Polymorphic Simple Sequence Repeat Primers Research Articles

Genetic diversity and geographic distribution patterns of Malus landraces from 5 cultivated species under climate change in China

China is the largest center of genetic diversity of apple (Malus Mill.), with rich germplasm resources. In the process of evolution and domestication, a large number of apple landraces appeared, and they played a very important role before the introduction of modern cultivated apple varieties to China. However, the habitat of Malus landraces is under serious threat. In this study, 19 pairs of polymorphic simple sequence repeat primers were used to analyze the genetic structure and genetic relationships of Malus landraces and the potential distribution was predicted according to eight environmental variables using MaxEnt modeling. The results showed that the genetic variation of Malus landraces was high (Ho = 0.728 and He = 0.884), and the genetic differentiation was mainly distributed within respective populations and with almost no differentiation among populations. The five population groups were ranked, from highest genetic diversity level to lowest, as follows: Malus domestica subsp. chinensis > Malus prunifolia > Malus robusta > Malus asiatica > Malus micromalus. The 216 germplasms were divided into five groups based on Nei's genetic distance, whereas the Bayesian structure analysis categorized them into two groups. Population structure and principal coordinate analyses showed that Malus domestica subsp. chinensis, Malus asiatica and Malus prunifolia may be derived from similar genetic sources. In the modeling analysis, the cumulative contribution of temperature to the distribution of Malus landraces in suitable growth areas reached 66.1 %, and BIO4 (temperature seasonality) was identified as the major factor influencing the distribution of Malus landraces. Under four future emission scenarios, the potential distribution range of Malus landraces basically unchanged, but the total potential distribution area is projected to grow greatly over time and will tend to shift to higher latitudes.

Bulk Segregant Analysis for the Identification of Shoot Fly Resistance Linked Molecular Marker in Sorghum [Sorghum bicolor (L.) Moench

Sorghum (Sorghum bicolor (L.) Moench) is one of the most important crops in the semi-arid regions of the world. One of the important biotic constraints to sorghum production in India is the shoot fly which attacks sorghum at the seedling stage. The study was undertaken during 2021–22 at the Centre for Millets Research, Sardarkrushinagar Dantiwada Agricultural University, Deesa, Gujarat, India to assess linked molecular markers for sorghum shoot fly resistance using the bulk segregant analysis (BSA) method with simple sequence repeat (SSR) marker from developed F2 mapping population with two genetically diverse parental lines, SWARNA (susceptible to shoot fly) and IS 18551 (resistant to shoot fly). Sixty-five SSRs primers pair were used for the parental polymorphism survey using two contrasting parents to detect the primers exhibiting polymorphism. Eight out of sixty-five primers showed polymorphism (12.30%) between two contrasting parental lines in sorghum. Two out of eight polymorphic SSRs primer pairs i.e., Xtxp 67 and Xgap 88 were found polymorphic between resistance and susceptibility in parents and bulks and thus reported to be putatively linked with shoot fly. Bulk segregant analysis (BSA) was extended to identify the traits controlled by minor genes with additive effects, which increased the power and efficiency of this molecular technique to construct genetic map in the sorghum crop improvement program. The identified SSRs markers i.e., Xtxp 67 and Xgap 88 might be useful to screen resistance for shoot fly infestation in future sorghum improvement program.

Characterisation of bell pepper (Capsicum annuum L. var. grossum Sendt.) accessions for genetic diversity and population structure based on agro-morphological and microsatellite markers

