Taq polymerase system is one of the key success factors for genotyping by using semi-random marker types. Start codon targeted (SCoT) and sequence-related amplified polymorphism (SRAP) are semi-random markers that amplify more than one target at a time, like in multiplex PCR. Here, we compared the performance of five multiplex PCR kits (QIAGEN®, Thermo Scientific™, New England Biolabs, KAPA Biosystem and iNtRON Biotechnology) and Taq DNA polymerase from Vivantis as a representative common Taq polymerase. The PCR using three SCoT and three SRAP markers was performed in six plants, which were different in genome size, including three monocotyledons (rice, oil palm and onion) and three dicotyledons (holy basil, chilli pepper and eggplant). The overall results demonstrated consistent high performance of the Taq polymerase system with the multiplex PCR kit from KAPA Biosystem. Considered by plant, the multiplex PCR kit from KAPA Biosystem showed the best performance in holy basil, eggplant, oil palm and chilli pepper, whereas in rice, the Taq DNA polymerase from Vivantis generated better results than the KAPA Biosystem. In onion, multiplex PCR kit from Thermo Scientific™ showed the best performance among those tested, even if it did not perform well in the other plans. Different Taq polymerase systems might generate different band patterns, therefore using the same Taq polymerase system within one experimental dataset is recommended. The multiplex PCR kit from KAPA Biosystem is the best starting choice.

Khasi mandarin (Citrus reticulata Blanco) is a commercial mandarin variety grown in northeast India and one of the 175 Indian food items included in the global first food atlas. The cultivated plantations of Khasi mandarin grown prominently in the lower Brahmaputra valley of Assam, northeast India, have been genetically eroded. The lack in the efforts for conservation of genetic variability in this mandarin variety prompted diversity analysis of Khasi mandarin germplasm across the region. Thus, the study aimed to investigate genetic diversity and partitioning of the genetic variations within and among 92 populations of Khasi mandarin collected from 10 cultivated sites in Kamrup and Kamrup (M) districts of Assam, India, using Inter-Simple Sequence Repeat (ISSR) markers. The amplification of genomic DNA with 17 ISSR primers yielded 216 scorable DNA amplicons of which 177 (81.94%) were polymorphic. The average polymorphism information content was 0.39 per primer. The total genetic diversity (HT = 0.28 ± 0.03) was close to the diversity within the population (HS = 0.20 ± 0.01). A high mean coefficient of gene differentiation (GST = 0.29) reflected a high level of gene flow (Nm = 1.22), indicating high genetic differentiation among the populations. Analysis of Molecular Variance (AMOVA) showed 78% of intra-population differentiation, 21% among the population and 1% among the districts. The obtained results indicate the existence of a high level of genetic diversity in the cultivated Khasi mandarin populations, indicating the need for preservation of each existing population to revive the dying out orchards in northeast India.

High-molecular-weight glutenin subunit (HMW-GS) is a key factor affecting dough-processing quality. 1Sty13 is a novel HMW-GS found in the tetraploid species, Elymus sibiricus L. 1Sty13 has faster electrophoretic mobility than the 1Dy12 subunit on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. The gene encoding the 1Sty13 subunit was composed of 1803 nucleotide base pairs with an open reading frame that was 599 amino acids in length. Analysis of the predicted amino acid sequence of 1Sty13 indicated that the N-terminal domain was similar to the y-type subunit, whereas the C-terminal domains were similar to the x-type subunit. Five cysteine residues were found in 1Sty13, which is one less than the published HMW-GS in the St genome. The 1Sty13 protein was purified at a scale sufficient for incorporation into flour for the SDS sedimentation test, which indicated that incorporating 1Sty13 improved dough quality.

Powdery mildew (PM) is one of the most severe fungal diseases of cucumber that limits its production worldwide. In this study, 140 genotypes of cucumber were screened for disease resistance under field and artificial conditions, and then validated with eight known SSR markers linked to PM resistance. Among these genotypes, genotype GS140 was found resistant (R), whereas GS148, GS16 and GS70 were moderately resistant, and GS169 was found to be tolerant. Of all the eight markers, only C31, C80, C162, SSR16472 and SSR16881 amplified the reported linked allele. The 127 bp allele of SSR16881 was found to be associated with the lowest disease severity of 37.65%. The associated markers could further be verified for their usability using linkage studies and the contrast genotypes in the present study could serve as a tool for selection in future marker-assisted selection breeding strategies for PM resistance.

