Simple Sequence Repeat Primer Pairs Research Articles

Development of SSRs Based on the Whole Genome and Screening of Bolting-Resistant SSR Marker in Brassica oleracea L.

Simple sequence repeats (SSRs), also known as microsatellites, stand out as the most crucial molecular markers in both animals and plants owing to their high polymorphism, extensive information content, ease of detection through polymerase chain reaction (PCR) assays, and widespread distribution across the genome. In this study, a total of 125,443 SSR loci were identified from the whole-genome sequence of B. oleracea, and 82,948 primer pairs for SSR have been designed. Furthermore, each primer pair is designated with a unique identifier (ranging from BolSSR00001 to BolSSR82984). Our findings indicated that certain markers within them could be transferred to other cruciferous crops. In addition, a total of 336 pairs of SSR primers have been used to screen the polymorphism between the bolting-resistant and bolting-easy gene pools. After the test of verification with F2 generation individual plants, we obtained an SSR dominant marker, BolSSR040196, linked with bolting-resistant locus in cabbage, and the genetic distance between this SSR marker and the bolting-resistant locus was 10.69 cM. Moreover, BolSSR040196 is located on the C05 chromosome with a CT motif, characterized by a repeat of 9 in bolting-easy plants and 11 in bolting-resistant plants. Haplotype analysis showed that the correct prediction rate reached 82.35%. The BolSSR040196 marker can be used in marker-assisted selection (MAS) breeding, offering a straightforward and efficient approach for bolting-resistant cabbage breeding in the future.

Genetic background of lily germplasm resources based on SSR markers

To study the genetic background of lily (Lilium spp.) germplasm resources, and accurately evaluate and select excellent germplasm for genetic improvement of lily, we analyzed the genetic background of 62 lily germplasm accessions from 11 provinces of China by using simple sequence repeat (SSR) molecular markers. The results showed that 15 out of 83 pairs of lily SSR primers were polymorphic. A total of 157 allelic loci were amplified, with the number of alleles per locus ranging from 5 to 19 and the average number of effective alleles per locus being 4.162 8. The average observed heterozygosity and expected heterozygosity were 0.228 2 and 0.694 1, respectively. The average polymorphic information content was 0.678 8. The average Nei's diversity index and Shannon's information index were 0.694 1 and 1.594 9, respectively, indicating that the tested lily germplasm had high genetic diversity. The 62 germplasm accessions were classified into 5 groups by the unweighted pair group method with arithmetic mean (UPGMA) and into 3 groups by the principal component analysis. The two analyses revealed a geographic correlation among different groups. The majority of lily germplasm accessions from the same source tended to cluster together. The population structure analysis classified the lily accessions into 4 populations and 1 mixed population. The above results provide a theoretical basis and genetic resources for the precise identification and breeding of lily germplasm resources.

Directional Breeding Generates Distinct Genetic Diversity in Hybrid Turf Bermudagrass as Probed with Simple Sequence Repeat Markers

Bermudagrass (Cynodon spp.) is a drought-resistant warm-season turfgrass adapted to the southern and transitional zones in the United States. Multiple hybrid cultivars have been developed and released for use as turfgrass, and others are still undergoing development. Increasing genetic diversity of commercial cultivars is vital to stress tolerance. A DNA profiling study of 21 experimental selections from the Oklahoma State University turfgrass breeding program and 11 cultivars was conducted using 51 simple sequence repeat primer pairs across the bermudagrass genome. A pairwise genetic relationship analysis of the genotypes using 352 polymorphic bands showed genetic similarity coefficients ranging from 0.59 to 0.89. The average pairwise population differentiation values were 0.012 for the 11 cultivars and 0.169 for the 21 selections. A cluster analysis using the unweighted paired group with the arithmetic average method grouped the entries into six clusters. A correlation analysis identified different levels of pairwise genetic relationships among the entries that largely reflected parental relationship. Directional breeding and selection for cold hardiness or drought resistance created progeny that had distinct genetic diversity in the tested bermudagrasses. It is evident that an increase in genetic diversity of the existing cultivar pool with the release of one or more experimental selections for commercial use will strengthen and improve bermudagrass systems.