Bell pepper (Capsicum annuum L. var. grossum Sendt.) is a leading Solanaceous vegetable crop across the world with immense therapeutic and export potential. Such wide cultivation exposes the crop to various biotic and abiotic stresses. Genetic diversity is one of the major prerequisites for any crop breeding programme. Hence, in this study, an attempt was made to evaluate the genetic diversity and population structure of 24 bell pepper genotypes using 25 agro-morphological [9 distinctness, uniformity and stability (DUS) descriptors; 13 quantitative traits; and 3 quality traits] and 82 microsatellite or simple sequence repeat (SSR) markers. Eight DUS descriptors showed polymorphism with maximum morphological variation (H = 1.29) for fruit shape in longitudinal section. Multivariate analysis using Mahalanobis D2 statistic distinguished the genotypes into 11 clusters. With a mean of 4.08 alleles per locus, a total of 151 alleles were generated from 37 polymorphic SSR primers. The polymorphic information content (PIC) ranged from 0.14 (CAMS 451) to 0.76 (CA 525390) with an average of 0.54. The genotypes were divided into two major clusters based on cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) algorithm, whereas the cluster tree using the neighbour-joining (NJ) method identified three major groups. The population was classified into three groups based on structure analysis. On comparing agro-morphological and molecular clusters, 11 genotypes viz., BWT-39, BWT-39-BR, BWT-39-DR, BWT-29, BWT-3Y, BWT-3Y-4L, Kandaghat Selection, BWT-48-AC, BWT-49-AC, EC-464107, and EC-464115, were found common. These genotypes also exhibited considerable resistance to bacterial wilt disease. Mantel's test showed a moderate positive correlation between agro-morphological (DUS descriptors) and molecular data. Therefore, a combined approach of agro-morphological and molecular evaluation can facilitate the selection of diverse parental lines followed by subsequent hybridisation between them to introduce new traits in bell pepper.

Genetic diversity and population structure analysis in a large collection of Vicia amoena in China with newly developed SSR markers

Vicia amoena is a high-nutritional quality forage similar to alfalfa. However, studies on the genetic background of V. amoena are scarce. In the present study, the genetic variation of 24 V. amoena populations was assessed with newly developed simple sequence repeat (SSR) markers. A total of 8799 SSRs were identified in the V. amoena genomic-enriched sequences, and the most abundant repeat number was four. A total of 569 sampled individuals were assayed to evaluate the genetic diversity of the V. amoena populations based on 21 polymorphic SSR primers. The polymorphism information content (PIC) ranged from 0.896 to 0.968, with an average of 0.931, which indicated that the markers were highly informative. Based on analysis of molecular variance, 88% of the variance occurred within populations, and the remaining 12% of the variance occurred among populations. The high degree of gene flow (Nm= 4.958) also showed slight differentiation among the V. amoena populations. The V. amoena populations were mainly clustered by steppe and mountain habitats based on principal coordinate analysis (PCoA) and STRUCTURE analysis. This indicated that the elevation and special habitat of geographical origins may be important factors affecting the clustered pattern of V. amoena populations. Neighbour-joining (NJ) analysis did not separate the populations well by geographical origin, which indicated that the genetic structure of V. amoena was complex and needs further study. Overall, our results showed that the newly developed SSR markers could benefit the V. amoena research community by providing genetic background information to help establish a foundation for breeding improvement and germplasm resource conservation.

Genome-wide identification of microsatellite and development of polymorphic SSR markers for spotted sea bass (Lateolabrax maculatus)

The spotted sea bass (Lateolabrax maculatus) is a valuable commercial mariculture fish in China and has huge potential for future genetic improvement. However, only limited number of SSR (simple sequence repeat) markers have been developed for L. maculatus. In this study, a total of 220,709 SSRs were identified from the L. maculatus genome. The total length, density and frequency of SSRs were 5,119,809 bp, 9898.28 bp/Mb and 425.06 loci/Mb, respectively. Dinucleotide repeats were predominant among SSRs (85.00 %) and AC was the most abundant SSR motifs. Both the number and length of SSRs in each chromosome were positively associated with the chromosome length. A total of 155,905 SSR markers were successfully designed (with an average of 6496 SSRs per chromosome). The frequency of SSR markers varied from 245.63 per Mb to 346.60 per Mb. 14 polymorphic SSR primers were used to evaluate the genetic diversity of L. maculatus wild population from Zhuhai. A total of 74 alleles were detected and most SSR markers showed high polymorphism information content (PIC). The cross-species transferability of 11 out of 67 tested loci were proven successful in largemouth bass Micropterus salmoides. The genome-wide SSRs and newly developed SSR markers made available in this study will facilitate future genetic breeding and genomic studies of spotted sea bass.