Wild soybean (Glycine soja) is a valuable genetic resource for soybean improvement. Seed composition profiles provide beneficial information for the effective conservation and utilization of wild soybeans. Therefore, this study aimed to assess the variation in free amino acid abundance in the seeds of wild soybean germplasm collected in Japan. The free amino acid content in the seeds from 316 accessions of wild soybean ranged from 0.965 to 5.987 mg/g seed dry weight (DW), representing a 6.2-fold difference. Three amino acids had the highest coefficient of variation (CV): asparagine (1.15), histidine (0.95) and glutamine (0.94). Arginine (0.775 mg/g DW) was the predominant amino acid in wild soybean seeds, whereas the least abundant seed amino acid was glutamine (0.008 mg/g DW). A correlation network revealed significant positive relationships among most amino acids. Wild soybean seeds from different regions of origin had significantly different levels of several amino acids. In addition, a significant correlation between latitude and longitude of the collection sites and the total free amino acid content of seeds was observed. Our study reports diverse phenotypic data on the free amino acid content in seeds of wild soybean resources collected from throughout Japan. This information will be useful in conservation programmes for Japanese wild soybean and for the selection of accessions with favourable characteristics in future legume crop improvement efforts.

Finger millet is gaining importance as a food crop with the increasing emphasis on nutritional aspects and drought resilience. However, yield improvement has stagnated. Therefore, popular varieties have been examined for the purpose of whether direct selection for grain yield can be continued or an alternate trait-based approach using the germplasm is necessary. Direct selection for grain yield over the ruling variety, cv. GPU-28 (Germplasm Unit) has not been satisfactory. The path analysis has revealed a high direct effect of mean ear weight on grain yield followed by a moderate direct effect of photosynthetic rate and leaf area index. Furthermore, backward stepwise regression analysis revealed that among the independent traits, the mean ear weight made a significant contribution (60.8%) towards grain yield, followed by the photosynthetic rate (39.2%). The regression equation predicts the inclusion of mean ear weight by 1.0 g extra (as in GE-2672) to cv. GPU-28 will increase grain yield by 4.74%. The trait-specific genotypes are superior to the cv. GPU-28 were GPU-67 (photosynthetic rate) and GE-2672 (mean ear weight) and they could be used as donors for yield improvement. Future selection would aim for genotypes having 70–75 days for flowering with 4−5 productive tillers and mean ear weight of more than 8−9 g/ear. The possible approaches for enhancing grain yield are also discussed.

Milk thistle, Silybum marianum (L.) Gaertn. (Asteraceae), is an economically important medicinal plant utilized for silymarin production. Moreover, the species has been positively evaluated for vegetable oil and biomass production. Despite these positive characteristics, milk thistle is still marked by traits that are typical of undomesticated species (most importantly natural fruit dispersal at maturity) and requires further genetic improvement for its complete exploitation. This manuscript summarizes all the information collected through time about a collection of nine milk thistle wild and mutant lines and it discusses the possible further utilization of these genotypes. The accessions are characterized by interesting traits related to: fruit silymarin composition (S. marianum chemotype A and B), fruit fatty acid composition (high oleic and high stearic acid lines), fruit condensed tannins content, vegetative biomass composition (modification of xylans or lignin content), vegetative biomass structure (dwarf and tall lines), modifications of leaf variegation (hypervareigated line) and different types of fruit shatter resistance at maturity. All the lines underwent subsequent generations of selfing and are stable for all the described traits. The accessions will be made available at the Genebank of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK, Gatersleben) and may prove to be a useful genetic material for the improvement of qualitative fruit traits (silymarin quality, fatty acid composition) and for the further development of shatter-resistant S. marianum genotypes towards the complete domestication of this promising species.

