study of important traits and the molecular genetics of tea plants and other Camellia species.     

Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids

A collection of 142 accessions of 23 Malus species, derived hybrids and cultivar accessions from the USDA-ARS Plant Genetic Resources Unit's core collection, which represents an extensive range of Malus species, was screened with a set of previously described SSR (simple sequence repeat) markers. The markers were used to determine genetic identities, estimate genetic diversity, identify genetic relationships among the accessions, and determine the utility of SSR primers developed from Malus ×domestica for making genetic assessments across the whole Malus genus. All eight primer pairs amplified multiple fragments when used in polymerase chain reactions with DNA from these accessions. High levels of variation were detected with a mean of 26.4 alleles per locus and a mean direct count heterozygosity across all eight loci equal to 0.623. The eight primer pairs used in this study unambiguously differentiated all but five pairs of accessions in this collection of 142 accessions of 23 Malus species, derived hybrids and cultivars. These SSR data were not useful in identifying genetic relationships among this diverse collection of accessions, with the majority of the accessions not clustering in ways concordant with taxonomic information and/or geographic origin. The resulting phenogram resolved only two meaningful clusters, for the taxonomically isolated Section Chloromeles and for M. fusca accessions, reflecting genetic relationships arising from geographic origin. The detection of identical accessions in the collection, which were previously considered to be unique, highlights the critical need to further bolster collections of certain Malus species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic characterization of rainfed upland New Rice for Africa (NERICA) varieties

A total of 18 rainfed upland New Rice for Africa (NERICA) varieties were categorized as the heavy panicle and low tillering types and early heading, in compared with 32 different varieties. These chromosome components were clarified using 243 SSR markers which showed polymorphism among NERICA varieties and their parents, CG 14 (O. glaberrima Steud.) and one of the recurrent parents, WAB-56-104 (O. sativa L.). NERICA varieties were classified into three groups, which corresponded with these parents’ continuation including two exceptions, NERICAs 14 and 17, by a cluster analysis using polymorphism data of SSR markers and 14 differential markers among them were selected to classify NERICA varieties. However, three groups: NERICAs, 3 and 4, NERICAs, 8, 9 and 11 and NERICAs, 15 and 16 were not distinguishable. Association analysis was carried out for characterization of NERICA varieties by using SSR markers genotype and phenotype of agronomic traits. A total of 131 quantitative trait loci between SSR markers and 11 agronomic traits were detected. The characteristics of early maturity and heavy panicle of upland NERICA varieties were succeeded from Asian rice varieties and the characteristics of high dry matter production and late heading were introduced from CG 14 and the other varieties.