Search of the new SSR-loci of DNA for development of effective technology for soybean genotyping

Soybean is the major protein-oil crop of a huge economic importance. Currently, to describe the new cultivars being applied for a patent there are used the modern methods based on an analysis of microsatellite (SSR) loci of DNA. The purposes of this work were a search of the new microsatellite markers to optimize the existing technology of soybean cultivars certification and identification as well as selection of conditions for PCR analysis and to test them on cultivars from the VIR’s collection. Seven microsatellite loci demonstrated the high polymorphism level on soybean cultivars and located in the different chromosomes were chosen in the literary sources and librarian data bases. The optimal temperatures for annealing were selected empirically for all the pairs of SSR-markers. The results of DNA amplification of 20 soybean genotypes showed all seven studied SSR-loci were polyallel. In general, we revealed 22 alleles that on average are 3.1 per a locus. The effective number of alleles Ne for the studied soybean genotypes varied from 1.69 to 2.27 and on average was equal to 2.01. An average meaning of an index of the polymorphic information content (PIC) was 0.50. All the investigated soybean samples have the unique sets of alleles by the studied loci. Seven approbated loci can be used in development of an effective technology for identification and certification of the soybean genotypes.

Selection of Contrasting Parents for Drought Tolerance in Sunflower (Helianthus Annuus L.)

The aim of this research was select the best combination of contrasting parents to develop a mapping population for drought tolerance, based on phenotypic and genotypic data. Phenotyping was conducted in a greenhouse during 16 days at vegetative stage under well-watered (WW) and water-deficit (WD) conditions. Traits evaluated were: gain of leaf area (GLA), total water use (TWU), net assimilation rate (NAR), water use efficiency (WUE) and transpiration rate (TR) response to vapor pressure deficit (VPD) (slope and breakpoint). Genotyping was performed with 127 SSR markers and a cluster analyses was conducted. An important interaction was observed for NAR, WUE and breakpoint in the VPD response. Under WD conditions, all genotypes showed lower GLA and TWU, whereas NAR and WUE increased its values. All genotypes showed reduction of the slope and breakpoint in high VPD response on WD. PCA analysis explains the 80% of the total variability. PC1 discriminated HA89 and R419 due to a lower slope and higher breakpoint, while PC2 separated by water treatment based on the WUE and TWU values. Nighty nine SSR marker were amplified detecting 262 alleles. Cluster analyzes showed two main groups, one including HAR4 and B59 and the other one including five remaining genotypes. According to these results, only R419xHA64 and HA89xHAR4 had a greater genetic distance (1.08), besides a high polymorphism level between ILs (about 60%). Therefore, we conclude that these would be the best combination of contrasting parents to develop mapping populations for drought tolerance in sunflower.

Genetic Diversity of Peach Cultivars from the Collection of the Nikita Botanical Garden Based on SSR Markers

The Nikita Botanical Garden (NBG) has a unique Prunus L. collection (peach, apricot, plum, cherry) comprising more than 3000 accessions. NBG is also a breeding center for stone fruits, including peach (Prunus persica (L.) Batsch). In the present study a set of 85 peach cultivars bred in NBG, Europe, and North America was analyzed using 12 SSR markers to assess their genetic diversity and relatedness. The detected polymorphism level was comparable to the previous estimates of genetic variability in peach cultivars. The average number of alleles per locus was 5.67, PIC value averaged 0.49, expected, and observed heterozygosity averaged 0.52 and 0.31, respectively. Among the detected alleles, 19 (27.94%) were rare and 12 (17.65%) were unique. All studied accessions except two could be identified with the used marker set. Cluster analysis revealed some groups according to the cultivars’ pedigrees. No clear differentiation of the studied sample according to geographic origin or fruit characteristics of peach cultivars was revealed. The results provide valuable information for identification and rational management of the material preserved in the NBG peach collection.

TU-DAMD employment for molecular characterization of Salvia judaica and Salvia palaestina species

Genetic diversity in perennial Salvia judaica Boiss (Judean sage) and Salvia palaestina Benth (Palestinian sage) species using touch-up directed amplification of minisatellite region DNA (TU-DAMD) has been performed in two separated sets; in the first set (set A) the initial annealing temperature was increased from 50 °C to 55 °C, whereas, in the second one (set B), it increased from 55 °C to 60 °C by 0.5 °C/cycle during the first 10 PCR amplification cycles. Fifteen DAMD primers have been tested for each set. Set (A) produced 89.39% polymorphism level (P%) with polymorphic information content (PIC) average of 0.33 and marker index (MI) average of 3.96. Whereas, in set (B) these values were recorded to be 94.02%, 0.34 and 3.98 for P%, PIC and MI, respectively. Data showed that the two mentioned sets successfully highlighted high polymorphism level between the two studied Salvia sp. This work studies genetic diversity of S. judaica and S. palaestina species using TU-DAMD test as a novel molecular marker.

De novo transcriptomic analysis and identification of EST-SSR markers in Stephanandra incisa

Stephanandra incisa is a wild-type shrub with beautiful leaves and white flowers and is commonly used as a garden decoration accessory. However, the limited availability of genomic data of S. incisa has restricted its breeding process. Here, we identified EST-SSR markers using de novo transcriptome sequencing. In this study, a transcriptome database containing 35,251 unigenes, having an average length of 985 bp, was obtained from S. incisa. From these unigene sequences, we identified 5,555 EST-SSRs, with a distribution density of one SSR per 1.60 kb. Dinucleotides (52.96%) were the most detected SSRs, followed by trinucleotides (34.64%). From the EST-SSR loci, we randomly selected 100 sites for designing primer and used the DNA of 60 samples to verify the polymorphism. The average value of the effective number of alleles (Ne), Shannon’s information index (I), and expective heterozygosity (He) was 1.969, 0.728, and 0.434, respectively. The polymorphism information content (PIC) value was in the range of 0.108 to 0.669, averaging 0.406, which represented a middle polymorphism level. Cluster analysis of S. incisa were also performed based on the obtained EST-SSR data in our work. As shown by structure analysis, 60 individuals could be classified into two groups. Thus, the identification of these novel EST-SSR markers provided valuable sequence information for analyzing the population structure, genetic diversity, and genetic resource assessment of S. incisa and other related species.