Development of a high-density SSR genetic linkage map in sweet potato

Simple sequence repeat (SSR) markers have been proved to be a very powerful tool for quantitative trait locus (QTL) mapping, marker-assisted selection and comparative genomics research in many crop species. However, a high-density SSR genetic linkage map is still lacking because there are only a few SSR markers available in sweet potato. In this study, a total of 2545 simple sequence repeat (SSR) primer pairs, including 1215 genomic SSR (gSSR) primer pairs and 1330 BES-SSR (bSSR) primer pairs designed from the genome sequence and BAC-end sequence of sweet potato, respectively, were screened with sweet potato cultivars Luoxushu 8 and Zhengshu 20 and their randomly sampled two F1 individuals and 571 of them generated polymorphic bands. The selected 571 polymorphic SSR primer pairs and 35 EST-based SSR (eSSR) primer pairs developed at our laboratory were used to genotype 240 F1 individuals derived from a cross between Luoxushu 8 and Zhengshu 20. A double pseudo-test-cross strategy was applied for linkage analysis. The Luoxushu 8 map included 90 linkage groups with 5057 SSR markers and covered 13,299.9 cM with a marker density of 2.6 cM, and the Zhengshu 20 map contained 90 linkage groups with 3009 SSR markers and covered 11,122.9 cM with a marker density of 3.7 cM. Fifteen homologous groups were identified in both parent maps. These are the first SSR linkage maps consisting of the complete 90 linkage groups and 15 homologous groups, which are consistent with the autohexaploid nature of sweetpotato, and are also the linkage maps with the highest SSR marker density reported to date. These results provide a basis for QTL mapping, marker-assisted breeding and comparative genomics research of sweet potato.

Development of 18 microsatellite markers for Atractylodes japonica.

Premise Atractylodes japonica (Asteraceae) is endemic to East Asia, where its rhizomes are used in traditional medicine. To investigate the genetic diversity of this species, we developed polymorphic microsatellite markers.Methods and ResultsWe obtained a total of 175,825 simple sequence repeat (SSR) loci using the Illumina HiSeq 2500 system. Eighteen polymorphic SSR primer pairs were selected to determine heterozygosity levels and allele numbers in 80 individuals from four A. japonica populations. The levels of observed and expected heterozygosity ranged from 0.000 to 1.000 and from 0.133 to 0.892, respectively. Cross‐amplification in the related species A. macrocephala and A. lancea was successful in 15 and 14 of the 18 markers, respectively.ConclusionsThese microsatellite markers will be useful for future studies involving A. japonica population genetics and breeding.

SSR fingerprinting of 203 sweetpotato (Ipomoea batatas (L.) Lam.) varieties

Simple sequence repeat (SSR) markers have been shown to be a powerful tool for varieties identification in plants. However, SSR fingerprinting of sweetpotato varieties has been a little reported. In this study, a total of 1294 SSR primer pairs, including 1215 genomic-SSR and 79 expressed sequence tag (EST)-SSR primer pairs, were screened with sweetpotato varieties Zhengshu 20 and Luoxushu 8 and their 2 F1 individuals randomly sampled, and 273 and 38 of them generated polymorphic bands, respectively. Four genomic-SSR and 3 EST-SSR primer pairs, which showed good polymorphism, were selected to amplify 203 sweetpotato varieties and gave a total of 172 bands, 85 (49.42%) of which were polymorphic. All of the 203 sweetpotato varieties showed unique fingerprint patterns, indicating the utility of SSR markers in variety identification of this crop. Polymorphism information content (PIC) ranged from 0.5824 to 0.9322 with an average of 0.8176. SSR-based genetic distances varied from 0.0118 to 0.6353 with an average of 0.3100 among these varieties. Thus, these sweetpotato varieties exhibited high levels of genetic similarity and had distinct fingerprint profiles. The SSR fingerprints of the 203 sweetpotato varieties have been successfully constructed. The highly polymorphic SSR primer pairs developed in this study have the potential to be used as core primer pairs for variety identification, genetic diversity assessment and linkage map construction in sweetpotato and other plants.

