High resolution mapping of QTLs for fruit color and firmness in Amrapali/Sensation mango hybrids.

Introduction: Mango (Mangifera indica L.), acclaimed as the 'king of fruits' in the tropical world, has historical, religious, and economic values. It is grown commercially in more than 100 countries, and fresh mango world trade accounts for ~3,200 million US dollars for the year 2020. Mango is widely cultivated in sub-tropical and tropical regions of the world, with India, China, and Thailand being the top three producers. Mango fruit is adored for its taste, color, flavor, and aroma. Fruit color and firmness are important fruit quality traits for consumer acceptance, but their genetics is poorly understood. Methods: For mapping of fruit color and firmness, mango varieties Amrapali and Sensation, having contrasting fruit quality traits, were crossed for the development of a mapping population. Ninety-two bi-parental progenies obtained from this cross were used for the construction of a high-density linkage map and identification of QTLs. Genotyping was carried out using an 80K SNP chip array. Results and discussion: Initially, we constructed two high-density linkage maps based on the segregation of female and male parents. A female map with 3,213 SNPs and male map with 1,781 SNPs were distributed on 20 linkages groups covering map lengths of 2,844.39 and 2,684.22cM, respectively. Finally, the integrated map was constructed comprised of 4,361 SNP markers distributed on 20 linkage groups, which consisted of the chromosome haploid number in Mangifera indica (n =20). The integrated genetic map covered the entire genome of Mangifera indica cv. Dashehari, with a total genetic distance of 2,982.75 cM and an average distance between markers of 0.68 cM. The length of LGs varied from 85.78 to 218.28 cM, with a mean size of 149.14 cM. Phenotyping for fruit color and firmness traits was done for two consecutive seasons. We identified important consistent QTLs for 12 out of 20 traits, with integrated genetic linkages having significant LOD scores in at least one season. Important consistent QTLs for fruit peel color are located at Chr 3 and 18, and firmness on Chr 11 and 20. The QTLs mapped in this study would be useful in the marker-assisted breeding of mango for improved efficiency.

Comparative transcriptome profiling of Polianthes tuberosa during a compatible interaction with root-knot nematode Meloidogyne incognita.

BACKGROUND: The root-knot nematode (RKN; Meloidogyne spp.) is the most destructive plant parasitic nematode known to date. RKN infections, especially those caused by Meloidogyne incognita, are one of the most serious diseases of tuberose. METHODS AND RESULTS: To investigate the molecular mechanism in the host-pathogen interactions, the Illumina sequencing platform was employed to generate comparative transcriptome profiles of uninfected and Meloidogyne incognita-infected tuberose plants, during early, mid, and late infection stage. A total of 7.5 GB (49 million reads) and 9.3 GB (61 million reads) of high-quality data was generated for the control and infected samples, respectively. These reads were combined and assembled using the Trinity assembly program which clustered them into 1,25,060 unigenes. A total of 85,360 validated CDS were obtained from the combined transcriptome whereas 6,795 CDS and 7,778 CDS were found in the data for the control and infected samples, respectively. Gene ontology terms were assigned to 958 and 1,310 CDSs from the control and infected data, respectively. The KAAS pathway analysis revealed that 1,248 CDS in the control sample and 1,482 CDS in the infected sample were enriched with KEGG pathways. The major proportions of CDS were annotated for carbohydrate metabolism, signal transduction and translation related pathways in control and infected samples. Of the 8,289 CDS commonly expressed between the control and infected plants, 256 were significantly upregulated and 129 were significantly downregulated in the infected plants. CONCLUSIONS: Collectively, our results provide a comprehensive gene expression changes in tuberose during its association with RKNs and point to candidate genes that are involved in nematode stress signaling for further investigation. This is the first report addressing genes associated with M. incognita-tuberose interaction and the results have important implications for further characterization of RKN resistance genes in tuberose.

Genome wide annotation and characterization of young, intact long terminal repeat retrotransposons (In-LTR-RTs) of seven legume species.

Availability of genome sequence of different legume species has provided an opportunity to characterize the abundance, distribution, and divergence of canonical intact long terminal retrotransposons (In-LTR-RT) superfamilies. Among seven legume species, Arachis ipaensis (Aip) showed the highest number of full-length canonical In-LTR-RTs (3325), followed by Glycine max (Gma, 2328), Vigna angularis (Van, 1625), Arachis durensis (Adu, 1348), Lotus japonicus (Lja, 1294), Medicago truncatula (Mtr, 788), and Circer arietinum (Car, 124). Divergence time analysis demonstrated that the amplification timeframe of LTR-RTs dramatically varied in different families. The average insertion time of Copia element varied from 0.51 (Van) to 1.37 million years ago (Mya) (Adu, and Aip), whereas that of Gypsy was between 0.22 (Mtr) and 1.82 Mya (Adu). Bayesian phylogenetic tree analysis suggested that the 1397 and 1917 reverse transcriptase (RT) domains of Copia and Gypsy families of the seven legume species were clustered into 7 and 14 major groups, respectively. The highest proportion (approximately 94.79-100%) of transposable element (TE)-associated genes assigned to pathways was mapped to metabolism-related pathways in all species. The results enabled the structural understanding of full-length In-LTR-RTs and will be valuable resource for the further study of the impact of TEs on gene structure and expression in legume species.

