Elaeis oleifera Genomic-SSR Markers: Exploitation in Oil Palm Germplasm Diversity and Cross-Amplification in Arecaceae

Species-specific simple sequence repeat (SSR) markers are favored for genetic studies and marker-assisted selection (MAS) breeding for oil palm genetic improvement. This report characterizes 20 SSR markers from an Elaeis oleifera genomic library (gSSR). Characterization of the repeat type in 2000 sequences revealed a high percentage of di-nucleotides (63.6%), followed by tri-nucleotides (24.2%). Primer pairs were successfully designed for 394 of the E. oleifera gSSRs. Subsequent analysis showed the ability of the 20 selected E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The average Polymorphism Information Content (PIC) value for the SSRs was 0.402, with the tri-repeats showing the highest average PIC (0.626). Low values of observed heterozygosity (Ho) (0.164) and highly positive fixation indices (Fis) in the E. oleifera germplasm collection, compared to the E. guineensis, indicated an excess of homozygosity in E. oleifera. The transferability of the markers to closely related palms, Elaeis guineensis, Cocos nucifera and ornamental palms is also reported. Sequencing the amplicons of three selected E. oleifera gSSRs across both species and palm taxa revealed variations in the repeat-units. The study showed the potential of E. oleifera gSSR markers to reveal genetic diversity in the genus Elaeis. The markers are also a valuable genetic resource for studying E. oleifera and other genus in the Arecaceae family

Integration of physical and genetic maps in apple confirms whole-genome and segmental duplications in the apple genome

A total of 355 simple sequence repeat (SSR) markers were developed, based on expressed sequence tag (EST) and bacterial artificial chromosome (BAC)-end sequence databases, and successfully used to construct an SSR-based genetic linkage map of the apple. The consensus linkage map spanned 1143 cM, with an average density of 2.5 cM per marker. Newly developed SSR markers along with 279 SSR markers previously published by the HiDRAS project were further used to integrate physical and genetic maps of the apple using a PCR-based BAC library screening approach. A total of 470 contigs were unambiguously anchored onto all 17 linkage groups of the apple genome, and 158 contigs contained two or more molecular markers. The genetically mapped contigs spanned ∼421 Mb in cumulative physical length, representing 60.0% of the genome. The sizes of anchored contigs ranged from 97 kb to 4.0 Mb, with an average of 995 kb. The average physical length of anchored contigs on each linkage group was ∼24.8 Mb, ranging from 17.0 Mb to 37.73 Mb. Using BAC DNA as templates, PCR screening of the BAC library amplified fragments of highly homologous sequences from homoeologous chromosomes. Upon integrating physical and genetic maps of the apple, the presence of not only homoeologous chromosome pairs, but also of multiple locus markers mapped to adjacent sites on the same chromosome was detected. These findings demonstrated the presence of both genome-wide and segmental duplications in the apple genome and provided further insights into the complex polyploid ancestral origin of the apple

High levels of genetic differentiation and selfing in the Brazilian cerrado fruit tree Dipteryx alata Vog. (Fabaceae)

Dipteryx alata is a native fruit tree species of the cerrado (Brazilian savanna) that has great economic potential because of its multiple uses. Knowledge of how the genetic variability of this species is organized within and among populations would be useful for genetic conservation and breeding programs. We used nine simple sequence repeat (SSR) primers developed for Dipteryx odorata to evaluate the genetic structure of three populations of D. alata located in central Brazil based on a leaf sample analysis from 101 adults. The outcrossing rate was evaluated using 300 open-pollinated offspring from 25 seed-trees. Pollen dispersal was measured by parentage analysis. We used spatial genetic structure (SGS) to test the minimal distance for harvesting seeds in conservation and breeding programs. Our data indicate that the populations studied had a high degree of genetic diversity and population structure, as suggested by the high level of divergence among populations . The estimated outcrossing rate suggested a mixed mating system, and the intrapopulation fixation index was influenced by SGS. We conclude that seed harvesting for genetic conservation and breeding programs requires a minimum distance between trees of 196 m to avoid collecting seeds from related seed-trees

Design and Implementation of Degenerate Microsatellite Primers for the Mammalian Clade

Microsatellites are popular genetic markers in molecular ecology, genetic mapping and forensics. Unfortunately, despite recent advances, the isolation of de novo polymorphic microsatellite loci often requires expensive and intensive groundwork. Primers developed for a focal species are commonly tested in a related, non-focal species of interest for the amplification of orthologous polymorphic loci; when successful, this approach significantly reduces cost and time of microsatellite development. However, transferability of polymorphic microsatellite loci decreases rapidly with increasing evolutionary distance, and this approach has shown its limits. Whole genome sequences represent an under-exploited resource to develop cross-species primers for microsatellites. Here we describe a three-step method that combines a novel in silico pipeline that we use to (1) identify conserved microsatellite loci from a multiple genome alignments, (2) design degenerate primer pairs, with (3) a simple PCR protocol used to implement these primers across species. Using this approach we developed a set of primers for the mammalian clade. We found 126,306 human microsatellites conserved in mammalian aligned sequences, and isolated 5,596 loci using criteria based on wide conservation. From a random subset of ∼1000 dinucleotide repeats, we designed degenerate primer pairs for 19 loci, of which five produced polymorphic fragments in up to 18 mammalian species, including the distinctly related marsupials and monotremes, groups that diverged from other mammals 120–160 million years ago. Using our method, many more cross-clade microsatellite loci can be harvested from the currently available genomic data, and this ability is set to improve exponentially as further genomes are sequenced

Genes, gene flow and adaptation of Diabrotica virgifera virgifera

Diabrotica virgifera virgifera has emerged as a major pest of cultivated maize, due to a combination of its high capacity to inflict economic damage, adaptability to pest management techniques and invasiveness. This review presents a survey of the current state of knowledge about the genetics of D. v. virgifera. In addition, the tools and resources currently available to Diabrotica geneticists are identified, as are areas where knowledge is lacking and research should be prioritized. A substantial amount of information has been published concerning the molecular phylogenetic relationships of D. v. virgifera to other chrysomelids. There is a growing literature focused on the population genetics and evolution of the species. Several adaptations to anthropogenic selection pressure have been studied, with resistance to synthetic insecticides providing some particularly well-characterized examples. A notable deficiency is a lack of studies directed toward the formal genetics of D. v. virgifera

Breeding without Breeding: Is a Complete Pedigree Necessary for Efficient Breeding?