Low-quality water and soil salinization are increasingly becoming limiting factors for food production, including olive – a major fruit crop in several parts of the world. Identifying putative salinity-stress tolerance in olive would be helpful in the future development of new tolerant varieties. In this study, novel salinity-responsive biomarkers (SRBs) were characterized in the species, namely, monooxygenase 1 (OeMO1), cation calcium exchanger 1 (OeCCX1), salt tolerance protein (OeSTO), proteolipid membrane potential modulator (OePMP3), universal stress protein (OeUSP2), adaptor protein complex 4 medium mu4 subunit (OeAP-4), WRKY1 transcription factor (OeWRKY1) and potassium transporter 2 (OeKT2). Unique structural features were highlighted for encoded proteins as compared with other plant homologues. The expression of olive SRBs was investigated in leaves of young plantlets of two cultivars, ‘Nabali’ (moderately tolerant) and ‘Picual’ (tolerant). At 60 mM NaCl stress level, OeMO1, OeSTO, OePMP3, OeUSP2, OeAP-4 and OeWRKY1 were up-regulated in ‘Nabali’ as compared with ‘Picual’. On the other hand, OeCCX1 and OeKT2 were up-regulated at three stress levels (30, 60 and 90 mM NaCl) in ‘Picual’ as compared to ‘Nabali’. Salinity tolerance in olive presumably engages multiple sets of responsive genes triggered by different stress levels.

Germplasm characterization is essential to estimate variation and identify desirable genotypes for crop improvement. The current study was conducted on eight qualitative and 14 quantitative traits related to growth and floral attributes for characterization and grouping of 54 chrysanthemum genotypes. Wide variability was observed among most of the traits and principal component analysis revealed that the first two principal components explained most of the existing variation (>98%) in germplasm. Genotypic coefficient of variation exhibited a wide range from 9.41% (ratio of leaf length/width) to 105.99% (corolla tube length of ray florets), while phenotypic coefficient of variation varied from 10.49% (ratio of leaf length/width) to 106.38% (corolla tube length of ray florets). Broad sense heritability estimates were higher (>96%) for most of the characters, except for traits such as ratio of leaf length/width and peduncle thickness. Forty-four pair-wise inter-trait combinations showed positive genotypic and phenotypic correlations for 91 possible combinations. D2 analysis revealed that genotypes of clusters I and IV to be highly distinct, and hybridization between them might have better chance to obtain desirable types. D2 analysis confirmed that the cultivars in cluster IV namely, Red D Spoon, Tokyo Soldier, Yellow Reflex and hierarchical clustering showed that the cultivar Tokyo Soldier to be highly diverse. It is recommended to use the above genotypes in crossing programme for obtaining better progenies.

Pigeonpea is an important grain legume. It contributes to the improvement of soil fertility through biological nitrogen (N) fixation. However, the symbiotic efficiency of pigeonpea with native soil rhizobia has not been determined adequately. This study was designed to determine the variation in the N fixation ability of pigeonpea inoculated with the native rhizobia. Forty soil samples were collected from diverse locations across South Africa and used for inoculating pigeonpea seed. Each pigeonpea genotype was inoculated separately with each soil sample and raised in a nitrogen-depleted growth medium in the greenhouse. A split-plot experimental design was used in the study. Several N fixation variables of pigeonpea were measured. There was >40.0% difference in the number of nodules between genotypes ‘Ex-PP-MD-321’ and ‘Mpuma-B-Spot’ but the nodule dry weight between the two genotypes was >80.0%. In contrast, the heaviest dry shoots (0.4513 g), weighed 52.0% heavier than those that were observed for ‘Mpuma-B-Spot’. Pigeonpea showed differential N fixation ability with the nodules, suggesting that there was potential to select for optimum host × rhizobial isolate combinations for the process and to expand the production area of the crop.

