A pipeline for effectively developing highly polymorphic SSR markers based on multi-sample genomic data

Simple sequence repeats (SSRs) are widely used genetic markers in ecology, evolution and conservation even in the genomics era, while a general limitation to their application is the difficulty of developing polymorphic SSR markers. Next-generation sequencing (NGS) offers the opportunity for the rapid development of SSRs; however, previous studies developing SSRs using genomic data from only one individual need redundant experiments to test the polymorphisms of SSRs. In this study, we designed a pipeline for the rapid development of polymorphic SSR markers from multi-sample genomic data. We used bioinformatic software to genotype multiple individuals using resequencing data, detected highly polymorphic SSRs prior to experimental validation, significantly improved the efficiency and reduced the experimental effort. The pipeline was successfully applied to a globally threatened species, the brown-eared pheasant (Crossoptilon mantchuricum), which showed very low genomic diversity. The 20 newly developed SSR markers were highly polymorphic, the average number of alleles was much higher than the genomic average. We also evaluated the effect of the number of individuals and sequencing depth on the SSR mining results, and we found that ten individuals and ~10X sequencing data were enough to obtain a sufficient number of polymorphic SSRs, even for species with low genetic diversity. Furthermore, the genome assembly of NGS data from the optimal number of individuals and sequencing depth can be used as an alternative reference genome if a high-quality genome is not available. Our pipeline provided a paradigm for the application of NGS technology to mining and developing molecular markers for ecological and evolutionary studies.

Genome-wide identification of simple sequence repeats and development of polymorphic SSR markers in swamp eel (Monopterus albus)

Objectives: Swamp eel is one model species for sexual reversion and an aquaculture fish in China. One local strain with deep yellow and big spots of Monopterus albus has been selected for consecutive selective breeding. The objectives of this study were characterizing the Simple Sequence Repeats (SSRs) of M. albus in the assembled genome obtained recently, and developing polymorphic SSRs for future breeding programs. Methods: The genome wide SSRs were mined by using MISA software, and their types and genomic distribution patterns were investigated. Based on the available flanking sequences, primer pairs were batched developed, and Polymorphic SSRs were identified by using Polymorphic SSR Retrieval tool. The obtained polymorphic SSRs were validated by using e-PCR and capillary electrophoresis, then they were used to investigate genetic diversity of one breeding population. Results: A total of 364,802 SSRs were identified in assembled M. albus genome. The total length, density and frequency of SSRs were 8,204,641 bp, 10,259 bp/Mb, and 456.16 loci/Mb, respectively. Mononucleotide repeats were predominant among SSRs (33.33%), and AC and AAT repeats were the most abundant di- and tri-nucleotide repeats motifs. A total of 287,189 primer pairs were designed, and a high-density physical map was constructed (359.11 markers per Mb). A total of 871 polymorphic SSRs were identified, and 38 SSRs of 101 randomly selected ones were validated by using e-PCR and capillary electrophoresis. Using these 38 polymorphic SSRs, 201 alleles were detected and genetic diversity level (Na, PIC, HO, and He) was evaluated. Conclusions: The genome-wide SSRs and newly developed SSR markers will provide a useful tool for genetic mapping, diversity analysis studies in swamp eel in the future. The high level of genetic diversity (Na = 5.29, PIC = 0.5068, HO = 0.4665, He = 0.5525) but excess of homozygotes ( FIS = 0.155) in one breeding population provide baseline information for future breeding program.

Application of Microsatellite and Inter Simple Sequence Markers for Certifying Trueness to Type of Grafted Ramets and Clustering of Persian Walnut Varieties

The utility of seventeen Microsatellite (SSR) markers and fifteen inter simple sequence repeats (ISSR) markers for the identification of twenty eight ramets of 11 varieties of walnut (Juglans regia) was explored. Thirty nine individual genomes were screened using 61 and 38 scorable fragments from SSR and ISSR markers, respectively. The least polymorphic SSR locus was WGA004 (two alleles) and the most polymorphic (5 alleles) was WGA276. Polymorphism information content values ranged from 0.08 (WGA004) to 0.43 (WGA032) in SSR markers and from 0.11 (AGA (AC)7) to 0.49 (CAC(TGT)5) in ISSR markers, with an average of 0.29 and 0.19, respectively. In most cases, grafted varieties with identical names also had the same microsatellites profile. The principal coordinate analysis and clustering (UPGMA) based on the combined marker set emphasized two failures in grafting or off-types, ramets identified as Serr 4 (S4) and Vina 1 (V1). The presence of two off-type ramets in the walnut research orchard emphasizes the importance of using molecular certification for proving true-to-type of walnut orchards. Using 13 polymorphic SSRs, we tabulated a DNA fingerprint chart of 11 walnut varieties. Except for ‘Chandler’, each cultivar could be distinguished using a combination of only two SSR loci. The 13 SSRs markers evaluated in this study could be used in future to identify clones produced from the varieties.

Heterotic pools in African and Asian origin populations of pearl millet [Pennisetum glaucum (L.) R. Br.]