Microsatellites for the Neotropical Ant, Odontomachus chelifer (Hymenoptera: Formicidae)

Odontomachus chelifer (Latreille) (Ponerinae) is a ground-dwelling, predominantly carnivorous ant whose colonies may contain multiple egg-laying queens and are potentially susceptible to border effects in the Brazilian savanna known as Cerrado. The ecology and natural history of O. chelifer is well studied, but very little is known about the genetic diversity of O. chelifer colonies. In this study, we developed microsatellite markers for the study of genetic variation in O. chelifer. We created a microsatellite-enriched library that resulted in the development and characterization of 22 markers, of which 18 were found to be polymorphic in the population studied. The mean expected heterozygosity was 0.59, whereas the mean rarified allelic richness was determined as 4.27 alleles per locus. The polymorphism level detected was similar to genetic diversity estimates found in other poneromorph ant species. The microsatellites developed here are likely to be useful for the investigation of colony structure, functional polygyny, breeding system, and population genetics in O. chelifer. Moreover, the description of O. chelifer’s genetic diversity is crucial for its conservation and maintenance of its ecological role in the Cerrado savanna.

Transposable Elements are an evolutionary force shaping genomic plasticity in the parthenogenetic root-knot nematode Meloidogyne incognita

Despite reproducing without sexual recombination, the root-knot nematode Meloidogyne incognita is adaptive and versatile. Indeed, this species displays a global distribution, is able to parasitize a large range of plants and can overcome plant resistance in a few generations. The mechanisms underlying this adaptability without sex remain poorly known and only low variation at the single nucleotide polymorphism level have been observed so far across different geographical isolates with distinct ranges of compatible hosts. Hence, other mechanisms than the accumulation of point mutations are probably involved in the genomic dynamics and plasticity necessary for adaptability. Transposable elements (TEs), by their repetitive nature and mobility, can passively and actively impact the genome dynamics. This is particularly expected in polyploid hybrid genomes such as the one of M. incognita. Here, we have annotated the TE content of M. incognita, analyzed the statistical properties of this TE content, and used population genomics approach to estimate the mobility of these TEs across 12 geographical isolates, presenting phenotypic variations. The TE content is more abundant in DNA transposons and the distribution of TE copies identity to their consensuses sequence suggests they have been at least recently active. We have identified loci in the genome where the frequencies of presence of a TE showed variations across the different isolates. Compared to the M. incognita reference genome, we detected the insertion of some TEs either within genic regions or in the upstream regulatory regions. These predicted TEs insertions might thus have a functional impact. We validated by PCR the insertion of some of these TEs, confirming TE movements probably play a role in the genome plasticity with possible functional impacts.

131 Genetic variants and haplotype combination in the bovine SH2B2 gene and their associations with molecular breeding for body size traits in Qinchuan cattle (Bos taurus)

The Src homology 2B 2 (SH2B2) gene regulates energy balance and body weight at least partially by enhancing Janus kinase-2 (JAK2)-mediated cytokine signalling, including leptin or GH signalling. Leptin is an adipose hormone that controls body weight. The objective of the current study was to evaluate the association between body measurement traits and SH2B2 gene polymorphisms. For this purpose, we selected four single-nucleotide polymorphisms (SNPs) in the SH2B2 gene, including two in intron 5 (A20545G and G20570A), one synonymous SNP (T20693C) in exon 6, and one in intron 8 (C24070A), and genotyped these through DNA sequencing in Qinchuan cattle. The general linear model (GLM) in SPSS 20.0 software (SPSS Inc.) was used for the association analysis between SNPs and selected traits of carcass quality. The SNPs in sample populations were in medium polymorphism level (0.250<pic<0.500). This association study indicated that the G20570A, T20693C, and C24070A were significantly (P<0.05) associated with body length (BL) and chest circumference (CC) in Qinchuan cattle. In addition, the H4H3 and H5H5 diplotype were associated with significantly (P=0.01) greater BL and rump length than was H4H2. Our investigation will not only extend understanding of genetic variations in the bovine SH2B2 gene but also provide useful information for marker-assisted selection in beef cattle breeding programs.

Relationships among cultivated peas and their wild relatives: Molecular and morphological characterization

This study was conducted to determine relationship between some wild pea accessions (Pisum fulvum L., P. abyssinicum L., P. sativum var. elatius), local varieties (P. sativum var. sativum L. and P. sativum var. arvense L.) and commercial varieties “Boogie” and “Rondo”. The genetic diversity was evaluated with 14 simple sequence repeat markers and 50 morphological characters. The results of morphology indicated that, genotypes showed a clustering pattern based on the taxonomic groups when considering only flower characters and all morphological characters. During the molecular study, a total of 48 alleles were obtained. Used all primers showed polymorphism in accessions. The number of alleles varied between 2 - 6 among 14 SSR loci revealing the polymorphism level of markers. Similarity coefficient (Dice’s) ranged from 0.100 to 0.800 with an average of 0.378. A dendrogram grouped the 15 genotypes into two main clusters. This information can be utilized for genetic analysis, genotype identification from different sources and development of improved germplasm.