Development and application of SSR markers of Saposhnikovia divaricata based on transcriptome

Transcriptome sequencing was employed to mine the simple sequence repeat(SSR) locus information of Saposhnikovia divaricata and design specific primers, which aimed to provide a basis for the research on the genetic diversity of S. divaricata germplasm resources. The seed purity, 1 000-seed weight, germination rate, and seed vigor were determined. MISA was used to obtain the SSR locus information from 12 606 unigene longer than 1 kb in the transcriptome database. Forty-three pairs of SSR primers designed in Primer 3 were used to analyze the polymorphism of 28 S. divaricata samples of different sources. The results showed that there were differences in the seed purity, 1 000-seed weight, germination rate, vigor, and seed length and width among S. divaricata samples of different sources. Particularly, the germination rate and seed vigor had significant differences, and HB-ZJK1, NMG-CF4, NMG-BT, NMG-HLE1, and NMG-CF2 had significantly higher 1 000-seed weight, germination rate, and seed vigor than the samples of other sources. Among the 86 233 unigene, 12 606(14.62%) unigene contained 15 958 SSR loci, with one SSR locus every 5 009 bp on average. The SSR loci were mainly single nucleotide and dinucleotide repeats, which were dominated by G/C and TC/AG, respectively. All the primers were screened by using 28 S. divaricata sample from different habitats, and the primers corresponding to the amplification products with clear bands and stable polymorphism were obtained. The clustering results of the biological characteristics and genetic diversity of the 28 S. divaricata samples were basically consistent, and the samples of the same origin(HB-AG1, HB-AG2, HB-ZJK1, and HB-ZJK2) generally gathered together and had close genetic relationship. The SSRs in S. divaricata transcriptome has high frequency, rich types, and high polymorphism, which provides candidate molecular markers for the germplasm identification, genetic map construction, and molecular-assisted breeding.

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.

Changes in Allele Frequencies and Genetic Diversity in Red Clover after Selection for Cold Tolerance Using SSR Markers

The selection of red clover (Trifolium pratense L.) populations adapted to extreme environmental conditions is of great importance due to continuing climate change. The plant material analyzed with simple sequence repeat (SSR) markers included two parent populations, P1 (cultivar ‘Reichersberger’) and P3 (cultivar ‘Croatia’) and their reselections, which were created after one cycle of selection under cold temperature conditions. The reselections PS1 and PS3 were produced by intercrossing 38 surviving plants of parent populations P1 and P3, respectively. A total of 48 plants from each cultivar and each reselection were randomly selected for SSR analysis. Sixteen SSR primer pairs were selected, taking into account the presence of loci on all seven pairs of red clover chromosomes. An increase in the average frequency of alleles from the initial populations to the populations after one cycle of selection was observed, followed by a decrease in the number of alleles. Out of a total of 16 loci, the Waples neutrality test revealed significant frequency changes at 12 loci from P1 to PS1 and 9 loci from P3 to PS3 populations. The genetic diversity in the studied populations did not change significantly after selection, leaving enough genetic variability as a prerequisite for the success of future selection.

Multiple-Genome-Based Simple Sequence Repeat Is an Efficient and Successful Method in Genotyping and Classifying Different Jujube Germplasm Resources.

Jujube (Ziziphus jujuba Mill.) is a commercially important tree native to China, known for its high nutritional value and widespread distribution, as well as its diverse germplasm resources. Being resilient to harsh climatic conditions, the cultivation of jujube could provide a solution to food insecurity and income for people of arid and semi-arid regions in and outside of China. The evaluation of germplasm resources and genetic diversity in jujube necessitates the use of Simple Sequence Repeat (SSR) markers. SSR markers are highly polymorphic and can be used to evaluate the genetic diversity within and between cultivars of Chinese jujube, and are important for conservation biology, breeding programs, and the discovery of important traits for Chinese jujube improvement in China and abroad. However, traditional methods of SSR development are time-consuming and inadequate to meet the growing research demands. To address this issue, we developed a novel approach called Multiple-Genome-Based SSR identification (MGB-SSR), which utilizes the genomes of three jujube cultivars to rapidly screen for polymorphic SSRs in the jujube genome. Through the screening process, we identified 12 pairs of SSR primers, which were then used to successfully classify 249 jujube genotypes. Based on the genotyping results, a digital ID card was established, enabling the complete identification of all 249 jujube plants. The MGB-SSR approach proved efficient in rapidly detecting polymorphic SSRs within the jujube genome. Notably, this study represents the first successful differentiation of jujube germplasm resources using 12 SSR markers, with 4 markers successfully identifying triploid jujube genotypes. These findings offer valuable information for the classification of Chinese jujube germplasm, thereby providing significant assistance to jujube researchers and breeders in identifying unknown jujube germplasm.

