Sequencing and de novo transcriptome assembly for discovering regulators of gene expression in Jack (Artocarpus heterophyllus).

Jack (Artocarpus heterophyllus) is a multipurpose fruit-tree species with minimal genomic resources. The study reports developing comprehensive transcriptome data containing 80,411 unigenes with an N50 value of 1265 bp. We predicted 64,215 CDSs from the unigenes and annotated and functionally categorized them into the biological process (23,230), molecular function (27,149), and cellular components (17,284). From 80,411 unigenes, we discovered 16,853 perfect SSRs with 192 distinct repeat motif types reiterating 4 to 22 times. Besides, we identified 2741 TFs from 69 TF families, 53 miRNAs from 19 conserved miRNA families, 25,953 potential lncRNAs, and placed three functional eTMs in different lncRNA-miRNA pairs. The regulatory networks involving genes, TFs, and miRNAs identified several regulatory and regulated nodes providing insight into miRNAs' gene associations and transcription factor-mediated regulation. The comparison of expression patterns of some selected miRNAs vis-à-vis their corresponding target genes showed an inverse relationship indicating the possible miRNA-mediated regulation of the genes.

iTRAQ based proteomic analysis of rice lines having single or stacked blast resistance genes: Pi54/Pi54rh during incompatible interaction with Magnaporthe oryzae.

Deployment of single or multiple blast resistance (R) genes in rice plant is considered to be the most promising approach to enhance resistance against blast disease caused by fungus Magnaporthe oryzae. At the proteome level, relatively little information about R gene mediated defence mechanisms for single and stacking resistance characteristics is available. The overall objective of this study is to look at the proteomics of rice plants that have R genes; Pi54, Pi54rh and stacked Pi54 + Pi54rh in response to rice blast infection. In this study 'isobaric tag for relative and absolute quantification' (iTRAQ)-based proteomics analysis was performed in rice plants at 72-h post inoculation with Magnaporthe oryzae and various differentially expressed proteins were identified in these three transgenic lines in comparison to wild type during resistance response to blast pathogen. Through STRING analysis, the observed proteins were further examined to anticipate their linked partners, and it was shown that several defense-related proteins were co-expressed. These proteins can be employed as targets in future rice resistance breeding against Magnaporthe oryzae. The current study is the first to report a proteomics investigation of rice lines that express single blast R gene Pi54, Pi54rh and stacked (Pi54 + Pi54rh) during incompatible interaction with Magnaporthe oryzae. The differentially expressed proteins indicated that secondary metabolites, reactive oxygen species-related proteins, phenylpropanoid, phytohormones and pathogenesis-related proteins have a substantial relationship with the defense response against Magnaporthe oryzae. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01327-3.

Smut fungi as a stratagem to characterize rust effectors: opportunities and challenges.

The rust pathogens are one of the most complex fungi in the Basidiomycetes. The development of genomic resources for rust and other plant pathogens has opened the opportunities for functional genomics of fungal genes. Despite significant progress in the field of fungal genomics, functional characterization of the genome components has lacked, especially for the rust pathogens. Their obligate nature and lack of standard stable transformation protocol are the primary reasons for rusts to be one of the least explored genera despite its significance. In the recently sequenced rust genomes, a vast catalogue of predicted effectors and pathogenicity genes have been reported. However, most of these candidate genes remained unexplored due to the lack of suitable characterization methods. The heterologous expression of putative effectors in Nicotiana benthamiana and Arabidopsis thaliana has proved to be a rapid screening method for identifying the role of these effectors in virulence. However, no fungal system has been used for the functional validation of these candidate genes. The smuts, from the evolutionary point of view, are closely related to the rust pathogens. Moreover, they have been widely studied and hence could be a suitable model system for expressing rust fungal genes heterologously. The genetic manipulation methods for smuts are also well standardized. Complementation assays can be used for functional validation of the homologous genes present in rust and smut fungal pathogens, while the species-specific proteins can be expressed in the mutant strains of smut pathogens having reduced or no virulence for virulence analysis. We propose that smuts, especially Ustilago maydis, may prove to be a good model system to characterize rust effector proteins in the absence of methods to manipulate the rust genomes directly.

