Genome-wide development of lncRNA-derived-SSR markers for Dongxiang wild rice (Oryza rufipogon Griff.)

Dongxiang wild rice (Oryza rufipogon Griff.) (DXWR) is the northernmost distributed wild rice found in the world. Similar to other populations of O. rufipogon, DXWR contains a large number of agronomically valuable genes, which makes it a natural gene pool for rice breeding. Molecular markers, especially simple repeat sequence (SSR) markers, play important roles in plant breeding. Although a large number of SSR markers have been developed, most of them are derived from the genome coding sequences, rarely from non-coding sequences. Meanwhile, long non-coding RNAs (lncRNAs), which are derived from the transcription of non-coding sequences, play vital roles in plant growth, development and stress responses. In our previous study, we obtained 1655 lncRNA transcripts from DXWR using strand-specific RNA sequencing. In this study, 1878 SSR loci were detected from the lncRNA sequences of DXWR, and 1258 lncRNA-derived-SSR markers were developed on the genome-wide scale. To verify the validity and applicability of these markers, 72 pairs of primers were randomly selected to test 44 rice accessions. The results showed that 42 (58.33%) pairs of primers have abundant polymorphism among these rice materials; the polymorphism information content values ranged from 0.04 to 0.87 with an average of 0.50; the genetic diversity index of SSR loci varied from 0.04 to 0.88 with an average of 0.56; and the number of alleles per marker ranged from 2 to 11 with an average of 4.36. Thus, we concluded that these lncRNA-derived-SSR markers are a very useful source for future basic and applied research.

Genetic combining, heterosis analysis for horticultural traits in tomato (Solanum lycopersicum L.) using ToLCV-resistant lines and molecular validation of Ty genes

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.

Assessing the leaf shape dynamic through marker–trait association under drought stress in a rice germplasm panel

The flag leaf acts as a functional leaf in rice, Oryza sativa L., primarily supplying photosynthate to the developing grains and influencing yields to a certain extent. Drought stress damages the leaf physiology, severely affecting grain fertility. Autumn rice of northeast India is called locally as ‘ahu’ rice, and is known for its drought tolerance. Exploring diverse germplasm resources at the morphological level using an association mapping approach can aid in identifying the genomic regions influencing leaf shape dynamics. A marker–trait association (MTA) study was carried out using 95 polymorphic SSR markers and a panel of 273 ahu rice germplasm accessions in drought stress and irrigated conditions. The trials suggest that at the vegetative stage, drought stress significantly affects leaf morphology. The leaf physiology of some tolerant accessions was relatively little affected by stress and these can be considered as ideal varieties for drought conditions. The phenotypic coefficient of variance and genotypic coefficient of variance values implied moderate to high variability for the leaf traits studied. Analysis of molecular variance inferred that 11% of variation in the germplasm panel was due to differences between populations, while the remaining 89% may be attributed to a difference within subgroups formed through STRUCTURE analysis. Using the mixed linear model approach revealed 11 MTAs explaining between 4.5 and 20.0% of phenotypic variance at P > 0.001 for all the leaf traits. The study concludes that ahu rice germplasm is extremely diverse and can serve as a valuable resource for mining desirable alleles for drought tolerance.

Evaluation of resistant genotypes and their characterization using molecular markers linked for powdery mildew resistance in cucumber (Cucumis sativus L.)

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.

Identification of elite germplasm of medicinally important Andrographis paniculata (Burm. f.) Nees with high content of four active diterpenoids in aerial parts from wild populations of eastern India

Andrographis paniculata is an Indian medicinal plant with tremendous therapeutic values due to the presence of active diterpenoids in its aerial parts. However, high domestic and export demand has led to overexploitation of wild populations of this species. With a view to bringing A. paniculata into cultivation and to reduce the pressure on wild populations, the present study was undertaken to identify elite germplasm from different locations of eastern India by analysing intraspecific variation in the content of four major active diterpenoids. A total of 166 wild accessions of A. paniculata analysed through high-performance liquid chromatography (HPLC) revealed remarkable variation in the sum of four active diterpenoids in the aerial parts, ranging from 0.41 to 8.55% on a dry weight basis. Three elite accessions (AP-6, AP-8, AP-46) having respectively 8.02, 8.36 & 8.55% of the sum of four major active diterpenoids were identified. These germplasm could be used for commercial cultivation and genetic improvement of A. paniculata.