Construction of Longan (Dimocarpus longan Lour.) Simple Sequence Repeat Fingerprints and Application in Distinctness, Uniformity and Stability Testing

Simple sequence repeat (SSR) markers are highly polymorphism, good reproducibility, abundant number and co-dominant inheritance, and were considered to be one of the preferred molecular markers for DNA fingerprints in distinctness, uniformity and stability (DUS) testing. In this study, 10 representative Dimocarpus longan Lour (longan) varieties with significant differences were selected from 63 longan varieties according to the morphological characteristics. Based on PCR amplifications of the 10 selected varieties, 24 SSR primers pairs were screened from total 300 SSR primers pairs, to establish SSR fingerprints for all 63 longan varieties. The results showed that a total of 127 alleles were detected in 63 longan varieties, with an average of 5.29 alleles for each pair of primers. The Shannon’s index of the 24 pairs of SSR markers ranged from 0.64 to 1.58, with an average of 1.20. The polymorphism information content of each locus ranged from 0.32 to 0.72, with an average of 0.58. Clustering analysis indicated that most of the varieties with close genetic relationships tended to fall in the same cluster, and only a few in different clusters or sub-clusters. The 63 longan varieties were completely identified by the optimal combination of 6 pairs of SSR primers (LY161, LY252, LY137, LY130, LY25 and LY34). Overall, DNA fingerprints of the 63 longan varieties were constructed based on these 24 pairs of SSR primers. This study may provide a technical support for variety identification and similar variety screening in DUS detection in longan.

Genetic Diversity and Association Mapping of Grain-Size Traits in Rice Landraces from the Honghe Hani Rice Terraces System in Yunnan Province.

The Honghe Hani Rice Terraces System (HHRTS) of Yunnan Province is an important agricultural and cultural heritage landscape. Until now, a large number of local rice landraces have been planted. Mining excellent genes contained in these landraces provides a reference for variety improvement and new variety breeding. In this study, 96 rice landraces collected from the Hani terraces were planted in Honghe Mengzi, Yunnan Province, in 2013, 2014, 2015, and 2021, and five major grain traits were measured and analyzed. The genomic variation of 96 rice landraces was scanned by 201 simple sequence repeat (SSR) markers. The genetic diversity, population structure, and genetic relationships of the natural population were analyzed. The mixed linear model (MLM) method of the TASSEL software was used to analyze the associations between markers and traits. A total of 936 alleles were amplified by 201 pairs of SSR primers. The average number of observed alleles (Na), the effective number of alleles (Ne), Shannon's information index (I), heterozygosity (H), and the polymorphism information content (PIC) per marker were 4.66, 2.71, 1.08, 0.15, and 0.55, respectively. Ninety-six landraces were divided into two groups by population structure, clustering, and principal component analysis, and indica rice was the main group. The coefficients of variation of the five traits ranged from 6.80 to 15.24%, and their broad heritabilities were more than 70%. In addition, there were positive correlations among the same grain traits between different years. Through MLM analysis, 2, 36, 7, 7, and 4 SSR markers were significantly associated with grain length (GL), grain width (GW), grain thickness (GT), grain length-width ratio (LWR), and thousand-grain weight (TGW), respectively. The explanation rates of phenotypic variation were 16.31 (RM449, Chr. 1)-23.51% (RM316, Chr. 9), 10.84 (RM523, Chr. 3; RM161/RM305, Chr. 5)-43.01% (RM5496, Chr. 1), 11.98 (RM161/RM305, Chr. 5)-24.72% (RM275, Chr. 6), 12.68 (RM126, Chr. 8)-36.96% (RM5496, Chr. 1), and 17.65 (RM4499, Chr. 2)-26.32% (RM25, Chr. 8), respectively. The associated markers were distributed on 12 chromosomes of the genome.