Transcriptomic analysis, genic SSR development, and genetic diversity of proso millet (Panicum miliaceum; Poaceae).

Premise of the study:Proso millet (Panicum miliaceum; Poaceae) is a minor crop with good nutritional qualities and strong tolerance to drought stress and soil infertility. However, studies on genetic diversity have been limited due to a lack of efficient genetic markers.Methods:Illumina sequencing technology was used to generate short read sequences of proso millet, and de novo transcriptome assemblies were used to develop a de novo assembly of proso millet. Genic simple sequence repeat (SSR) markers were identified and used to detect polymorphism among 56 accessions. Population structure and genetic similarity coefficient were estimated.Results:In total, 25,341 unique gene sequences and 4724 SSR loci were obtained from the transcriptome, of which 229 pairs of SSR primers were validated, which resulted in 14 polymorphic genic SSR primers exhibiting 43 total alleles. According to the ratio of polymorphic markers (6.1%, 14/229), there are potentially 288 polymorphic genic SSR markers available for genetic assay development in the future. Bayesian population analyses showed that the 56 accessions comprised two distinct groups.Discussion:A genetic structure and cluster assay indicated that the accessions from the Loess Plateau of China shared a high genetic similarity coefficient with those from other regions and that there was no correlation between genetic diversity and geographic origin. The transcriptome sequencing data and millet-specific SSR markers developed in this study establish an excellent resource for gene discovery and may improve the development of breeding programs in proso millet in the future.

Polymorphism of microsatellite markers in barley varieties contrasting in response to drought stress

Drought is one of the most serious abiotic stresses limiting plant growth and crop productivity worldwide. The long arm of the barley chromosome 4H is often implicated in adaptation to drought. Therefore, the aim of the present study was to explore the polymorphism of microsatellite markers localized mostly on chromosome 4H in eight barley varieties (Hordeum vulgare L.) representing the wide range of drought tolerance. The differences in physiological responses related to drought stress classified these genotypes in two groups based on the Euclidean distances. The susceptible varieties are gathered in the same cluster recording an average Euclidean distance of 27.2, and an average dissimilarity of approximately 52.4 regarding the drought-tolerant group. Tolerance to drought stress of the Tunisian and Jordanian barley cultivars was associated with lower changing ratios of malondialdehyde contents, smaller decrease in leaf water content and significant increase in proline concentration compared to other analyzed genotypes. In simple sequence repeat (SSR) analyses, from a total of 17 analyzed SSR primers, 12 microsatellites showed clear patterns with high level of polymorphism. Among the 12 polymorphic SSR primers, four were found to be polymorphic across drought susceptible and drought-tolerant cultivars (Bmag808, EBMAC624, WMS6 and BMAC0577). High level of diversity was observed in the chosen genotypes through analyzing both physiological traits and SSR markers. A significant correlation was observed between the distance matrices based on SSR markers and physiological data, as determined by the Pearson’s correlation coefficient (r = 0.63; Mantel test P < 0.05).

Molecular and agronomic analysis of intraspecific variability in Capsicum baccatum var. pendulum accessions.

Capsicum baccatum is one of the most important chili peppers in South America, since this region is considered to be the center of origin and diversity of this species. In Brazil, C. baccatum has been widely explored by family farmers and there are different local names for each fruit phenotype, such as cambuci and dedo-de-moça (lady's finger). Although very popular among farmers and consumers, C. baccatum has been less extensively studied than other Capsicum species. This study describes the phenotypic and genotypic variability in C. baccatum var. pendulum accessions. Twenty-nine accessions from the Universidade Estadual do Norte Fluminense Darcy Ribeiro gene bank, and one commercial genotype ('BRS-Mari') were evaluated for 53 morphoagronomic descriptors (31 qualitative and 22 quantitative traits). In addition, accessions were genotyped using 30 microsatellite primers. Three accessions from the C. annuum complex were included in the molecular characterization. Nine of 31 qualitative descriptors were monomorphic, while all quantitative descriptors were highly significant different between accessions (P < 0.01). Using the unweighted pair group method using arithmetic averages, four groups were obtained based on multicategoric variables and five groups were obtained based on quantitative variables. In the genotyping analysis, 12 polymorphic simple sequence repeat primers amplified in C. baccatum with dissimilarity between accessions ranging from 0.13 to 0.91, permitting the formation of two distinct groups for Bayesian analysis. These results indicate wide variability among the accessions comparing phenotypic and genotypic data and revealed distinct patterns of dissimilarity between matrices, indicating that both steps are valuable for the characterization of C. baccatum var. pendulum accessions.

