Simple Sequence Repeat Primer Pairs Research Articles

Development of Simple Sequence Repeat of Monochamus alternatus (Coleoptera: Cerambycidae) Based on Restriction Site-Associated DNA Sequencing.

Monochamus alternatus, a pest posing a serious threat to coniferous species, such as Pinus massoniana, has had devastating effects on pine forests due to its association with Bursaphelenchus xylophilus. The creation of unique simple sequence repeat (SSR) primers for M. alternatus is crucial, as there has been little study of the species' phylogeography. The aim of this study was to identify and create polymorphic SSR primers by sequencing samples of M. alternatus obtained from three different sampling points using the restriction site-associated DNA sequencing (Red-seq) approach. Subsequently, supplementary samples were integrated, and genetic typing was performed utilizing the identified polymorphic primers. Through comprehensive analysis, a total of 95,612 SSR loci were identified. Among these, mononucleotide repeats (51.43%), dinucleotide repeats (28.79%), and trinucleotide repeats (16.74%) predominated among the SSR motif types. Ultimately, 18 pairs of SSR primers were screened out, demonstrating stable amplification and high polymorphism. Genetic typing revealed that the mean number of alleles (Na) for these primer pairs ranged from 3 to 8, observed heterozygosity (Ho) ranged from 0.133 to 0.733, polymorphic information content (PIC) ranged from 0.294 and 0.783, and Shannon's index (I) ranged from 0.590 to 1.802. This study effectively produced 16 pairs of SSR primers that can be applied to different populations of M. alternatus. As a result, important tools for furthering studies on the phylogeography of pine wood nematodes, creating genetic maps, gene mapping, and carrying out in-depth investigations into gene function have been made available.

Simple Sequence Repeat Marker-Based Genetic Diversity and Chemical Composition Analysis of Ancient Camellia sinensis in Jiulong County, Sichuan Province, China.

The ancient tea plant germplasm resources are rich in genetic diversity and provide an important basis for the genetic diversity in tea germplasm resources. To explore the genetic diversity of ancient tea plant germplasm resources in Jiulong County, Sichuan Province. 59 ancient tea tree germplasm resources were analyzed using simple sequence repeat (SSR) molecular markers and chemical composition analysis. The results showed that a total of 83 alleles were amplified by 23 pairs of SSR primers, with an average observed allele number (Na) of 3.6 and an effective allele number (Ne) of 2.335. The average Shannon information index (I) and the polymorphic information content (PIC) of the primers were 0.896 and 0.446, respectively. The results of the UPGMA cluster analysis showed that 59 ancient tea tree samples could be classified into five different subgroups. Based on the results of chemical composition analysis, two specific tea germplasm resources with high amino acid content, 10 excellent germplasm resources with tea polyphenol content over 20% and some other tea germplasm resources were identified. This study reveals that Jiulong's ancient tea tree germplasm exhibits significant genetic diversity and includes valuable tea tree planting resources. These findings provide a foundational framework for the conservation, detailed exploration and sustainable utilization of these resources.

Genetic Diversity Analysis and Fingerprint Construction for 87 Passionfruit (Passiflora spp.) Germplasm Accessions on the Basis of SSR Fluorescence Markers

A comprehensive genetic diversity analysis of 87 Passiflora germplasm accessions domesticated and cultivated for several years in the karst region of Guizhou, China, was conducted utilizing simple sequence repeat (SSR) fluorescent markers. These Passiflora species, renowned for their culinary and medicinal value, could bring significant economic and ecological benefits to the region. This study aimed to assess the genetic resources of these species and facilitate the selection of superior cultivars adapted to the karst environment. Our analysis revealed an abundance of SSR loci within the Passiflora transcriptome, with single-base repeats being the most prevalent type. Through rigorous primer screening and amplification, we successfully identified 27 SSR primer pairs exhibiting robust polymorphisms. Further interrogation at eight microsatellite loci revealed 68 alleles, underscoring the high level of genetic diversity present in the cultivated accessions. The average expected heterozygosity was 0.202, with the ssr18 locus exhibiting the highest value of 0.768, indicating significant genetic variation. The mean polymorphic information content (PIC) of 0.657 indicates the informativeness of these SSR markers. Comparative analyses of the cultivated and potential wild progenitors revealed distinct genetic variations among the different Passiflora types. Genetic structure and clustering analyses of the 87 accessions revealed seven distinct groups, suggesting gene flow and similarities among the resources. Notably, a DNA fingerprinting system was established using eight SSR primer pairs, effectively distinguishing the selected cultivars that had adapted to the karst mountainous region. This study not only deepens our understanding of Passiflora genetic resources in the karst environment but also provides a valuable reference for conservation, genetic improvement, and cultivar selection. The rich genetic diversity of the Passiflora germplasm underscores their potential for sustainable utilization in breeding programs aimed at enhancing the economic and ecological viability of these valuable plant species.

Establishment of Novel Simple Sequence Repeat (SSR) Markers from Chimonanthus praecox Transcriptome Data and Their Application in the Identification of Varieties.

Chimonanthus praecox, a member of the Calycanthaceae family, is a unique, traditional, and famous flowering economic tree species in China. Despite the existence of several varieties, only a few cultivars have been formally named. Currently, expression sequence tag-simple sequence repeat (EST-SSR) markers are extensively used to identify different species and varieties; a large number of microsatellites can be identified from transcriptome databases. A total of 162,638 unigenes were assembled using RNA-seq; 82,778 unigenes were annotated using the Nr, Nt, Swiss-Prot, Pfam, GO, KOG, and KEGG databases. In total, 13,556 SSR loci were detected from 11,691 unigenes, with trinucleotide repeat motifs being the most abundant among the six repeat motifs. To develop the markers, 64,440 pairs of SSR primers with polymorphism potential were designed, and 75 pairs of primers were randomly selected for amplification. Among these markers, seven pairs produced amplified fragments of the expected size with high polymorphism. Using these markers, 12 C. praecox varieties were clustered into two monophyletic clades. Microsatellites in the transcriptome of C. praecox exhibit rich types, strong specificity, and great polymorphism potential. These EST-SSR markers serve as molecular technical methods for identifying different varieties of C. praecox and facilitate the exploration of a large number of candidate genes associated with important traits.

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.