De novo assembly, annotation and molecular marker identification from the leaf transcriptome of Ocimum gratissimum L

Ocimum gratissimum L. is a well-known medicinal plant with several therapeutic properties, but molecular studies on this species are lacking. Therefore, we have sequenced the whole transcriptome from the leaves of O. gratissimum and assembled 121,651 transcripts. The transcriptome of O. gratissimum was closely related to Sesamum indicum and Erythranthe guttata in congruence with the molecular phylogenetic relationships among these species. Further, 62,194 transcripts were annotated and classified according to the GO terms concerning the biological process, cellular component and metabolic function. In the KEGG pathway analysis, 34,876 transcripts were mapped to 149 pathways and 1410 of them were involved in the biosynthesis of secondary metabolites. In the phenylpropanoid pathway, 101 transcripts were associated with the biosynthesis of eugenol, the principal constituent of the essential oil of O. gratissimum. In the caffeine metabolism pathway, none of the transcripts was related to caffeine biosynthesis, supportive of the caffeine-free nature of Ocimum. Transcripts coding for the metallothionein were abundant in the leaves, supporting the observation that O. gratissimum is an accumulator of heavy metals. We also identified the 930 transcripts coding for 59 transcription factors families with myeloblastosis transcription factors being the most predominant. About 6500 simple sequence repeats were identified, which will be useful in DNA marker-based applications. This is the first report of the leaf transcriptome of O. gratissimum, which will serve as an essential resource for further molecular studies in this important medicinal species.

QTL mapping of drought tolerance at germination stage in wheat using the 50 K SNP array

Drought is a major concern among abiotic stresses in wheat (Triticum aestivum L.) production. Breeding resistant cultivars are the most effective means to manage drought stress. F6 recombinant inbred lines (RIL) derived from the cross of Berkut/Worrakatta were used to identify quantitative trait loci (QTL) for drought tolerance at germination stage under treatment of PEG6000 using the wheat 50 K single nucleotide polymorphism (SNP) array. Twenty-eight linkage groups were constructed, covering a length of 2220.26 cM. Eighteen QTL were detected based on the drought tolerance coefficients and D-value, explaining 2.7–6.5% of the phenotypic variances, in which 15 were likely to be novel. Three QTL, QGR.xjau-5AS, QCL.xjau-5AS and QD.xjau-5AS for GR, CL and D-value, respectively, at physical positions of 11.70–20.61 Mb between markers AX-111258240 and AX-94458300 on chromosome 5AS accounted for 3.4–4.8% of the phenotypic variances. Three QTL, QGP.xjau-5DL, QSH.xjau-5DL and QD.xjau-5DL for GP, SH and D-value, respectively, were flanked by markers AX-94524442 and AX-110998507 at 560.42–567.39 Mb on chromosome 5DL, accounting for 4.4–6.5% of the phenotypic variances. In addition, the candidate genes TraesCS5A02G022100, TraesCS5B02G014200 and TraesCS5D02G563900 were predicted. Based on transcriptional expression data, the results showed that the expression level of TaGATAs-5A, TaUbox-5B and TaGSTP-5D changed with the increase of treatment time under drought stress in tolerant and sensitive varieties. These are interesting targets in mining drought tolerance genes and the improvement of drought tolerance in wheat.