Effect of fragmentation on the natural genetic diversity of Theobroma speciosum Willd. ex Spreng. populations.

Forest fragmentation reduces the effective size of natural populations, isolates individuals in the landscape, and, consequently, changes species' mating systems by increasing the degree of relatedness between individuals and inbreeding. Investigating the impact of habitat degradation on forest fragments helps to assess the genetic and ecological consequences of these changes, and allows the development of effective and sustainable conservation strategies to manage the genetic resources of species living in degraded landscapes. The aim of the present study was to assess the genetic diversity of fragmented Theobroma speciosum populations using microsatellite markers. Three urban forest fragments were selected in the municipality of Alta Floresta, Mato Grosso State, Brazil, namely C/E park, J park, and Zoo Botanical park. Seventy-five individuals (25 in each fragment) were sampled by collecting their leaves for genomic DNA extraction. Polymerase chain reaction amplifications were performed using nine polymorphic simple sequence repeat primers, which amplified 84 alleles. The mean expected heterozygosity was 0.970, and it was always higher than the observed heterozygosity. Analysis of molecular variance revealed that most variability occurred within populations (64%) rather than between them (36%). The Structure software and an unweighted pair group method with arithmetic mean dendrogram revealed three distinct groups, showing that individuals were allocated to their correct populations. Genotype number 3 from C/E park, number 45 from J park, and number 51 from Zoo Botanical park could be used as stock plants in breeding programs, because they were the most dissimilar within the populations studied. The high genetic diversity levels detected in all three populations studied emphasize the importance of protecting this species in its natural habitat.

Molecular Identification of Dominant Cultivars of Soybean Using Simple Sequence Repeat Markers

Molecular identity of dominant cultivars of soybean needs to complement the morphological descriptors, which are insufficient and influenced by environment, for protecting the interest of breeders and maintaining the genetic purity. In the present investigation, genomic DNA of six commercial Indian soybean varieties viz. JS95-60, JS93-05, NRC7, JS97-52, NRC37, JS335 was amplified using ten simple sequence repeat (SSR) markers, which were selected based upon the relatively higher polymorphic information content (PIC) value observed after polymorphism survey of 46 soybean genotypes through 58 SSR markers. PCR analysis showed nine polymorphic SSR primers which could differentiate unambiguously all the six varieties. Maximum similarity was observed between NRC7, JS93-05 and JS95-60. Two unique alleles amplified in each of the two varieties namely JS335 and NRC37 and four unique alleles in JS97-52 unambiguously differentiated them from the remaining varieties. Morphologically similar varieties JS93-05 and JS95-60 could be differentiated distinctly by primer pair Satt181, while NRC7 could be distinguished from both JS93-05 and JS95-60 through Sct_199. Results also showed that these nine polymorphic markers could distinguish the dominant cultivars from six non-trading varieties, which further confirmed the utility of nine SSR primers for protecting the breeders’ rights and maintaining the purity of dominant cultivars during seed production programme.

Pl(17) is a novel gene independent of known downy mildew resistance genes in the cultivated sunflower (Helianthus annuus L.).

