The effects of foliar amino acid and Zn applications on agronomic traits and Zn biofortification in soybean (Glycine max L.).

The production and consumption of soybeans are widespread due to their nutritional and industrial value. Nutrient enrichment is vital for improving the nutritional quality of soybeans. This study aimed to evaluate the effect of foliar application of amino acids (AA) and zinc (Zn) on agronomic traits and the accumulation of grain Zn in soybeans. The experimental design comprised 16 treatment combinations involving four levels of amino acid application (0, 50, 100, and 150 ml 100 L-1) and Zn (0, 2, 4, and 6 mg L-1) following a randomized complete block design with three replications in field conditions. The results demonstrated that the application of foliar Zn and AA did not affect the yield, whereas that of AA50*Zn2 and AA150*Zn2 affected the number of pods and branches. The effects of AA application on N and the protein content in grains were determined to be significant. The application of AA100*Zn6 emerged as the most effective treatment for the enhancement of Zn biofortification in soybean grains. The combined foliar application of AA and Zn contributed to enhanced Zn accumulation in the grains.

Construction of a high-density genetic linkage map and QTL mapping for bioenergy-related traits in sweet sorghum [Sorghum bicolor (L.) Moench].

Sorghum is an important but arguably undervalued cereal crop, grown in large areas in Asia and Africa due to its natural resilience to drought and heat. There is growing demand for sweet sorghum as a source of bioethanol as well as food and feed. The improvement of bioenergy-related traits directly affects bioethanol production from sweet sorghum; therefore, understanding the genetic basis of these traits would enable new cultivars to be developed for bioenergy production. In order to reveal the genetic architecture behind bioenergy-related traits, we generated an F2 population from a cross between sweet sorghum cv. 'Erdurmus' and grain sorghum cv. 'Ogretmenoglu'. This was used to construct a genetic map from SNPs discovered by double-digest restriction-site associated DNA sequencing (ddRAD-seq). F3 lines derived from each F2 individual were phenotyped for bioenergy-related traits in two different locations and their genotypes were analyzed with the SNPs to identify QTL regions. On chromosomes 1, 7, and 9, three major plant height (PH) QTLs (qPH1.1, qPH7.1, and qPH9.1) were identified, with phenotypic variation explained (PVE) ranging from 10.8 to 34.8%. One major QTL (qPJ6.1) on chromosome 6 was associated with the plant juice trait (PJ) and explained 35.2% of its phenotypic variation. For fresh biomass weight (FBW), four major QTLs (qFBW1.1, qFBW6.1, qFBW7.1, and qFBW9.1) were determined on chromosomes 1, 6, 7, and 9, which explained 12.3, 14.5, 10.6, and 11.9% of the phenotypic variation, respectively. Moreover, two minor QTLs (qBX3.1 and qBX7.1) of Brix (BX) were mapped on chromosomes 3 and 7, explaining 8.6 and 9.7% of the phenotypic variation, respectively. The QTLs in two clusters (qPH7.1/qBX7.1 and qPH7.1/qFBW7.1) overlapped for PH, FBW and BX. The QTL, qFBW6.1, has not been previously reported. In addition, eight SNPs were converted into cleaved amplified polymorphic sequences (CAPS) markers, which can be easily detected by agarose gel electrophoresis. These QTLs and molecular markers can be used for pyramiding and marker-assisted selection studies in sorghum, to develop advanced lines that include desirable bioenergy-related traits.

Breeding history for shattering trait in sesame: classic to genomic approach.

Sesame is an important oilseed crop that has high oil and protein content and unique antioxidant lignans. Capsule shattering at harvest is one of the most important problems affecting sesame production, with seed losses of up to 50%, making the crop unsuitable for mechanized harvesting. This paper provides an overview of breeding approaches addressing the capsule shattering trait in sesame and gives an outlook about the future perspectives of improvement for this trait. Sesame research has proceeded along the following parallel tracks: breeding for additional shatter resistance for manual harvest, breeding for mechanized harvest, and using molecular biology to improve the shatter resistance trait. In the future, genes controlling the shattering trait should be studied with techniques like RNA interference (RNAi), site-oriented mutagenesis, and gene editing with zinc finger nucleases (ZFNs) or CRISPR/Cas9, to develop new sesame varieties with capsules suitable for fully mechanized harvest.