Complete pedigree information is a prerequisite for modern breeding and the ranking of parents and offspring for selection and deployment decisions. DNA fingerprinting and pedigree reconstruction can substitute for artificial matings, by allowing parentage delineation of naturally produced offspring. Here, we report on the efficacy of a breeding concept called “Breeding without Breeding” (BwB) that circumvents artificial matings, focusing instead on a subset of randomly sampled, maternally known but paternally unknown offspring to delineate their paternal parentage. We then generate the information needed to rank those offspring and their paternal parents, using a combination of complete (full-sib: FS) and incomplete (half-sib: HS) analyses of the constructed pedigrees. Using a random sample of wind-pollinated offspring from 15 females (seed donors), growing in a 41-parent western larch population, BwB is evaluated and compared to two commonly used testing methods that rely on either incomplete (maternal half-sib, open-pollinated: OP) or complete (FS) pedigree designs. BwB produced results superior to those from the incomplete design and virtually identical to those from the complete pedigree methods. The combined use of complete and incomplete pedigree information permitted evaluating all parents, both maternal and paternal, as well as all offspring, a result that could not have been accomplished with either the OP or FS methods alone. We also discuss the optimum experimental setting, in terms of the proportion of fingerprinted offspring, the size of the assembled maternal and paternal half-sib families, the role of external gene flow, and selfing, as well as the number of parents that could be realistically tested with BwB

Characterization of the sesame (Sesamum indicum L.) global transcriptome using Illumina paired-end sequencing and development of EST-SSR markers

<p>Abstract</p> <p>Background</p> <p>Sesame is an important oil crop, but limited transcriptomic and genomic data are currently available. This information is essential to clarify the fatty acid and lignan biosynthesis molecular mechanism. In addition, a shortage of sesame molecular markers limits the efficiency and accuracy of genetic breeding. High-throughput transcriptomic sequencing is essential to generate a large transcriptome sequence dataset for gene discovery and molecular marker development.</p> <p>Results</p> <p>Sesame transcriptomes from five tissues were sequenced using Illumina paired-end sequencing technology. The cleaned raw reads were assembled into a total of 86,222 unigenes with an average length of 629 bp. Of the unigenes, 46,584 (54.03%) had significant similarity with proteins in the NCBI nonredundant protein database and Swiss-Prot database (E-value < 10^-5). Of these annotated unigenes, 10,805 and 27,588 unigenes were assigned to gene ontology categories and clusters of orthologous groups, respectively. In total, 22,003 (25.52%) unigenes were mapped onto 119 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Furthermore, 44,750 unigenes showed homology to 15,460 <it>Arabidopsis </it>genes based on BLASTx analysis against The Arabidopsis Information Resource (TAIR, Version 10) and revealed relatively high gene coverage. In total, 7,702 unigenes were converted into SSR markers (EST-SSR). Dinucleotide SSRs were the dominant repeat motif (67.07%, 5,166), followed by trinucleotide (24.89%, 1,917), tetranucleotide (4.31%, 332), hexanucleotide (2.62%, 202), and pentanucleotide (1.10%, 85) SSRs. AG/CT (46.29%) was the dominant repeat motif, followed by AC/GT (16.07%), AT/AT (10.53%), AAG/CTT (6.23%), and AGG/CCT (3.39%). Fifty EST-SSRs were randomly selected to validate amplification and to determine the degree of polymorphism in the genomic DNA pools. Forty primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among 24 sesame accessions.</p> <p>Conclusions</p> <p>This study demonstrates that Illumina paired-end sequencing is a fast and cost-effective approach to gene discovery and molecular marker development in non-model organisms. Our results provide a comprehensive sequence resource for sesame research.</p

Investigating mating system of white pine weevil, Pissodes strobi (Coleoptera: Curculionaidae) using microsatellite DNA markers

White pine weevil is one of the most destructive and economically important forest pests in British Columbia because it causes damage to natural forest and plantation. The mating system, which is essential information for assessing possibility of sterile insect release programs and determining genetic diversity of insect populations, was studied from breeding experiments with varying number of females and males. Four polymorphic microsatellite markers were used for the mating system analysis. All microsatellite loci segregated in a Mendenlian fashion. The results of 1 Female: 2 Males mating showed that sperm precedence occurs in 82% of the studied replications. The 1 Female: 4 Males mating revealed not only mixed paternity, but supported the occurrences of sperm precedence as well. In this experiment, female weevils mated to four different males had a mean paternity of 2.8. The existences of sperm mixing were observed in both 1 Female: 2 Males and 1 Female: 4 Males matings. In addition, the possibility of sperm depletion was also observed in both of the 2 Females: 1 Male and 4 Females: 1 Male matings. The evidences of sperm precedence and multiple paternity will influence the style of "the Integrated Pest Management". Sperm precedence has important implications on the ability of sterile insect techniques. Also, the incidence of multiple paternity seems likely to affect the long-term outcome of tree breeding program via the adaptation of white pine weevils to overcome resistant trees.Forestry, Faculty ofGraduat