Leaf pubescence is widespread among higher plants. In bread wheat, a relationship was found between this trait and the efficiency of photosynthetic processes and productivity. In this work, we established the chromosomal localization of the gene for leaf pubescence introgressed from Triticum timopheevii into a bread wheat line 821 and studied its expression in the genetic background of two wheat cultivars differing in genetic control and phenotypic expression of pubescence. To obtain quantitative characteristics of pubescence in cultivars and hybrid populations, the LHDetect2 program was used, which makes it possible to estimate the length and number of trichomes on a leaf fold. A genetic analysis showed the dominant inheritance of the gene. Monosomic analysis F2 was used to establish chromosome localization and investigate the expression of the gene in cultivars Saratovskaya S29 (S29) and Diamant 2 (Dm2). As a result, the gene Hltt, introgressed from T. timopheevii, was identified and localized in the distal region of the long arm of 5A chromosome for the first time. In both F2 populations, the gene reduced the density of trichomes and formed long trichomes, uncharacteristic for the two recipient cultivars S29 and Dm2. A larger number of long trichomes was formed in the genetic background of S29, which carry the bread wheat gene Hl1 and Hl3 for leaf pubescence, than in Dm2. Development of substitution and isogenic lines with the fragment of introgression carrying the gene Hltt will allow determining function and assessing the adaptive significance of the gene more precisely.

A diverse set of 107 rice genotypes was evaluated for yield, shattering and dormancy traits. Analysis of variance revealed sizable variation while skewness and kurtosis values indicated near-normal distribution for most of the traits, thus quantitative nature controlled by many genes. A highly significant deviation from a normal distribution for dormancy and shattering % indicated their qualitative nature of inheritance. Four promising genotypes ‘IRGC1723’ (early with 65 days to flowering), ‘IRGC 11108’ and ‘RNR 15459’ (high grain number – 358 and low average shattering – <5%), ‘RNR 11718’ (high single plant yield – 56.73 g, low average shattering – <5% and dormancy period – 21 days) are identified. A significant positive correlation between shattering and dormancy confirms inter-relationship among domestication-related characteristics. The principal component analysis revealed the contribution of four PCs to maximum variability and hierarchical clustering grouped the genotypes into 18 divergent clusters. Five cultivars (Karimnagar Samba, Sheetal, PR 121, Pranahitha and Jagitial Samba) with a combination of low shattering ability (3.35–5.7%) and considerable dormancy period (13–20 days) falling in the same cluster can be used as donors for the improvement of rice genotypes with low shattering ability and incorporating a considerable period of dormancy so as to avoid pre-harvest sprouting due to delayed harvesting. Further, they can be crossed with ‘Pratyumna’ having less than 1 week dormancy period, a genotype of the cluster XVII with which they have a maximum genetic divergence of 51.4 and may serve as parents in the development of mapping populations for the identification of QTLs/genes for shattering and dormancy traits.

Tomato is a pivotal vegetable crop worldwide concerning human nutrition, economy and in service to biotechnology. Tomato leaf curl virus (ToLCV) is a begomovirus transmitted through the whitefly (Bemisia tabaci) and is responsible for severe losses in tomato production. In this context, the current investigation was carried out to determine heterotic combiners with high yield, resistance to ToLCV and mode of gene action for economically important traits. For this, 11 prescreened inbred lines were crossed in a half diallel fashion to generate 55 F1 hybrids. The 55 crosses with 11 parents and commercial checks were evaluated for different horticultural traits and resistance to ToLCV. The molecular validation with SCAR markers TG0302 and SCAR1 confirmed the presence of ToLCV-resistant genes in parents and their crosses. The hybrid IIHR-2902 × IIHR-2852 showed the presence of both Ty-2 and Ty-3 alleles in the homozygous state. The estimation of σSCA2 and σGCA2 was significant. It also indicated that the genetic control of target traits was under additive and non-additive gene effects. The values of σA/D2 along with σGCA2/σSCA2 found to be less than unity indicates the preponderance of non-additive gene action in the expression of the studied traits except for percent disease incidence. The parental line IIHR-2919 was the best combiner for fruit and yield traits. The cross combinations IIHR-2913 × IIHR-2898 exhibited significantly higher economic heterosis for yield along with the presence of Ty-2 and Ty-3 genes. The study paves the way for breeding high yielding and ToLCV-resistant hybrids in tomato.