Validation of reference genes for qRT-PCR data normalisation in lentil (Lens culinaris) under leaf developmental stages and abiotic stresses.

Lentil (Lens culinaris) is one of the most important staple food crops of developing countries. Transcriptome based global gene expression profiling followed by validation of expression of important genes through quantitative real time-PCR (qRT-PCR) has achieved significance in recent years. However, there is a severe scarcity of information regarding stable reference genes in lentil, which is mandatory for qRT-PCR data normalisation. Hence, the present study was under-taken to identify the most stable reference gene(s) in lentil. Expression stability of eight candidate genes viz. ribulose 1,5-bisphosphate carboxylase large subunit (Rbcl), ribosomal protein L2 (RPL2), 18S rRNA, tubulin (Tub), elongation factor 1α (EF1α), glyceraldehydes-3-phosphate dehydrogenase (GAPDH), heat shock protein (HSP70), and Maturase (mat K) was evaluated in five varieties of lentil at three different stages of leaf development and abiotic stress conditions using qRT-PCR. The results were analysed using four types of statistical software viz., geNorm, BestKeeper, NormFinder and RefFinder; all softwares identified RPL2 as most stable under abiotic stress conditions and developmental stages followed by Tub and Rbcl; while, HSP70 was identified as least stable. Relative expression of the target genes, defensin and PR4, was evaluated under abiotic stress conditions and data normalisation was done using two stable reference genes, RPL2 and Tub, either alone or in combination and with two least stable genes, HSP70 and 18S. The present work provides a list of potential reference genes in lentil, which will help in selection of appropriate reference gene for qRT-PCR data normalization depending upon the experiment.

Status and Prospects of Next Generation Sequencing Technologies in Crop Plants.

The history of DNA sequencing dates back to 1970s. During this period the two first generation nucleotide sequencing techniques were developed. Subsequently the Sanger's dideoxy method of sequencing gained popularity over Maxam and Gilbert's chemical method of sequencing. However, in the last decade, we have observed revolutionary changes in DNA sequencing technologies leading to the emergence of next-generation sequencing (NGS) techniques. NGS technologies have enhanced the throughput and speed of sequencing combined with bringing down the overall cost of the process over a time. The major applications of NGS technologies being genome sequencing and resequencing, transcriptomics, metagenomics in relation to plant-microbe interactions, exon and genome capturing, development of molecular markers and evolutionary studies. In this review, we present a broader picture of evolution of NGS tools, its various applications in crop plants, and future prospects of the technology for crop improvement.

Oscillating Transcriptome during Rice-Magnaporthe Interaction.

Rice blast disease caused by the fungus, Magnaporthe oryzae, is one of the most devastating diseases of rice. Deciphering molecular mechanism of host-pathogen interactions is of great importance in devising disease management strategies. Transcription being the first step for gene regulation in eukaryotes, basic understanding of the transcriptome is sine qua non for devising effective management strategy. The availability of genome sequences of rice and M. oryzae has facilitated the process to a large extent. The current review summarizes recent understanding of rice-blast pathosystem, application of transcriptomics approaches to understand the interactions employing different platforms, major determinants in the interaction and possibility of using certain candidate for conditioning enhanced disease resistance (Effector Triggered Immunity and PAMP Triggered Immunity) and downstream signalling in rice. A better understanding of the interaction elements and effective strategies hold potential to reduce yield losses in rice caused by M. oryzae.

Tagging and mapping of SSR marker for rust resistance gene in lentil (Lens culinaris Medikus subsp. culinaris).