Forty-five African or Asian origin pearl millet populations bred either in Africa or Asia were investigated to generate information on heterotic pools. They were clustered into seven groups (G1 to G7) when genotyped, using 29 highly polymorphic SSRs. Fourteen parental populations representing these seven marker-based groups were crossed in diallel mating design to generate 91 population hybrids. The hybrids evaluated at three locations in India showed mean panmictic mid-parent heterosis (PMPH) and better-parent heterosis (PBPH) for grain yield ranging from − 21.7 to 62.08% and − 32.51 to 42.99%, respectively. Higher grain yield and heterosis were observed in G2 × G6 (2462 kg ha−1, 43.2%) and G2 × G5 (2455 kg ha−1, 42.8%) marker group crosses compared to the most popular Indian open-pollinated variety (OPV) ICTP 8203. Two heterotic groups, Pearl millet Population Heterotic Pool-1 (PMPHP-1) comprising G2 populations and Pearl millet Population Heterotic Pool-2 (PMPHP-2) comprising G5 and G6 populations, were identified based on hybrid performance, heterosis and combining ability among marker group crosses. Population hybrids from two heterotic groups, PMPHP-1 × PMPHP-2 demonstrated PMPH of 14.75% and PBPH of 6.8%. Populations of PMPHP-1 had linkages with either African or Asian origin populations, whereas PMPHP-2 composed of populations originating in Africa and later bred for Asian environments. Results indicated that parental populations from the two opposite heterotic groups can be used as base populations to derive superior inbred lines to develop high yielding hybrids/cultivars.

Comprehensive Characterization and Validation of Chromosome-Specific Highly Polymorphic SSR Markers From Pomegranate (Punica granatum L.) cv. Tunisia Genome

The simple sequence repeat (SSR) survey of ‘Tunisia’ genome (296.85 Mb) identified a total of 365,279 perfect SSRs spanning eight chromosomes, with a mean marker density of 1,230.6 SSRs/Mb. We found a positive trend in chromosome length and the SSR abundance as marker density enhanced with a shorter chromosome length. The highest number of SSRs (60,708) was mined from chromosome 1 (55.56 Mb), whereas the highest marker density (1,294.62 SSRs/Mb) was recorded for the shortest chromosome 8 (27.99 Mb). Furthermore, we categorized all SSR motifs into three major classes based on their tract lengths. Across the eight chromosomes, the class III had maximum number of SSR motifs (301,684, 82.59%), followed by the class II (31,056, 8.50%) and the class I (5,003, 1.37%). Examination of the distribution of SSR motif types within a chromosome suggested the abundance of hexanucleotide repeats in each chromosome followed by dinucleotides, and these results are consistent with ‘Tunisia’ genome features as a whole. Concerning major repeat types, AT/AG was the most frequent (14.16%), followed by AAAAAT/AAAAAG (7.89%), A/C (7.54%), AAT/AAG (5.23%), AAAT/AAAG (4.37%), and AAAAT/AAAAG (1.2%) types. We designed and validated a total of 3,839 class I SSRs in the ‘Tunisia’ genome through electronic polymerase chain reaction (ePCR) and found 1,165 (30.34%) SSRs producing a single amplicon. Then, we selected 906 highly variable SSRs (> 40 nt) from the ePCR-verified class I SSRs and in silico validated across multiple draft genomes of pomegranate, which provided us a subset of 265 highly polymorphic SSRs. Of these, 235 primers were validated on six pomegranate genotypes through wet-lab experiment. We found 221 (94%) polymorphic SSRs on six genotypes, and 187 of these SSRs had ≥ 0.5 PIC values. The utility of the developed SSRs was demonstrated by analyzing genetic diversity of 30 pomegranate genotypes using 16 HvSSRs spanning eight pomegranate chromosomes. In summary, we developed a comprehensive set of highly polymorphic genome-wide SSRs. These chromosome-specific SSRs will serve as a powerful genomic tool to leverage future genetic studies, germplasm management, and genomics-assisted breeding in pomegranate.

Genome-Wide Characterization and Comparative Analyses of Simple Sequence Repeats among Four Miniature Pig Breeds

Simple sequence repeats (SSRs) are commonly used as molecular markers in research on genetic diversity and discrimination among taxa or breeds because polymorphisms in these regions contribute to gene function and phenotypically important traits. In this study, we investigated genome-wide characteristics, repeat units, and polymorphisms of SSRs using sequencing data from SSR-enriched libraries created from Wuzhishan (WZS), Bama (BM), inbred Luchuan (LC) and Zangxiang (ZX) miniature pig breeds. The numbers and types of SSRs, distributions of repeat units and polymorphic SSRs varied among the four breeds. Compared to the Duroc pig reference genome, 2518 polymorphic SSRs were unique and common to all four breeds and functional annotation revealed that they may affect the coding and regulatory regions of genes. Several examples, such as FGF23, MYF6, IGF1R, and LEPROT, are associated with growth and development in pigs. Three of the polymorphic SSRs were selected to confirm the polymorphism and the corresponding alleles through fluorescence polymerase chain reaction (PCR) and capillary electrophoresis. Together, this study provides useful insights into the discovery, characteristics and distribution of SSRs in four pig breeds. The polymorphic SSRs, especially those common and unique to all four pig breeds, might affect associated genes and play important roles in growth and development.

SSREnricher: a computational approach for large-scale identification of polymorphic microsatellites based on comparative transcriptome analysis

Microsatellite (SSR) markers are the most popular markers for genetic analyses and molecular selective breeding in plants and animals. However, the currently available methods to develop SSRs are relatively time-consuming and expensive. One of the most factors is low frequency of polymorphic SSRs. In this study, we developed a software, SSREnricher, which composes of six core analysis procedures, including SSR mining, sequence clustering, sequence modification, enrichment containing polymorphic SSR sequences, false-positive removal and results output and multiple sequence alignment. After running of transcriptome sequences on this software, a mass of polymorphic SSRs can be identified. The validation experiments showed almost all markers (>90%) that were identified by the SSREnricher as putative polymorphic markers were indeed polymorphic. The frequency of polymorphic SSRs identified by SSREnricher was significantly higher (P < 0.05) than that of traditional and HTS approaches. The software package is publicly accessible on GitHub (https://github.com/byemaxx/SSREnricher).