Microsporogenesis in the triploid hybrid ‘Beilinxiongzhu 1#’ and detection of primary trisomy in 2x × 3 × Populus hybrids

BackgroundPrimary trisomy is a powerful genetic tool in plants. However, trisomy has not been detected in Populus as a model system for tree and woody perennial plant biology.ResultsIn the present study, a backcross between Populus alba × Populus glandulosa ‘YXY 7#’ (2n = 2x = 38) and the triploid hybrid ‘Beilinxiongzhu 1#’ (2n = 3x = 57) based on the observation of microsporogenesis and an evaluation of the variations in pollen was conducted to create primary trisomy. Many abnormalities, such as premature migration of chromosomes, lagging of chromosomes, chromosome bridges, asymmetric separation, micronuclei, and premature cytokinesis, have been detected during meiosis of the triploid hybrid clone ‘Beilinxiongzhu 1#’. However, these abnormal behaviors did not result in completely aborted pollen. The pollen diameter of the triploid hybrid clone ‘Beilinxiongzhu 1#’ is bimodally distributed, which was similar to the chromosomal number of the backcross progeny. A total of 393 progeny were generated. We provide a protocol for determining the number of chromosomes in aneuploid progeny, and 19 distinct simple sequence repeat (SSR) primer pairs covering the entire Populus genome were developed. Primary trisomy 11 and trisomy 17 were detected in the 2x × 3 x hybrid using the SSR molecular markers and counting of somatic chromosomes.ConclusionsNineteen distinct SSR primer pairs for determining chromosomal number in aneuploid individuals were developed, and two Populus trisomies were detected from 2x × 3 x hybrids by SSR markers and somatic chromosome counting. Our findings provide a powerful genetic tool to reveal the function of genes in Populus.

Identification of F1 Hybrid Progenies in Mango Based on Fluorescent SSR Markers

Mango (Mangifera indica L.) belongs to the genus Mangifera and family Anacardiaceae, and is an important tropical fruits. Artificial cross breeding (hand pollination) is an important method for breeding new mango cultivars. It is easy to produce false hybrids in the process of artificial pollination breeding. Therefore, it is necessary to establish rapid and accurate molecular detection methods to identify the authenticity of hybrids. Mango ‘Jinhuang’ and ‘Renong No.1′ and 65 individual plants of their F1 hybrids were used as experimental materials, eight SSRs (simple sequence repeats) primer pairs with polymorphism in parents were used to identify the F1 hybrids by capillary electrophoresis. The results showed that PCR product size (bp) for eight primers ranged from 108 bp (ES55) to 176 bp (ES63) in 65 samples. A total of 62 true hybrids were identified from 65 hybrid progenies, and the true hybrid rate was 95.38%. A total of 18 alleles were amplified by eight SSRs, seven SSR loci showed binary segregations, whereas only one SSR locus ES83 showing ab:ac:bb:bc segregation fitted to the expected segregation ratio of 1:1:1:1. The value of expected heterozygosity (He), ranged from 0.34 to 0.62, whereas the value of observed heterozygosity ranged from 0.44 to 0.81. Chi-square test showed that eight SSR loci were in accordance with Mendel’s segregation law. The results of cluster analysis showed that the parents and 62 true hybrids could be classified into two categories at 0.58: the first category contained 27 offspring, clustered with ‘Jinhuang’ and showed a maternal genetic tendency. The second category contained 35 offspring, clustered with ‘Renong No.1′ and showed a partial paternal genetic tendency. DNA fingerprint of hybrids from ‘Jinhuang’ × ‘Renong No.1′ cross were constructed using eight SSR primers for variety protection.

Occurrence of simple sequence repeats in cDNA sequences of safflower (Carthamus tinctorius) reveals the importance of SSR-containing genes for cell biology and dynamic response to environmental cues.