Lentil, as an economical source of protein, minerals and vitamins, plays important role in nutritional security of the common man. Grown mainly in West Asia, North Africa (WANA) region and South Asia, it suffers from several biotic stresses such as wilt, rust, blight and broomrape. Lentil rust caused by autoecious fungus Uromyces viciae fabae (Pers.) Schroet is a serious lentil disease in Algeria, Bangladesh, Ethiopia, India, Italy, Morocco, Pakistan and Nepal. The disease symptoms are observed during flowering and early podding stages. Rust causes severe yield losses in lentil. It can only be effectively controlled by identifying the resistant source, understanding its inheritance and breeding for host resistance. The obligate parasitic nature of pathogen makes it difficult to maintain the pathogen in culture and to apply it to screen segregating progenies under controlled growth conditions. Hence, the use of molecular markers will compliment in identification of resistant types in different breeding programs. Here, we studied the inheritance of resistance to rust in lentil using F1, F2 and F2:3 from cross PL 8 (susceptible) x L 4149 (resistant) varieties. The phenotyping of lentil population was carried out at Sirmour, India. The result of genetic analysis revealed that a single dominant gene controls rust resistance in lentil genotype L 4149. The F2 population from this cross was used to tag and map the rust resistance gene using SSR and SRAP markers. Markers such as 270 SRAP and 162 SSR were studied for polymorphism and 101 SRAP and 33 SSRs were found to be polymorphic between the parents. Two SRAP and two SSR markers differentiated the resistant and susceptible bulks. SSR marker Gllc 527 was estimated to be linked to rust resistant locus at a distance of 5.9 cM. The Gllc 527 marker can be used for marker assisted selection for rust resistance; however, additional markers closer to rust resistant locus are required. The markers linked to the rust resistance gene can serve as starting points for map-based cloning of the rust resistance gene.

Development and evaluation of near-isogenic lines for major blast resistance gene(s) in Basmati rice.

KEY MESSAGE: A set of NILs carrying major blast resistance genes in a Basmati rice variety has been developed. Also, the efficacy of pyramids over monogenic NILs against rice blast pathogen Magnaporthe oryzae has been demonstrated. Productivity and quality of Basmati rice is severely affected by rice blast disease. Major genes and QTLs conferring resistance to blast have been reported only in non-Basmati rice germplasm. Here, we report incorporation of seven blast resistance genes from the donor lines DHMASQ164-2a (Pi54, Pi1, Pita), IRBLz5-CA (Pi2), IRBLb-B (Pib), IRBL5-M (Pi5) and IRBL9-W (Pi9) into the genetic background of an elite Basmati rice variety Pusa Basmati 1 (PB1). A total of 36 near-isogenic lines (NILs) comprising of 14 monogenic, 16 two-gene pyramids and six three-gene pyramids were developed through marker-assisted backcross breeding (MABB). Foreground, recombinant and background selection was used to identify the plants with target gene(s), minimize the linkage drag and increase the recurrent parent genome (RPG) recovery (93.5-98.6 %), respectively, in the NILs. Comparative analysis performed using 50,051 SNPs and 500 SSR markers revealed that the SNPs provided better insight into the RPG recovery. Most of the monogenic NILs showed comparable performance in yield and quality, concomitantly, Pusa1637-18-7-6-20 (Pi9), was significantly superior in yield and stable across four different environments as compared to recurrent parent (RP) PB1. Further, among the pyramids, Pusa1930-12-6 (Pi2+Pi5) showed significantly higher yield and Pusa1633-7-8-53-6-8 (Pi54+Pi1+Pita) was superior in cooking quality as compared to RP PB1. The NILs carrying gene Pi9 were found to be the most effective against the concoction of virulent races predominant in the hotspot locations for blast disease. Conversely, when analyzed under artificial inoculation, three-gene pyramids expressed enhanced resistance as compared to the two-gene and monogenic NILs.

Phenotypic expression of blast resistance gene Pi54 is not affected by its chromosomal position.