Allelic sequence variation in the Sub1A, Sub1B and Sub1C genes among diverse rice cultivars and its association with submergence tolerance.

Erratic rainfall leading to flash flooding causes huge yield losses in lowland rice. The traditional varieties and landraces of rice possess variable levels of tolerance to submergence stress, but gene discovery and utilization of these resources has been limited to the Sub1A-1 allele from variety FR13A. Therefore, we analysed the allelic sequence variation in three Sub1 genes in a panel of 179 rice genotypes and its association with submergence tolerance. Population structure and diversity analysis based on a 36-plex genome wide genic-SNP assay grouped these genotypes into two major categories representing Indica and Japonica cultivar groups with further sub-groupings into Indica, Aus, Deepwater and Aromatic-Japonica cultivars. Targetted re-sequencing of the Sub1A, Sub1B and Sub1C genes identfied 7, 7 and 38 SNPs making 8, 9 and 67 SNP haplotypes, respectively. Haplotype networks and phylogenic analysis revealed evolution of Sub1B and Sub1A genes by tandem duplication and divergence of the ancestral Sub1C gene in that order. The alleles of Sub1 genes in tolerant reference variety FR13A seem to have evolved most recently. However, no consistent association could be found between the Sub1 allelic variation and submergence tolerance probably due to low minor allele frequencies and presence of exceptions to the known Sub1A-1 association in the genotype panel. We identified 18 cultivars with non-Sub1A-1 source of submergence tolerance which after further mapping and validation in bi-parental populations will be useful for development of superior flood tolerant rice cultivars.

A 62K genic-SNP chip array for genetic studies and breeding applications in pigeonpea (Cajanus cajan L. Millsp.).

Pigeonpea is the second most important pulse legume crop for food and nutritional security of South Asia that requires accelerated breeding using high throughput genomic tools. Single nucleotide polymorphisms (SNPs) are highly suitable markers for this purpose because of their bi-allelic nature, reproducibility and high abundance in the genome. Here we report on development and use of a pigeonpea 62 K SNP chip array 'CcSNPnks' for Affymetrix GeneTitan® platform. The array was designed after filtering 645,662 genic-SNPs identified by re-sequencing of 45 diverse genotypes and has 62,053 SNPs from 9629 genes belonging to five different categories, including 4314 single-copy genes unique to pigeonpea, 4328 single-copy genes conserved between soybean and pigeonpea, 156 homologs of agronomically important cloned genes, 746 disease resistance and defense response genes and 85 multi-copy genes of pigeonpea. This fully genic chip has 28.94% exonic, 33.04% intronic, 27.56% 5'UTR and 10.46% 3'UTR SNPs and incorporates multiple SNPs per gene allowing gene haplotype network analysis. It was used successfully for the analysis of genetic diversity and population structure of 95 pigeonpea varieties and high resolution mapping of 11 yield related QTLs for number of branches, pod bearing length and number of seeds per pod in a biparental RIL population. As an accurate high-density genotyping tool, 'CcSNPnks' chip array will be useful for high resolution fingerprinting, QTL mapping and genome wide as well as gene-based association studies in pigeonpea.

Identification of putative flowering genes and transcription factors from flower de novo transcriptome dataset of tuberose (Polianthes tuberosa L.).

Polianthes tuberosa is commercially popular because of their economic importance in floriculture for cut and loose flowers and in perfume industry because of the unique fragrance. Despite its commercial importance, no ready-to-use transcript sequence information is available in the public database. We have sequenced the RNA obtained from tuberose flowers using the Illumina HiSeq. 2000 platform and have carried out a de novo analysis of the transcriptome data. The de novo assembly generated 11,100 transcripts. These transcripts represent a total of 7876 unigenes that were considered for downstream analysis. These 7876 unigenes, which was further annotated using blast2go and KEGG pathways, were also assigned. Tuberose transcripts were also assigned to metabolic pathways using the Kyoto Encyclopedia of Genes and Genomes database to determine their biochemical functions. 4591 of the tuberose transcripts matched to genes in KEGG pathways and 66 transcripts were mapped to the Flavonoid biosynthesis pathway. 21 flowering genes have been identified in this tuberose transcriptome. Transcription factor analysis helped in the identification of a large number of transcripts similar to key genes in the flowering regulation network of Arabidopsis thaliana. Among the transcription factors identified "NAC" which is associated with plant stress response represented the most abundant category followed by APETALA2 (AP2)/ethylene-responsive element binding proteins (EREBPs) which plays various role in floral organ identity and respond to different biotic and abiotic stress.