Safflower (Carthamus tinctorius) is a diploid crop plant belonging to the family Asteraceae and is well known as one of important oilseed crops due to edible oil containing unsaturated fatty acids. In recent years it is gaining increased attention for food, pharmaceutical and industrial uses, and hence the updating its breeding methods is necessary. Genic simple sequence repeats (SSRs) in addition of being desire molecular markers, are supposed to influence gene function and the respective phenotype. This study aimed to identify SSRs in cDNA sequences and further analysis of the functional features of the SSR-containing genes to elucidate their role in biological and cellular processes. We identified 1,841 SSR regions in 1,667 cDNA sequences. Among all types of repeats, trinucleotide repeats were the most abundant (35.7%), followed by hexanucleotide (29.6%) and dinucleotide repeats (22.0%). Thirty five SSR primer pairs were validated by PCR reaction, detected a high rate of polymorphism (>57%) among safflower accessions, physically mapped on safflower genome and could clearly discriminate the cultivated accessions from wild relatives. The cDNA-derived SSR markers are suitable for evaluation of genetic diversity, linkage and association mapping studies and genome-based breeding programmes. Occurrence of SSR repeats in biologically-important classes of proteins such as kinases, transferases and transcription factors was inferred from functional analyses, which along with variability of their repeat copies, can endow the cell and whole organism the flexibility of facing with continuously changing environment, and indicate a structure-based evolution mechanism of the genome which acts as an up-to-dating tool for the cell and whole origanism, which is realized in GO terms such as involvement of most SSR-containing genes in biological, cellular and metabolic processes, especially in response to stimulus, response to stress, interaction to other organisms and defense responses.

Genetic Diversity and Population Structure of Hemiculter leucisculus (Basilesky, 1855) in Xinjiang Tarim River.

Hemiculter leucisculus is an invasive fish and widely distributed in the Xinjiang Tarim River. In this study, RAD-seq was used to explore the genetic diversity and population subgroup structure of H. leucisculus in the Tarim River and develop relevant Simple Sequence Repeat (SSR) markers. The study collected 40 samples distributed at four different sites of the Tarim River. A total of 7,291,260 single nucleotide polymorphisms (SNPs) were obtained. The genetic diversity results showed that the population genetic diversity level of H. leucisculus was low. The population pairwise FST values ranged from 0.231 to 0.258, indicating that there was moderate genetic differentiation among these populations. AMOVA showed that the genetic variation within populations accounted for 92.31% of the total variation. The principal component analysis (PCA) and neighbor joining (NJ) tree revealed that the four populations could be separated into two clusters (upper-middle and downstream populations) and the individuals from Taitema Lake (TTMH) showed differences and had a bigger geographic distance than the others. There is the probability that the H. leucisculus from Bosten Lake entered Taitema Lake to breed and then expanded into the Tarim River due to the water diversion projects in location. In addition, 147,705 SSRs loci were detected and 22,651 SSR primer pairs were developed. This study will contribute to providing valuable molecular data for the management of wild populations, marker-assisted selection and resource exploitation of H. leucisculus.

Genetic Evaluation of In Vitro Micropropagated and Regenerated Plants of Cannabis sativa L. Using SSR Molecular Markers.

Simple sequence repeat (SSR) markers were used to evaluate the genetic stability of the acclimatized micropropagated and regenerated plants of a high cannabidiol (H-CBD) and a high cannabigerol (H-CBG) variety of Cannabis sativa L. Shoot regeneration and proliferation were achieved by culturing calli in Murashige and Skoog basal medium (MS) supplemented with several concentrations of 6-benzyladenine (BA) or thidiazuron (TDZ). Calli derived mostly from stem explants, rather than leaves, cultured on MS supplemented with 2,4-Dichlorophenoxyacetic acid (2,4-D) or combination of kinetin (KIN) with 1-Naphthaleneacetic acid (NAA) or 2,4-D. Rooting of the regenerated plantlets accomplished on half-strength MS medium supplemented with indole-3-butyric acid (IBA). Previous studies performed have developed an efficient in vitro micropropagation protocol for mass production. Both in vitro methodologies can be employed in genetic breeding via molecular techniques. The genetic stability of micropropagated and regenerated plants was accomplished using twelve SSR primer pairs that produced reproducible and clear bands, ranging from 90 to 330 bp in size, and resulted in amplification of one or two alleles, corresponding to homozygous or heterozygous individuals. The SSR amplification products were monomorphic across all the micropropagated and regenerated plants and comparable to mother plants. The monomorphic banding pattern confirmed the genetic homogeneity of the in vitro cultured acclimatized and mother plants as no somaclonal variation was detected in clones for these specific SSRs. Our results evidently suggest that the developed culture protocols for in vitro multiplication is appropriate and applicable for clonal mass propagation of the C. sativa varieties and demonstrate the reliability of this in vitro propagation system.