Pl 17, a novel downy mildew resistance gene independent of known downy mildew resistance genes in sunflowers, was genetically mapped to linkage group 4 of the sunflower genome. Downy mildew (DM), caused by Plasmopara halstedii (Farl.). Berl. et de Toni, is one of the serious sunflower diseases in the world due to its high virulence and the variability of the pathogen. DM resistance in the USDA inbred line, HA 458, has been shown to be effective against all virulent races of P. halstedii currently identified in the USA. To determine the chromosomal location of this resistance, 186 F 2:3 families derived from a cross of HA 458 with HA 234 were phenotyped for their resistance to race 734 of P. halstedii. The segregation ratio of the population supported that the resistance was controlled by a single dominant gene, Pl 17. Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) primers were used to identify molecular markers linked to Pl 17. Bulked segregant analysis using 849 SSR markers located Pl 17 to linkage group (LG) 4, which is the first DM gene discovered in this linkage group. An F2 population of 186 individuals was screened with polymorphic SSR and SNP primers from LG4. Two flanking markers, SNP SFW04052 and SSR ORS963, delineated Pl 17 in an interval of 3.0 cM. The markers linked to Pl 17 were validated in a BC3 population. A search for the physical location of flanking markers in sunflower genome sequences revealed that the Pl 17 region had a recombination frequency of 0.59 Mb/cM, which was a fourfold higher recombination rate relative to the genomic average. This region can be considered amenable to molecular manipulation for further map-based cloning of Pl 17.

Prevalence of mating type idiomorphs in Pyrenophora teres f. teres and P. teres f. maculata populations from the Canadian prairies

The fungus Pyrenophora teres Drechs. occurs as two morphologically similar but genetically distinct forms, P. teres f. teres (Ptt) and P. teres f. maculata (Ptm), which cause the net form and spot form of net blotch of barley, respectively. A collection of 220 isolates from the Canadian prairie provinces (Alberta, Saskatchewan and Manitoba) was evaluated for mating type (MAT) idiomorph distribution and frequency. Fungal isolates were classified as Ptt or Ptm using form-specific polymerase chain reaction (PCR) primers. PCR analysis with MAT-specific primers indicated that the MAT1 and MAT2 idiomorphs of Ptt and Ptm could be identified within the same field, on the same plant, and on the same leaf. There was no significant departure from the expected 1:1 MAT1/MAT2 ratio for both forms in all three provinces or in the Canadian prairies population as a whole. Polymorphic simple sequence repeat primers were used to detect evidence of possible recombination between the two forms. Cluster analysis revealed that all P. teres isolates, including 30 isolates causing intermediate symptoms, clustered in two distinct groups conforming to either Ptt or Ptm. Therefore, hybridization was not detectable from the 220 isolates collected in western Canada. Pyrenophora teres f. teres is still the dominant form (58%) of the net blotch pathogen, and the data suggest both Ptt and Ptm go through regular cycles of sexual reproduction in the Canadian prairies.

Identification of microsatellite markers linked to thermotolerance in silkworm by bulk segregant analysis and in silico mapping

Silkworm, being a poikilothermic insect, its growth and development is affected by environmental factors especially, temperature. In tropical countries like India, it has considerable effect on silk production due to the prevailing of hot climatic conditions. Previous attempts to evolve silkworm breeds and hybrids tolerant to high temperature by traditional breeding methods have not yielded the desired results. Hence application of new strategies like marker assisted selection (MAS) could be the most effective strategy for developing a thermo-tolerant bivoltine silkworm for sustainable silk production in India. As a prelude, in this study it is aimed to identify simple sequence repeat (SSR) markers closely linked with thermotolerance in silkworm. To do so, 20 silkworm breeds were evaluated at high temperature (36?C) and based on pupation percentage, two multivoltines (Nistari and Cambodge) and two bivoltines (SK4C and BHR3) were identified as thermo-tolerant and one bivoltine (CSR2) was identified as the susceptible breed. These breeds were screened with 85 SSR markers drawn from different linkage groups and out of those, only 11 markers (12.9%) showed distinct polymorphism between thermo-tolerant and susceptible breeds. Further, bulked segregant analysis (BSA) was performed using 11 polymorphic SSR primers, by comparing the SSR profiles of the tolerant (Nistari) and susceptible (CSR2) parents, their F1 and F2 bulks. Nevertheless, only 5 markers generated clear differences in the amplified DNAs between the bulks corresponding to that of the parents suggesting that the DNA regions amplified by these SSR markers are closely linked to thermotolerance in B. mori. The results obtained through bulk segregant analysis was further confirmed by genotyping 5 linked SSR markers using 140 individual F2 progenies. Of these 5 markers, highest Spearman's rho correlation coefficient was shown by S0816 indicating a high degree of closeness between the genotypic and phenotype variations in F2 population. Furthermore, we have also attempted to locate the genes near to S0816 by in silico approach and upshot revealed 3 genes nearer to its sequence on the B. mori genome. The BGIBMGA005249 gene was found to be located nearest to S0816 at a distance of 14.8 Kb. But, further studies are required in this regard to derive a relationship between the thermotolerance and the functional role of identified genes nearer to the closest marker, so that the identified markers can be used to develop a thermo-tolerant silkworm breed through MAS.