Nipa (Nypa fruticans Wurmb.), aside from being part of the mangrove forest ecosystem that has been under assault at an alarming rate, lacks information on its genetic variation at the population level for conservation planning purposes. Microsatellites, or simple sequence repeats (SSRs), are molecular markers employed to detect genetic variations in population studies. Here, the primers for this palm were identified through whole-genome sequencing using the Illumina® Sequencing Technology platform. Over half (52.4%) of SSRs consisted of mononucleotide repeats, whereas the remainder mostly consisted of dinucleotide (22.7%) and trinucleotide (22.4%) repeats. The frequencies of longer repeats (tetranucleotides, pentanucleotides and hexanucleotides) were exceptionally rare (2.5%). At least 18 out of 75 genomic loci were polymorphic, based on 37 randomly selected samples assayed for screening. Polymorphic loci (P) reached a mean of 72% across the Indo-West Pacific region. Estimates of genetic diversity showed a lower mean observed heterozygosity (Ho) (0.195) than expected (He) (0.316), suggesting evolutionary forces were acting across nipa populations. The fixation (F) index (0.388) supported this result, which indicates considerable genetic differentiation among populations in the region. An average of 2.111 alleles (Na) was obtained. In conclusion, these microsatellite markers are promising tools for genetic evaluation of nipa populations to facilitate characterization of this species in its distribution range.

Melon (Cucumis melo L.) is an economically important vegetable crop worldwide. Karnataka state of southern India is rich in melon landraces and wild relatives, which are potential sources of genetic diversity for crop improvement. Here we report on the collection, characterization and documentation of previously unexplored landrace and wild material to broaden the availability of this genetic diversity for use in melon improvement. Accessions of botanical groups, momordica, kachri, chandalak, reticulatus, acidulus and indicus, intermediate forms of acidulus and momordica, and three melons of an unknown group were sourced directly from farmers of eight agro-ecological regions. Twenty-three representative melon landraces and wild relatives, along with four reference varieties of different botanical groups, were evaluated for agronomic traits during the year 2016. Very wide and distinctive genetic variations were observed among the melon accessions for ovary characters, sex expression, days taken to first fruit maturity, external and internal fruit traits, and fruit cracking (splitting). The remarkable variability displayed among the melon accessions as observed from the study demonstrates the importance of them as a valuable genetic reservoir for melon improvement.

During recent decades, global warming has intensified, altering crop growth, development and survival. To overcome changes in their environment, plants undergo transcriptional reprogramming to activate stress response strategies/pathways. To evaluate the genetic bases of the response to heat stress, Conserved DNA-derived Polymorphism (CDDP) markers were applied across tomato genome of eight cultivars. Despite scattered genotypes, cluster analysis allowed two neighbouring panels to be discriminate. Tomato CDDP-genotypic and visual phenotypic assortment permitted the selection of two contrasting heat-tolerant and heat-sensitive cultivars. Further analysis explored differential expression in transcript levels of genes, encoding heat shock transcription factors (HSFs, HsfA1, HsfA2, HsfB1), members of the heat shock protein (HSP) family (HSP101, HSP17, HSP90) and ascorbate peroxidase (APX) enzymes (APX1, APX2). Based on discriminating CDDP-markers, a protein functional network was built allowing prediction of candidate genes and their regulating miRNA. Expression patterns analysis revealed that miR156d and miR397 were heat-responsive showing a typical inverse relation with the abundance of their target gene transcripts. Heat stress is inducing a set of candidate genes, whose expression seems to be modulated through a complex regulatory network. Integrating genetic resource data is required for identifying valuable tomato genotypes that can be considered in marker-assisted breeding programmes to improve tomato heat tolerance.

Maize and Imperata cylindrica have been utilized globally as a pollen source for induction of haploids in wheat through chromosome elimination technique. Pollen parents with a higher haploid induction rate are desired for recovering the high frequency of haploids in wheat and related species. The present investigation was carried out with the aim to assess haploid induction efficiency of diverse germplasm of maize and I. cylindrica in different generations of intra and intergeneric crosses of hexaploid and tetraploid wheat and triticale-wheat derivatives. Crosses of twenty-six lines (female) with each of two I. cylindrica and twenty-one maize genotypes (testers) were evaluated for four haploid induction parameters viz., pseudoseed formation frequency (PFF), embryo formation frequency (EFF), haploid regeneration frequency (HRF) and haploid formation frequency (HFF). I. cylindrica outperformed maize in haploid induction rate with a frequency of embryos formed with I. cylindrica (18.39%) were significantly higher as compared to maize (4.08%). In the case of I. cylindrica genotype Ic-ye identified best with mean EFF of 30.55, 14.48 and 25.43% for hexaploids, tetraploids and triticale × wheat derivatives, respectively whereas in the case of maize genotype HPMC-60 performed best with EFF of 12.61% for hexaploids, HPMC-58 (12.58%) for tetraploids and HPMC-16 for triticale × wheat derivatives with EFF of 8.91%. I. cylindrica genotype Ic-ye and maize genotypes HPMC-14, HPMC-53, HPMC-60, HPMC-64 with significantly positive GCA for haploid induction parameters may be utilized as efficient pollen parents for recovering higher frequency of haploids in wheat.