Development and characterisation of SSR markers in the potato rot nematode Ditylenchus destructor

Summary The potato rot nematode, Ditylenchus destructor, causes serious disease limiting the production of many crops. This disease usually decreases sweet potato yield by 20-50%, and in heavily infested fields the crop may be completely lost. Although the nematode has economic importance in China, its transmission route and genetic diversity are unknown. In this study, a collection of 1761 contigs of the D. destructor genome was mined for simple sequence repeat (SSR) markers, which resulted in the identification of 9745 SSRs. A total of 150 pairs of SSR primers were further developed and used for validation of the amplification rate and assessment of the polymorphism. Nine SSR markers were finally identified and analysed using 96 individual specimens of D. destructor sampled from four provinces in China. These loci were found to be moderately polymorphic with 2-8 alleles per locus. The observed and expected heterozygosity across the four populations ranged from 0.000 to 0.833 and from 0.000 to 0.666, respectively. This is the first report of the development and characterisation of genomic SSR markers in D. destructor. Our study demonstrated the obvious gene differentiation among different populations of D. destructor in China. This suggests that D. destructor in China may have been introduced from multiple origins. Much more work is needed on this species to identify patterns of spread, and the microsatellite loci we develop here should be useful in many regions for modelling range expansion, studying the evolution of resistance, and increasing the effectiveness of pest management strategies.

Use of SSR Markers for the Exploration of Genetic Diversity and DNA Finger-Printing in Early-Maturing Upland Cotton (Gossypium hirsutum L.) for Future Breeding Program

DNA fingerprinting and genetic diversity analysis of 79 early-maturing upland cotton (Gossypium hirsutum L.) cultivars were performed using Simple Sequence Repeat (SSR) molecular markers. Out of 126 pairs of SSR primers, we selected 71 pairs that gave good polymorphisms and clear bands, had good stability, and showed even distribution on the cotton chromosomes, and 142 polymorphic genotypes were amplified. The average number of alleles amplified with the SSR primers was 2.01. The polymorphism information content (PIC) of the markers ranged from 0.1841 to 0.9043, with an average of 0.6494. The results of fingerprint analysis showed that nine varieties had characteristic bands, and at least six primer pairs could be used to completely distinguish all 79 cotton accessions. Using NTSYS-pc 2.11 cluster analysis, the genetic similarity coefficients between the cotton genotypes ranged from 0.3310 to 0.8705, with an average of 0.5861. All cotton accessions were grouped into five categories at a similarity coefficient of 0.57, which was consistent with the pedigree sources. At the same time, the average genetic similarity coefficients of early-maturing upland cotton varieties in China showed a low-high-low pattern of variation over time, revealing the development history of early-maturing upland cotton varieties from the 1980s to the present. This also indirectly reflects that in recent years, China’s cotton breeders have focused on innovation and have continuously broadened the genetic resources for early-maturing upland cotton.

Development and Application of Fruit Color-Related Expressed Sequence Tag-Simple Sequence Repeat Markers in Abelmoschus esculentus on the Basis of Transcriptome Sequencing.

Abelmoschus esculentus is a medicinal and edible plant that contains large amounts of active ingredients, including anthocyanins, polysaccharides, flavonoids, and terpenoids. However, because of a relative lack of molecular research, there are few molecular markers applicable for this plant species. In this study, on the basis of A. esculentus fruit color-related transcriptome sequencing data, we analyzed the patterns of simple sequence repeats (SSRs) in differentially expressed genes (DEGs) and revealed the biological processes and metabolic pathways associated with the related genes. We also designed primers for SSR loci to develop SSR molecular markers. Primers were synthesized using a DEG associated with a protein–protein interaction network. Polymorphic SSR markers were screened for the subsequent examination of A. esculentus germplasm resources and fruit color association analysis. The results indicated that 24.98% of the unigenes contained SSR motifs. Single-base (mononucleotide) repeats were the main SSRs, followed by trinucleotide and dinucleotide repeats. We selected 47 expressed sequence tag (EST)-SSR primer pairs for the genotyping of 153 A. esculentus varieties/lines. We ultimately obtained 21 EST-SSR markers suitable for genotyping. A generalized linear model-based association analysis detected two EST-SSR markers significantly associated with A. esculentus fruit color. In conclusion, several EST-SSR and SSR molecular markers in A. esculentus were developed in this study. The fruit color-associated markers may be useful for the molecular marker-assisted breeding of new A. esculentus varieties.