Diversity of white Guinea yam (Dioscorea rotundata Poir.) cultivars from Benin as revealed by agro-morphological traits and SNP markers

White Guinea yam (Dioscorea rotundata Poir.) is indigenous to West Africa, a region that harbours the crop's tremendous landrace diversity. The knowledge and understanding of local cultivars’ genetic diversity are essential for properly managing genetic resources, conservation, sustainable use and their improvement through breeding. This study aimed to dissect phenotypic and molecular diversity of white yam cultivars from Benin using agro-morphological and single nucleotide polymorphism (SNP) markers. Eighty-eight Beninese white Guinea yam cultivars collected through a countrywide ethnobotanical survey were phenotyped with 53 traits and genotyped with 9725 DArT-SNP. Multivariate analysis using phenotypic traits revealed 30 traits as most discriminative and explained up to 80.78% of cultivars’ phenotypic variation. Assessment of diversity indices such as Shannon–Wiener (H′), inverse Shannon (H.B.), Simpson's (λ) index and Pilou evenness (J) based molecular and phenotypic data depicted a moderate genetic diversity in Beninese white Guinea yam cultivars. Genetic differentiation of cultivars among country production zones was low due to the high exchange of planting materials among farmers of different regions. However, there was high genetic diversity within regions. Hierarchical clusters (HCs) on phenotypic data revealed the presence of two groups while HCs based on the SNP markers and the combined analysis identified three genetic groups. Our result provided valuable insights into the Beninese white Guinea yam diversity for its proper conservation and improvement through breeding.

Storage duration increases oxidation products in peanut seeds

Seeds kept in long-term storage are essential for maintaining genetic resources of crops and other plant materials in seed banks and national germplasm systems. But seeds undergo chemical changes over time as part of the ageing process that result in reduced germination rates and seedling normalcy. For example, oilseed crops such as peanuts are particularly vulnerable to oxidation and rancidification due to their high oil content. To test the effect of storage time on seed oil in peanuts, we grew different accessions and harvested fresh seeds to compare the oil composition of new seeds with seeds aged in storage for varying lengths of time. Out of the nine fatty acids detected and measured by gas chromatography, five including oleic, gadoleic, behenic, lignoceric and cerotic acids differed significantly between new and old seeds. Additionally, old seeds accumulated up to four oxidation products together averaging about 1% of the total oil composition. The concentration of these oxidation products was positively correlated with the age of the seeds, increasing linearly up to more than 6% of the total in the oldest seeds. The presence and concentration of oxidation products measured using simple chromatography techniques can be used as an initial indication of quality and viability in older seed inventories stored in germplasm repositories.

Comparative assessment of SCoT and ISSR markers for analysis of genetic diversity and population structure in some Aegilops tauschii Coss. accessions

Aegilops tauschii, the diploid progenitor of the wheat D-genome, is a valuable genetic resource for wheat breeders. In this study, we compared the efficiency of inter-simple sequence repeat (ISSR) (as an arbitrary technique) and start codon targeted (SCoT) (as a gene-targeting technique) markers in determining the genetic diversity and population structure of 90 accessions of Ae. tauschii. SCoT markers indicated the highest values for polymorphism information content, marker index and effective multiplex ratio compared to ISSR markers. The total genetic diversity (Ht) and genetic diversity within populations (Hs) parameters were comparably modest for the two marker systems. The results of the analysis of molecular variance showed that the genetic variation within populations was significantly higher than among them (ISSR: 92 versus 8%; SCoT: 88 versus 12%). Furthermore, SCoT markers discovered a high level of genetic differentiation among populations than ISSRs (0.19 versus 0.05), while the amount of gene flow detected by ISSR was higher than SCoT (2.13 versus 8.62). Cluster analysis and population structure of SCoT and ISSR data divided all investigated accessions into two and four main clusters, respectively. Our results revealed that SCoT and ISSR fingerprinting could be used to further molecular analysis in Ae. tauschii and other wild species. The high-genetic variability found in this study also indicates the valuable genetic potential present in the investigated Ae. tauschii germplasm, which could be utilized for future genetic analysis and linkage mapping in breeding programmes.