KEY MESSAGE: This is a novel report in which chromosomal position of the rice blast resistance gene Pi54 was not found to affect significantly the resistance phenotype or morphological traits. Blast disease caused by Magnaporthe oryzae is a serious constraint in rice production at global level. Pi54 gene imparts resistance against M. oryzae. Three different transgenic lines containing Pi54 and its orthologue Pi54rh were shown to be resistant to different races of M. oryzae. To determine the chromosomal location of Pi54 gene in transgenic lines, inverse PCR was performed. Our analysis showed that in two transgenic lines, Pi54 gene was integrated on chromosomes 6 and 10 at 12.94 and 22.30 Mb, respectively. Similarly, Pi54rh allele was integrated on chromosome 1 at 16.25 Mb. The Pi54 gene present on chromosome 6 was located in a non-coding region whereas in the other TP-Pi54 line, the gene was introgressed on chromosome 10 in between the coding region of SAP domain gene. The Pi54rh was also located in the non coding region flanked by the retrotransposon genes. These rice lines were evaluated for eight different traits related to seed and plant morphology and agronomic features for two consecutive years. The transgenic lines containing Pi54 gene have higher tiller number, grain weight, epicotyl length, and yield compared to the non-transgenic control. Multivariate correlation analysis shows that blast resistance was positively correlated with the number of tillers; thousand grain weight and epicotyl length. These results will facilitate precise utilization of Pi54 gene and its orthologue in breeding programs for the development of rice cultivars with broad spectrum and durable resistance to M. oryzae.

Transposon-based high sequence diversity in Avr-Pita alleles increases the potential for pathogenicity of Magnaporthe oryzae populations.

Magnaporthe oryzae causes rice blast that is one of the most devastating diseases of rice worldwide. Highly variable nature of this fungus has evolved itself against major resistance genes in newly released rice varieties. Understanding the population structure of this fungus is essential for proper utilization of the rice blast resistance genes in rice crop plants. In the present study, we analyzed 133 isolates of M. oryzae from ten countries to find the allelic variation of Avr-Pita gene that is triggering Pita-mediated resistance in rice plant. The diversity analysis of these alleles showed higher level of nucleotide variation in the coding regions than the noncoding regions. Evolutionary analysis of these alleles indicates that Avr-Pita gene is under purifying selection to favor its major alleles in 133 isolates analyzed in this study. We hypothesize that the selection of favorable Avr-Pita allele in these isolates may occur through a genetic mechanism known as recurrent selective sweeps. A total of 22 functional Avr-Pita protein variants were identified in this study. Insertion of Pot3 transposable element into the promoter of Avr-Pita gene was identified in virulent isolates and was suggested that mobility of repeat elements in avirulence genes of M. oryzae seems to help in emergence of new virulent types of the pathogen. Allele-specific markers developed in this study will be helpful to identify a particular type of Avr-Pita allele from M. oryzae population which can form the basis for the deployment of Pita gene in different epidemiological regions.

Inhibitory role of copper and silver nanocomposite on important bacterial and fungal pathogens in rice (Oryza sativa).

Rice (Oryza sativa) being among the most important food crops in the world is also susceptible to various bacterial and fungal diseases that are the major stumbling blocks in the way of increased production and productivity. The bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae and the sheath blight disease caused by Rhizoctonia solani are among the most devastating diseases of the rice crop. In spite of the availability of array of chemical control, there are chances of development of resistance. Thus, there is a need for the nanotechnological intervention for management of disease in the form of copper and silver nano-composites. The copper (CuNPs) and silver nanoparticles (AgNPs) were synthesized using green route and characterized using different high throughput techniques, i.e., UV-Vis, FT-IR, DLS, XRD, FE-SEM, TEM. The particle size and zeta potential of synthesized CuNPs and AgNPs were found 273 nm and - 24.2 mV; 95.19 nm and - 25.5 mV respectively. The nanocomposite of CuNPs and AgNPs were prepared having particle size in the range of 375-306 nm with improved stability (zeta potential - 54.7 to - 39.4 mV). The copper and silver nanoparticle composites evaluated against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani were found to have higher antibacterial (inhibition zone 13 mm) and antifungal activities (77%) compared to only the copper nanoparticle (8 mm; 62% respectively). Net house trials of nano-composite formulations against the bacterial blight of rice also corroborated the potential of nanocomposite formulation. In silico studies were carried out selecting two disease-causing proteins, peptide deformylase (Xanthomonas oryzae) and pectate lyase (Rhizoctonia solani) to perform the molecular docking. Interaction studies indicatedthat both of these proteins generated better complex with CuNPs than AgNPs. The study suggested that the copper and silver nano-composites could be used for developing formulations to control these devastating rice diseases.

Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae.

Rice blast is one of the important diseases of rice which can be effectively managed by the deployment of resistance genes. Pi-ta is one of the major blast resistant genes effective against pathogen populations in different parts of India. We analysed allelic variants of Pi-ta from 48 rice lines selected after phenotyping of 529 rice landraces across three eco-geographical blast hot spot regions. Besides, Pi-ta orthologue sequences of 220 rice accessions belonging to wild and cultivated species of rice were also included in the study for a better evo-devo perspective of the diversity present in the gene and the selection pressures acting on this locus. We obtained high nucleotide variations (SNPs and insertion-deletions) in the intronic region. We also identified 64 haplotypes based on nucleotide polymorphism in these alleles. Pi-ta orthologues of Indian landraces were scattered in eight major haplotypes indicating its heterogenous nature. We identified a total of 47 different Pi-ta protein variants on the basis of deduced amino acid residues amongst the orthologues. Five unique and novel Pi-ta variants were identified for the first time in rice landraces exhibiting different reaction types against the Magnaporthe oryzae population. A high value of Pi(non/syn) was observed only in the leucine-rich domain of the alleles cloned from Indian landraces, indicating strong selective forces acting on this region. The detailed molecular analysis of the Pi-ta orthologues provides insights to a high degree of inter- and intraspecific relationships amongst the Oryza species. We identified rice landraces possessing the effective alleles of this resistance gene which can be used in future blast resistance breeding programmes.

Genetic and molecular analysis of leaf blast resistance in Tetep derived line RIL4 and its relationship to genes at Pita/Pita2 locus.

The Vietnamese indica landrace 'Tetep' is known worldwide for its durable and broad spectrum-resistance to blast. We performed genetic and molecular analyses of leaf blast resistance in a Tetep derived recombinant inbred line 'RIL4' which is resistant to both leaf and neck blast. Phenotypic analysis of segregating F2 progenies suggested that leaf blast resistance in RIL4 was controlled by a dominant gene tentatively designated as Pi-l(t). The gene was mapped to a 2.4 cm region close to the centromere of chromosome 12. The search for the gene content in the equivalent genomic region of reference cv. Nipponbare revealed the presence of five NBS-LRR genes, two of which corresponded to the alleles of Pita and Pi67 genes previously identified from Tetep. The two other genes, LOC_Os12g17090, and LOC_Os12g17490 represented the homologs of stripe rust resistance gene Yr10. The allelic tests with Pita2 and Pi67 lines suggested that the leaf blast resistance gene in RIL4 is either allelic or tightly linked to these genes. The genomic position of the leaf blast resistance gene in RIL4 perfectly coincided with the genomic position of a neck blast resistance gene Pb2 previously identified from this line suggesting that the same gene confers resistance to leaf and neck blast. The present results were discussed in juxtaposition with past studies on the genes of Pita/Pita2 resistance gene complex.

Spatio-temporal expression pattern of Raffinose Synthase genes determine the levels of Raffinose Family Oligosaccharides in peanut (Arachis hypogaea L.) seed.