Development and Characterization of Simple Sequence Repeat (SSR) Markers Based on RNA-Sequencing of Medicago sativa and In silico Mapping onto the M. truncatula Genome

Sufficient codominant genetic markers are needed for various genetic investigations in alfalfa since the species is an outcrossing autotetraploid. With the newly developed next generation sequencing technology, a large amount of transcribed sequences of alfalfa have been generated and are available for identifying SSR markers by data mining. A total of 54,278 alfalfa non-redundant unigenes were assembled through the Illumina HiSeqTM 2000 sequencing technology. Based on 3,903 unigene sequences, 4,493 SSRs were identified. Tri-nucleotide repeats (56.71%) were the most abundant motif class while AG/CT (21.7%), AGG/CCT (19.8%), AAC/GTT (10.3%), ATC/ATG (8.8%), and ACC/GGT (6.3%) were the subsequent top five nucleotide repeat motifs. Eight hundred and thirty- seven EST-SSR primer pairs were successfully designed. Of these, 527 (63%) primer pairs yielded clear and scored PCR products and 372 (70.6%) exhibited polymorphisms. High transferability was observed for ssp falcata at 99.2% (523) and 71.7% (378) in M. truncatula. In addition, 313 of 527 SSR marker sequences were in silico mapped onto the eight M. truncatula chromosomes. Thirty-six polymorphic SSR primer pairs were used in the genetic relatedness analysis of 30 Chinese alfalfa cultivated accessions generating a total of 199 scored alleles. The mean observed heterozygosity and polymorphic information content were 0.767 and 0.635, respectively. The codominant markers not only enriched the current resources of molecular markers in alfalfa, but also would facilitate targeted investigations in marker-trait association, QTL mapping, and genetic diversity analysis in alfalfa.

SSR FINGERPRINTING OF BLACK RASPBERRY CULTIVARS SHOWS DISCREPANCIES IN IDENTIFICATION

Breeding progress in black raspberry (Rubus occidentalis L.) has been limited by a lack of genetic diversity in elite germplasm. Black raspberry cultivars are noted for showing very few differences, and seedlings for a lack of segregation for important traits. Genetic fingerprinting using microsatellite, or simple sequence repeat (SSR) markers, can reliably identify unique clones and evaluate diversity in black raspberry cultivars. Twenty-one black raspberry cultivars were sampled from the USDA-ARS National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon. Black raspberry clones were also sampled from nurseries, grower’s fields, and other black raspberry breeding programs for comparison. These genotypes were compared using 18 polymorphic SSR primer pairs. The black raspberries 'Bristol,' 'Jewel,' and 'Mac Black' had consistent SSR fingerprints between sources. However, plants being sold as 'Black Hawk' and 'Cumberland' had the same fingerprint as 'Jewel'. Plants of 'Bristol', 'Cumberland', 'Munger', 'New Logan', 'Plum Farmer' and 'Shuttleworth' in the NCGR collection had identical fingerprints. Eleven unique SSR fingerprints were found among plants being grown or sold as ‘Munger’, though there was one predominant fingerprint for this cultivar. 'Allen' and 'John Robertson' were each represented by three different fingerprints from three different sources, and 'Earlysweet' and 'Jewel' had SSR alleles at multiple loci that cannot be explained by their reported pedigrees. While overall genetic diversity in black raspberry cultivars is low, discrepancies in the naming of clones appear to be widespread in commercial and research plantings. Future work in this area should focus on sampling additional independent sources of plant material and evaluating clones to determine the extent of performance differences. Further SSR development in black raspberry may be needed to fingerprint some unique clones.