Development of EMS-induced Mutagenized Groundnut Population and Discovery of Point Mutations in the ahFAD2 and Ara h 1 Genes by TILLING.

Reducing allergenicity and increasing oleic content are important goals in groundnut breeding studies. Ara h 1 is a major allergen gene and Delta(12)-fatty-acid desaturase (FAD2) is responsible for converting oleic into linoleic acid. These genes have homoeologues with one copy in each subgenome, identified as Ara h 1.01, Ara h 1.02, ahFAD2A and ahFAD2B in tetraploid groundnut. To alter functional properties of these genes we have generated an Ethyl Methane Sulfonate (EMS) induced mutant population to be used in Targeting Induced Local Lesions in Genomes (TILLING) approach. Seeds were exposed to two EMS concentrations and the germination rates were calculated as 90.1% (1353 plants) for 0.4% and 60.4% (906 plants) for 1.2% EMS concentrations in the M1 generation. Among the 1541 M2 mutants, 768 were analyzed by TILLING using four homoeologous genes. Two heterozygous mutations were identified in the ahFAD2B and ahFAD2A gene regions from 1.2% and 0.4% EMS-treated populations, respectively. The mutation in ahFAD2B resulted in an amino acid change, which was serine to threonine predicted to be tolerated according to SIFT analysis. The other mutation causing amino acid change, glycine to aspartic acid was predicted to affect protein function in ahFAD2A. No mutations were detected in Ara h 1.01 and Ara h 1.02 for both EMS-treatments after sequencing. We estimated the overall mutation rate to be 1 mutation every 2139 kb. The mutation frequencies were also 1/317 kb for ahFAD2A in 0.4% EMS and 1/466 kb for ahFAD2B in 1.2% EMS treatments. The results demonstrated that TILLING is a powerful tool to interfere with gene function in crops and the mutagenized population developed in this study can be used as an efficient reverse genetics tool for groundnut improvement and functional genomics.

A High-Density SNP Genetic Map Construction Using ddRAD-Seq and Mapping of Capsule Shattering Trait in Sesame.

The seed-bearing capsule of sesame shatters at harvest. This wildish trait makes the crop unsuitable for mechanized harvesting and also restricts its commercial potential by limiting the cultivation for countries that have no access to low-cost labor. Therefore, the underlying genetic basis of the capsule shattering trait is highly important in order to develop mechanization-ready varieties for sustainable sesame farming. In the present study, we generated a sesame F2 population derived from a cross between a capsule shattering cultivar (Muganli-57) and a non-shattering mutant (PI 599446), which was used to construct a genetic map based on double-digest restriction-site-associated DNA sequencing. The resulting high-density genetic map contained 782 single-nucleotide polymorphisms (SNPs) and spanned a length of 697.3 cM, with an average marker interval of 0.89 cM. Based on the reference genome, the capsule shattering trait was mapped onto SNP marker S8_5062843 (78.9 cM) near the distal end of LG8 (chromosome 8). In order to reveal genes potentially controlling the shattering trait, the marker region (S8_5062843) was examined, and a candidate gene including six CDSs was identified. Annotation showed that the gene encodes a protein with 440 amino acids, sharing ∼99% homology with transcription repressor KAN1. Compared with the capsule shattering allele, the SNP change and altered splicing in the flanking region of S8_5062843 caused a frameshift mutation in the mRNA, resulting in the loss of function of this gene in the mutant parent and thus in non-shattering capsules and leaf curling. With the use of genomic data, InDel and CAPS markers were developed to differentiate shattering and non-shattering capsule genotypes in marker-assisted selection studies. The obtained results in the study can be beneficial in breeding programs to improve the shattering trait and enhance sesame productivity.

Genome-Wide Discovery of InDel Markers in Sesame (Sesamum indicum L.) Using ddRADSeq.