Raffinose family oligosaccharides (RFOs) are known to have important physiological functions in plants. However, the presence of RFOs in legumes causes flatulence, hence are considered antinutrients. To reduce the RFOs content to a desirable limit without compromising normal plant development and functioning, the identification of important regulatory genes associated with the biosynthetic pathway is a prerequisite. In the present study, through comparative RNA sequencing in contrasting genotypes for seed RFOs content at different seed maturity stages, differentially expressed genes (DEGs) associated with the pathway were identified. The DEGs exhibited spatio-temporal expression patterns with high RFOs variety showing early induction of RFOs biosynthetic genes and low RFOs variety showing a late expression at seed maturity. Selective and seed-specific differential expression of raffinose synthase genes (AhRS14 and AhRS6) suggested their regulatory role in RFOs accumulation in peanut seeds, thereby serving as promising targets in low RFOs peanut breeding programs. Despite stachyose being the major seed RFOs fraction, differential expression of raffinose synthase genes indicated the complex metabolic regulation of this pathway. The transcriptomic resource and the genes identified in this study could be studied further to develop low RFOs varieties, thus improving the overall nutritional quality of peanuts.

The chromosome-scale genome assembly of cluster bean provides molecular insight into edible gum (galactomannan) biosynthesis family genes.

Cluster bean (Cyamopsis tetragonoloba (L.) Taub 2n = 14, is commonly known as Guar. Apart from being a vegetable crop, it is an abundant source of a natural hetero-polysaccharide called guar gum or galactomannan. Here, we are reporting a chromosome-scale reference genome assembly of a popular cluster bean cultivar RGC-936, by combining sequencing data from Illumina, 10X Genomics, Oxford Nanopore technologies. An initial assembly of 1580 scaffolds with an N50 value of 7.12 Mb was generated and these scaffolds were anchored to a high density SNP linkage map. Finally, a genome assembly of 550.31 Mb (94% of the estimated genome size of ~ 580 Mb (through flow cytometry) with 58 scaffolds was obtained, including 7 super scaffolds with a very high N50 value of 78.27 Mb. Phylogenetic analysis using single copy orthologs among 12 angiosperms showed that cluster bean shared a common ancestor with other legumes 80.6 MYA. No evidence of recent whole genome duplication event in cluster bean was found in our analysis. Further comparative transcriptomics analyses revealed pod-specific up-regulation of genes encoding enzymes involved in galactomannan biosynthesis. The high-quality chromosome-scale cluster bean genome assembly will facilitate understanding of the molecular basis of galactomannan biosynthesis and aid in genomics-assisted improvement of cluster bean.

A novel blast resistance gene, Pi54rh cloned from wild species of rice, Oryza rhizomatis confers broad spectrum resistance to Magnaporthe oryzae.

The dominant rice blast resistance gene, Pi54 confers resistance to Magnaporthe oryzae in different parts of India. In our effort to identify more effective forms of this gene, we isolated an orthologue of Pi54 named as Pi54rh from the blast-resistant wild species of rice, Oryza rhizomatis, using allele mining approach and validated by complementation. The Pi54rh belongs to CC-NBS-LRR family of disease resistance genes with a unique Zinc finger (C(3)H type) domain. The 1,447 bp Pi54rh transcript comprises of 101 bp 5'-UTR, 1,083 bp coding region and 263 bp 3'-UTR, driven by pathogen inducible promoter. We showed the extracellular localization of Pi54rh protein and the presence of glycosylation, myristoylation and phosphorylation sites which implicates its role in signal transduction process. This is in contrast to other blast resistance genes that are predicted to be intracellular NBS-LRR-type resistance proteins. The Pi54rh was found to express constitutively at basal level in the leaves, but upregulates 3.8-fold at 96 h post-inoculation with the pathogen. Functional validation of cloned Pi54rh gene using complementation test showed high degree of resistance to seven isolates of M. oryzae collected from different geographical locations of India. In this study, for the first time, we demonstrated that a rice blast resistance gene Pi54rh cloned from wild species of rice provides broad spectrum resistance to M. oryzae hence can be used in rice improvement breeding programme.

