Genome-wide discovery of di-nucleotide SSR markers based on whole genome re-sequencing data of Cicer arietinum L. and Cicer reticulatum Ladiz.

Simple sequence repeats (SSRs) are valuable genetic markers due to their co-dominant inheritance, multi-allelic and reproducible nature. They have been largely used for exploiting genetic architecture of plant germplasms, phylogenetic analysis, and mapping studies. Among the SSRs, di-nucleotide repeats are the most frequent of the simple repeats distributed throughout the plant genomes. In present study, we aimed to discover and develop di-nucleotide SSR markers by using the whole genome re-sequencing (WGRS) data from Cicer arietinum L. and C. reticulatum Ladiz. A total of 35,329 InDels were obtained in C. arietinum, whereas 44,331 InDels in C. reticulatum. 3387 InDels with 2 bp length were detected in C. arietinum, there were 4704 in C. reticulatum. Among 8091 InDels, 58 di-nucleotide regions that were polymorphic between two species were selected and used for validation. We tested primers for evaluation of genetic diversity in 30 chickpea genotypes including C. arietinum, C. reticulatum, C. echinospermum P.H. Davis, C. anatolicum Alef., C. canariense A. Santos & G.P. Lewis, C. microphyllum Benth., C. multijugum Maesen, C. oxyodon Boiss. & Hohen. and C. songaricum Steph ex DC. A total of 244 alleles were obtained for 58 SSR markers giving an average of 2.36 alleles per locus. The observed heterozygosity was 0.08 while the expected heterozygosity was 0.345. Polymorphism information content was found to be 0.73 across all loci. Phylogenetic tree and principal coordinate analysis clearly divided the accessions into four groups. The SSR markers were also evaluated in 30 genotypes of a RIL population obtained from an interspecific cross between C. arietinum and C. reticulatum. Chi-square (χ2) test revealed an expected 1:1 segregation ratio in the population. These results demonstrated the success of SSR identification and marker development for chickpea with the use of WGRS data. The newly developed 58 SSR markers are expected to be useful for chickpea breeders.

Molecular and morphological evidence for resistance to sugarcane aphid (Melanaphis sacchari) in sweet sorghum [Sorghum bicolor (L.) Moench].

Aphids are one of the devastating pests affecting the productivity of sorghum in many countries. The aim of the present investigation was to identify sweet sorghum genotypes resistant to the sugarcane aphid, Melanaphis sacchari (Zehntner). A Sequence Characterized Amplified Region (SCAR) marker linked to an aphid-resistance gene (RMES1) was first used to prescreen for resistant genotypes in 561 sorghum accessions. Molecular assays indicated that 91 sorghum accessions in the collection had the RMES1 resistance marker allele. Of those, 26 agronomically superior sweet sorghum accessions, along with three commercial cultivars and one susceptible check, were further evaluated in two locations (Antalya, a lowland province, and Konya, a highland province) under field conditions. These accessions were scored for resistance to aphid damage under natural aphid infestations. The number of aphids counted on the plant leaves and stalks in the accessions during the growing seasons was used to score resistant genotypes on a scale of 1-5, where 1 was highly resistant (plants having 0-50 aphids/plant) and 5 was highly sensitive (plants having 1000 + aphids/plant). Fumagine intensity on the leaves was also taken into consideration. Ten accessions from the lowland and one accession from the highland scored "1," indicating a high resistance to aphid infestation. A further 13 accessions scored "1" or "2" in both environments. Only two accessions scored "4," and no accession scored "5," indicating the utility of the RMES1 marker for prescreening purposes. One accession, BSS507, showed outstanding resistance to M. sacchari, with a score of "1" in both environments.

Multiplex Real-Time qPCR Assay for Simultaneous and Sensitive Detection of Phytoplasmas in Sesame Plants and Insect Vectors.

Phyllody, a destructive and economically important disease worldwide caused by phytoplasma infections, is characterized by the abnormal development of floral structures into stunted leafy parts and contributes to serious losses in crop plants, including sesame (Sesamum indicum L.). Accurate identification, differentiation, and quantification of phyllody-causing phytoplasmas are essential for effective management of this plant disease and for selection of resistant sesame varieties. In this study, a diagnostic multiplex qPCR assay was developed using TaqMan® chemistry based on detection of the 16S ribosomal RNA gene of phytoplasmas and the 18S ribosomal gene of sesame. Phytoplasma and sesame specific primers and probes labeled with different fluorescent dyes were used for simultaneous amplification of 16SrII and 16SrIX phytoplasmas in a single tube. The multiplex real-time qPCR assay allowed accurate detection, differentiation, and quantification of 16SrII and 16SrIX groups in 109 sesame plant and 92 insect vector samples tested. The assay was found to have a detection sensitivity of 1.8 x 10(2) and 1.6 x 10(2) DNA copies for absolute quantification of 16SrII and 16SrIX group phytoplasmas, respectively. Relative quantification was effective and reliable for determination of phyllody phytoplasma DNA amounts normalized to sesame DNA in infected plant tissues. The development of this qPCR assay provides a method for the rapid measurement of infection loads to identify resistance levels of sesame genotypes against phyllody phytoplasma disease.

Development of co-dominant SCAR markers linked to resistant gene against the Fusarium oxysporum f. sp. radicis-lycopersici.

We developed highly reliable co-dominant SCAR markers linked to the Frl gene. FORL testing is difficult. The marker is expected to be quickly adapted for MAS by tomato breeders. Fusarium oxysporum f. sp. radicis-lycopersici causes Fusarium crown and root rot (FCR), an economically important soil-borne disease of tomato. The resistance against FCR is conferred by a single dominant gene (Frl) located on chromosome 9. The aim of this study was to develop molecular markers linked to the Frl gene for use in marker-assisted breeding (MAS) programs. The FCR-resistant 'Fla. 7781' and susceptible 'B560' lines were crossed, and F1 was both selfed and backcrossed to 'B560' to generate segregating F2 and BC1 populations. The two conserved set II (COSII) markers were found linked to the Frl gene, one co-segregated with FCR resistance in both F2 and BC1 populations and the other was 8.5 cM away. Both COSII markers were converted into co-dominant SCAR markers. SCARFrl marker produced a 950 and a 1000 bp fragments for resistant and susceptible alleles, respectively. The linkage of SCARFrl marker was confirmed in BC2F3 populations developed by backcrossing the resistant 'Fla. 7781' to five different susceptible lines. The SCARFrl marker has been in use in the tomato breeding programs in BATEM, Antalya, Turkey, since 2012 and has proved highly reliable. The SCARFrl marker is expected to aid in the development of FCR-resistant lines via marker-assisted selection (MAS).