Embryogenic callus induction and regeneration are useful in many aspects of plant biotechnology, especially in the functional characterization of economically important genes. However, in sugarcane, callus induction and regeneration vary across genotypes. Saccharum spontaneum is an important wild germplasm that confers disease resistance and stress tolerance to modern sugarcane cultivars, and its genome has been completely sequenced. The aim of this study was to investigate the effect of genetic variations on embryogenic callus induction and regeneration in S. spontaneum and to screen genotypes having high tissue culture susceptibility. The study was performed using nine genotypes of S. spontaneum and the following five parameters were assessed to determine the response of genotypes to embryogenic callus induction and regeneration: callus induction, embryogenic callus ratio, embryogenic callus induction, embryonic callus regeneration and regeneration capacity. All the genotypes varied significantly (P < 0.01) in all the parameters, except for embryonic callus regeneration, which was high (>80%) for all the genotypes. High broad-sense heritability (86.1–96.8%) indicated that genetic differences are the major source of genotypic variations. Callus induction was found to be strongly positively correlated with embryogenic callus induction (r = 0.890, P < 0.01) and regeneration capacity (r = 0.881, P < 0.01). Among the nine tested genotypes, VN2 was found to be the most responsive to tissue culture and could therefore be used to characterize functional genes in S. spontaneum. We also suggested an approach with potential applications in facilitating the rapid identification of sugarcane genotypes susceptible to tissue culture.

Drought tolerance is a highly complex trait and one of the important components of yield stability in wheat. An experiment was therefore conducted to study the drought tolerance status of 14 high-yielding wheat varieties based on morphological characteristics under a randomized complete block design following factorial arrangement with three replications. Variety × Treatment × Year interactions of wheat genotypes were studied for various morphological traits under normal and drought stress conditions for 2 years in plants grown in pots. Significant differences were observed among the 14 varieties of wheat. Sutlej-86 was found to be highly affected by drought, with maximum reductions in plant height, spike length and number of spikelets/spike. The variety with the maximum tolerance to drought was Bahawalpur-97; this variety also had the lowest reductions in the number of fertile tillers/plant, the number of spikelets/spike, spike weight, the number of grains/spike, 1000-grain weight, grain yield/spike, total biomass and stress susceptibility and tolerance indices. Stronger correlations among the studied attributes under drought stress over normal conditions further highlighted drought stress adversities. The studied traits are thus recognized as drought tolerance indicators for varietal selection, and varieties showing less reduction under drought could be used as a standard check in breeding programmes to identify lineages with drought tolerance and could be recommended for drought-stressed areas.

Triticum monococcum L. and T. boeoticum L., diploid wild relatives of bread wheat (T. aestivum L.), possess resistance to leaf rust (also known as brown rust) caused by Puccinia triticina Eriks. Haustorium formation-based resistance mechanisms (i.e. pre-haustorial and post-haustorial resistance) to leaf rust have been studied and reported in various T. monococcum accessions. In the present study, the mechanism of leaf rust resistance in T. monococcum and T. boeoticum accessions was studied using confocal laser scanning microscopy. Components of resistance studied at a histological level against leaf rust pathotypes, a Mexican pathotype (TCB/TD) and a Swiss pathotype (97512-19), indicated different types of resistance mechanism operative in the two accessions. The resistance in T. monococcum ranged from pre-haustorial resistance against 97512-19 to post-haustorial resistance against TCB/TD. The response in T. boeoticum was post-haustorial with necrosis against the two pathotypes. Pre-haustorial resistance observed in T. monococcum could serve as a potential source of durable rust resistance in wheat breeding.