Reduced tillering in Basmati rice T-DNA insertional mutant OsTEF1 associates with differential expression of stress related genes and transcription factors.

A T-DNA insertional mutant OsTEF1 of rice gives 60-80% reduced tillering, retarded growth of seminal roots, and sensitivity to salt stress compared to wild type Basmati 370. The insertion occurred in a gene encoding a transcription elongation factor homologous to yeast elf1, on chromosome 2 of rice. Detailed transcriptomic profiling of OsTEF1 revealed that mutation in the transcription elongation factor differentially regulates the expression of more than 100 genes with known function and finely regulates tillering process in rice by inducing the expression of cytochrome P450. Along with different transcription factors, several stress associated genes were also affected due to a single insertion. In silico analysis of the TEF1 protein showed high conservation among different organisms. This transcription elongation factor predicted to interact with other proteins that directly or indirectly positively regulate tillering in rice.

Variation in immuno-reproductive milieu of testis in Clarias magur from pre-spawning to spawning phase: An indication towards non-canonical role of immune elements in testes.

Immune mechanisms are major players in ensuring the normal functioning of testicular functions. However, apart from their role in active defence against pathogens, prior studies have also suggested a possibility for reproduction-related (non-immune) functions of certain immune elements. This study employs a comparative transcriptomics approach followed by network analysis for tracking the variation in the immuno-reproductive milieu of Clarias magur testis in spawning versus pre-spawning phase. The results show a significant modulation of both reproduction and immune-relevant genes in spawning versus pre-spawning phase. The functional enrichment of the upregulated reproduction-relevant gene network also shows immune-related biological processes which indicates a probability of involvement of these candidates in spermatogenesis-related events for switching from pre-spawning to spawning phase. The upregulated immune network is highly dense with 40 hubs, 10 cluster sub-networks and 142 functionally enriched pathways in comparison to its downregulated counterpart with only 5 hubs, 1 cluster and 1 enriched pathway. These findings indicate that the synchronisation in modulation of both reproductive and immune-related factors is critical for progression of testicular events guiding the switch from pre-spawning to spawning phase. The reproductive phase-dependent variation in plasma sex steroid levels and the selected genes for quantitative PCR also corroborated this hypothesis. The study also serves as a preliminary screening step for probable immune candidates that may be involved in reproductive functions of testis in addition to defence.

Identification of genomic regions associated with early plant vigour in lentil (Lens culinaris).

Lentil is one of the most important food legume species, however its genetic and genomic resources remained largely uncharacterized and unexploited. In the past few years, a number of genetic maps have been constructed and marker resources have been developed in lentil. These resources could be exploited for understanding the extent and distribution of genetic variation in genus Lens and also for developing saturated and consensus genetic maps suitable for quantitative trait loci (QTL) mapping and marker-assisted selection. The present study aims to enrich polymerase chain reaction-based linkage map of F10 recombinant inbred lines (RILs) population of 94 individuals derived from cross WA8649090 9 Precoz and identification of QTLs linked to early plant vigour traits. Of the 268 polymorphic markers (93 simple sequence repeats (SSR), three inter-simple sequence repeats (ISSRs) and 172 random amplified polymorphic DNA (RAPDs)), 265 (90 SSRs, three ISSRs and 172 RAPDs) were mapped on seven linkage groups, varying in length between 25.6 and 210.3 cM, coverage of 809.4 cM with an average marker spacing of 3.05 cM. The study also reported assigning of 24 new cross-genera SSRs of Trifolium pratense on the present linkage map. The RILs along with the parents were screened for shoot length, root length, seedling length, dry weight, number of leaves and number of branches based on two replications under polyhouse conditions. A QTLhotspot consisting of six QTLs for shoot length (cm), root length (cm) and seedling length (cm) was observed between a map distances of56.61 and 86.81 cM on LG1.