Genome-Wide Development of Polymorphic Microsatellite Markers and Association Analysis of Major Agronomic Traits in Core Germplasm Resources of Tartary Buckwheat.

Tartary buckwheat (TB; Fagopyrum tataricum Gaertn.) is an important multigrain crop and medicinal plant, but functional genomics and molecular breeding research in this species have been lacking for quite some time. Here, genome-wide screening was performed to develop simple sequence repeat (SSR) markers associated with six major agronomic traits and the rutin contents of 97 core germplasm resources. A total of 40,901 SSR loci were identified; they were uniformly distributed throughout the TB genome, with a mean distance of 11 kb between loci. Based on these loci, 8,089 pairs of SSR primers were designed, and 101 primer pairs for polymorphic SSR loci were used to genotype the 97 core germplasm resources. The polymorphic SSR loci showed high genetic variation in these core germplasm resources, with an average polymorphic information content (PIC) value of 0.48. In addition, multiple SSR markers, such as SXAU8002 [100-grain weight (HGW)] and SXAU8006 [stem diameter (SD)], were found to be associated with agronomic traits in the two environments. Finally, based on gene functional annotation and homology analysis, a candidate gene, FtPinG0007685500, that may affect the node number and SD of the main stem by participating in lignin synthesis was identified. This study reports the mining of genome-wide SSR loci and the development of markers in TB, which can be used for molecular characterization of the germplasm in its gene pool. In addition, the detected markers and candidate genes could be used for marker-assisted breeding and functional gene cloning in TB.

Microsatellite analysis in some watermelon (Citrullus lanatus) genotypes

Conservation of genetic resources is essential for the continuation of future crop production. Watermelon (Citrul-lus lanatus), a member of Cucurbitaceae, is widely distributed in tropical and subtropical regions. The aim of this study was to reveal the genetic relationships with the help of microsatellite markers in a watermelon collection free of unnecessary repetitions, and to determine the success of SSR (Simple Sequence Repeats) primers developed in cucurbits. In this study, 96 watermelon genotypes with good agronomic characteristics were used among the geno types collected from different regions of Turkey and purified up to the S4-S6 (self-pollination) stage. In the study, 33 SSR primer pairs were used to determine the genetic relationship between watermelon genotypes. In the study, a total of 67 bands were obtained with SSR primers. As a result of UPGMA (Unweighted Pair Group Method Using Arithmetic Averages) analysis, genotypes showed similarity at the level of 0.84-1.00. The number of alleles detected per primer varied between 1 and 6. In terms of the total number of alleles obtained, CMCT44 (5 units) and Cgb4767 (6 units) loci produced the most alleles. Primers with high polymorphism rate and allele excess were determined, and the possibilities for use in genetic stability analyses, variety differentiation and other genetic analyses were determined.

Evaluation of genetic diversity and drought tolerance among thirty-three dichondra (<i>Dichondra repens</i>) genotypes

Dichondra (<italic>Dichondra repens</italic>) is an important ground cover plants and is also used as an herbal medicine in China. Objectives of this study were to evaluate phenotypic and genetic diversities among 33 genotypes by using 18 simple sequence repeat (SSR) markers and to further identify the drought tolerance of these germplasms based on five physiological parameters. Results showed that natural variations in phenotypes including plant height, leaf area, leaf thickness, and petiole length were observed among 33 genotypes under well-watered condition. All 18 SSR primer pairs were found to be polymorphic and significant genetic variation was found in these genotypes. In addition, there were obvious differences in leaf relative water content (RWC), electrolyte leakage (EL), chlorophyll (Chl) content, photochemical efficiency (Fv/Fm), and performance index on absorption basis (PI_ABS) among 33 genotypes in response to a prolonged period of drought stress (46 days). Drought tolerance of 33 genotypes was then ranked by using subordinate function value analysis (SFVA) and the most drought-tolerant or -sensitive genotypes were identified as Dr5 or Dr29, respectively. Principal component analysis (PCA) further classified 33 genotypes into group I (drought-tolerant), group II (drought-sensitive), and group III (medium types). Current findings showed that 18 selected SSR primers could be potentially used to analyze genetic diversity and varietal identification in dichondra species. Drought-tolerant wild dichondra resources provide a rich genetic base for breeding of new cultivars.