The development and validation of different types of molecular markers is crucial to conducting marker-assisted sesame breeding. Insertion-deletion (InDel) markers are highly polymorphic and suitable for low-cost gel-based genotyping. From this perspective, this study aimed to discover and develop InDel markers through bioinformatic analysis of double digest restriction site-associated DNA sequencing (ddRADSeq) data from 95 accessions belonging to the Mediterranean sesame core collection. Bioinformatic analysis indicated the presence of 7477 InDel positions genome wide. Deletions accounted for 61% of the InDels and short deletions (1-2 bp) were the most abundant type (94.9%). On average, InDels of at least 2 bp in length had a frequency of 2.99 InDels/Mb. The 86 InDel sites having length ≥8 bp were detected in genome-wide analysis. These regions can be used for the development of InDel markers considering low-cost genotyping with agarose gels. In order to validate these InDels, a total of 38 InDel regions were selected and primers were successfully amplified. About 13% of these InDels were in the coding sequences (CDSs) and in the 3'- and 5'- untranslated regions (UTRs). Furthermore, the efficiencies of these 16 InDel markers were assessed on 32 sesame accessions. The polymorphic information content (PIC) of these 16 markers ranged from 0.06 to 0.62 (average: 0.33). These results demonstrated the success of InDel identification and marker development for sesame with the use of ddRADSeq data. These agarose-resolvable InDel markers are expected to be useful for sesame breeders.

Genotyping of Low β -ODAP Grass Pea (Lathyrus sativus L. ) Germplasm with EST-SSR Markers

Abstract Grass pea (Lathyrus sativus L.) is an important protein source in arid regions as both human and animal food. Despite its significance, the use of grass pea is limited by the presence of β-N-oxalyl-L-a,b-diaminopropionic acid (β-ODAP) which can cause neurological disorders. Breeding studies in grass pea have therefore focused on developing high-yielding varieties with low β-ODAP content. However, the narrow range of genetic diversity and the restricted genomic tools in grass pea have slowed progress in such breeding. The present investigation was conducted to explore the genetic diversity of low β-ODAP germplasm consisting of 22 accessions with 31 EST-SSR markers. The molecular analyses revealed a total of 133 alleles ranging from 142 to 330 bp with a mean number of alleles per locus of 4.29. The mean polymorphic information content (PIC) value was calculated as 0.49, and the EST-SSRs in loci S5, S6 and S116 were of the most informative PICs. A dendrogram based on Nei's genetic distance matrix revealed that breeding lines were grouped in two main clusters. Genetic distances were higher between GP6/GP11, GP4/GP11 and GP5/GP8 accessions which could be further used in crop improvement studies for developing wider genetic diversity.

Genetic diversity and population structure of the Mediterranean sesame core collection with use of genome-wide SNPs developed by double digest RAD-Seq.

The Mediterranean sesame core collection contains agro-morphologically superior sesame accessions from geographically diverse regions in four continents. In the present investigation, the genetic diversity and population structure of this collection was analyzed with 5292 high-quality SNPs discovered by double-digest restriction site associated DNA (ddRAD) sequencing, a cost-effective and flexible next-generation sequencing method. The genetic distance between pairs of accessions varied from 0.023 to 0.524. The gene diversity was higher in accessions from Asia than from America, Africa, and Europe. The highest genetic differentiation was observed between accessions collected from America and Europe. Structure analysis showed the presence of three subpopulations among the sesame accessions, and only six accessions were placed in an admixture group. Phylogenetic tree and principal coordinate analysis clustered the accessions based on their countries of origin. However, no clear division was evident among the sesame accessions with regard to their continental locations. This result was supported by an AMOVA analysis, which revealed a genetic variation among continental groups of 5.53% of the total variation. The large number of SNPs clearly indicated that the Mediterranean sesame core collection is a highly diverse genetic resource. The collection can be exploited by breeders to select appropriate accessions that will provide high genetic gain in sesame improvement programs. The high-quality SNP data generated here should also be used in genome-wide association studies to explore qualitative trait loci and SNPs related to economically and agronomically important traits in sesame.

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.