Construction of Mungbean Genetic Linkage Map

A high-density genetic linkage map with informative markers is essential for plant genome analyses, such as gene mapping, identification of quantitative trait loci, map-based cloning, and physical map construction. A genetic linkage map of mungbean ( Vigna radiata, 2 n = 2 x = 22) was constructed using a recombinant inbred line (RIL) population derived from an inter-subspecific cross between Berken (a bruchid-susceptible cultivar) and ACC41 (a bruchid-resistant genotype belonging to V. radiata subsp. s ublobata). For enhancing the density of the existing linkage map, 104 pairs of polymorphic simple sequence repeat (SSR) primer, were screened out. In combination with the data from other markers, a new genetic linkage map consisting of 179 markers was obtained. They included 97 SSR markers (91 of which were derived from mungbean close relatives), 76 RFLP markers, and 2 STS markers. These markers were assigned to 12 linkage groups with an average distance of 10.2 cM between markers. The transferability of the SSR markers from these relatives of mungbean, including azuki bean ( V. angularis), black gram ( V. mungo), common bean ( Phaseolus vulgaris), and cowpea ( V. unguiculata) was also evaluated. Approximately 65% of SSR markers form azuki bean, 72% from black gram, 42% from common bean, and 30% from cowpea could be effectively amplified in mungbean. These different ratios might reflect different levels of genomic homology between mungbean and the four close relatives. A total of 98 pairs of polymorphic SSR primers from these close relatives were effective in genetic analysis of mungbean. The new linkage map of mungbean was compared with a published linkage map of azuki bean based on 32 common SSR markers. The majority of these markers were mapped in similar orders between these 2 species and a few markers with different orders suggested relative rearrangements between the 2 genomes. As previously reported, a major locus resistant to bruchid was located on linkage group I (9). In this new map, the distances between the bruchid locus and its flanking markers were 8.0 cM (C78) and 2.7 cM (C220), respectively.

Establishment of DNA Fingerprint Identity for Sugarcane Cultivars in Yunnan, China

The purpose of this study was to provide simple sequence repeat (SSR)-based DNA fingerprinting information for sugarcane ( Saccharum spp. Hybrids) cultivars that were developed in Yunan Province, China. Genomic DNAs from 27 cultivars were amplified with 8 pairs of polymorphic SSR primers. A total of 129 bands were obtained, including 123 polymorphic bands (95.35%). The mean value of polymorphic information content (PIC) was 0.9445, and the genetic similarity coefficient ranged from 0.269 to 0.767. Markers SMC1047HA and MSSCIR21 had the highest PIC values of 0.963 and could distinguish all the cultivars tested. Besides, primer combinations MSSCIR36/MSSCIR21, MSSCIR16/MSSCIR36, and MSSCIR36/SMC336BS were efficient in identifying the 27 cultivars, and their similar coefficients were lower than those of other primer combinations. These 3 primer combinations were validated with 27 cultivars and 10 staple commercial sugarcane cultivars in China, and MSSCIR16/MSSCIR36 was found to be optimal to construct DNA fingerprint identity (ID). Nomenclature of DNA fingerprint ID was suggested as serial number of National Nursery of Sugarcane Germplasm Resources (NNSGR) plus country and region code plus English initial abbreviation of breeding institution plus core primer name plus SSR marker data. This system is informative and useful in protecting new varieties and identifying cultivars because it consists of not only important information of a cultivar but also its specific molecular identify.