Characterization of OglDREB2A gene from African rice (Oryza glaberrima), comparative analysis and its transcriptional regulation under salinity stress.

In this study, AP2 DNA-binding domain-containing transcription factor, OglDREB2A, was cloned from the African rice (Oryza glaberrima) and compared with 3000 rice genotypes. Further, the phylogenetic and various structural analysis was performed using in silico approaches. Further, to understand its allelic variation in rice, SNPs and indels were detected among the 3000 rice genotypes which indicated that while coding region is highly conserved, yet noncoding regions such as UTR and intron contained most of the variation. Phylogenetic analysis of the OglDREB2A sequence in different Oryza as well as in diverse eudicot species revealed that DREB from various Oryza species were diversed much earlier than other genes. Further, structural features and in silico analyses provided insights into different properties of OglDREB2A protein. The neutrality test on the coding region of OglDREB2A from different genotypes of O. glaberrima showed the lack of selection in this gene. Among the different developmental stages, it was upregulated at tillering and flag leaf under salinity treatment indicating its positive role in seedling and reproductive stage tolerance. Real-time PCR analysis also indicated the conserve expression pattern of this gene under salinity stress across the three different Oryza species having different degree of salinity tolerance.

MiSNPDb: a web-based genomic resources of tropical ecology fruit mango (Mangifera indica L.) for phylogeography and varietal differentiation.

Mango is one of the most important fruits of tropical ecological region of the world, well known for its nutritive value, aroma and taste. Its world production is >45MT worth >200 billion US dollars. Genomic resources are required for improvement in productivity and management of mango germplasm. There is no web-based genomic resources available for mango. Hence rapid and cost-effective high throughput putative marker discovery is required to develop such resources. RAD-based marker discovery can cater this urgent need till whole genome sequence of mango becomes available. Using a panel of 84 mango varieties, a total of 28.6 Gb data was generated by ddRAD-Seq approach on Illumina HiSeq 2000 platform. A total of 1.25 million SNPs were discovered. Phylogenetic tree using 749 common SNPs across these varieties revealed three major lineages which was compared with geographical locations. A web genomic resources MiSNPDb, available at http://webtom.cabgrid.res.in/mangosnps/ is based on 3-tier architecture, developed using PHP, MySQL and Javascript. This web genomic resources can be of immense use in the development of high density linkage map, QTL discovery, varietal differentiation, traceability, genome finishing and SNP chip development for future GWAS in genomic selection program. We report here world's first web-based genomic resources for genetic improvement and germplasm management of mango.

A tree of life based on ninety-eight expressed genes conserved across diverse eukaryotic species.

Rapid advances in DNA sequencing technologies have resulted in the accumulation of large data sets in the public domain, facilitating comparative studies to provide novel insights into the evolution of life. Phylogenetic studies across the eukaryotic taxa have been reported but on the basis of a limited number of genes. Here we present a genome-wide analysis across different plant, fungal, protist, and animal species, with reference to the 36,002 expressed genes of the rice genome. Our analysis revealed 9831 genes unique to rice and 98 genes conserved across all 49 eukaryotic species analysed. The 98 genes conserved across diverse eukaryotes mostly exhibited binding and catalytic activities and shared common sequence motifs; and hence appeared to have a common origin. The 98 conserved genes belonged to 22 functional gene families including 26S protease, actin, ADP-ribosylation factor, ATP synthase, casein kinase, DEAD-box protein, DnaK, elongation factor 2, glyceraldehyde 3-phosphate, phosphatase 2A, ras-related protein, Ser/Thr protein phosphatase family protein, tubulin, ubiquitin and others. The consensus Bayesian eukaryotic tree of life developed in this study demonstrated widely separated clades of plants, fungi, and animals. Musa acuminata provided an evolutionary link between monocotyledons and dicotyledons, and Salpingoeca rosetta provided an evolutionary link between fungi and animals, which indicating that protozoan species are close relatives of fungi and animals. The divergence times for 1176 species pairs were estimated accurately by integrating fossil information with synonymous substitution rates in the comprehensive set of 98 genes. The present study provides valuable insight into the evolution of eukaryotes.

The draft genome of Corchorus olitorius cv. JRO-524 (Navin).

Here, we present the draft genome (377.3 Mbp) of Corchorus olitorious cv. JRO-524 (Navin), which is a leading dark jute variety developed from a cross between African (cv. Sudan Green) and indigenous (cv. JRO-632) types. We predicted from the draft genome a total of 57,087 protein-coding genes with annotated functions. We identified a large number of 1765 disease resistance-like and defense response genes in the jute genome. The annotated genes showed the highest sequence similarities with that of Theobroma cacao followed by Gossypium raimondii. Seven chromosome-scale genetically anchored pseudomolecules were constructed with a total size of 8.53 Mbp and used for synteny analyses with the cocoa and cotton genomes. Like other plant species, gypsy and copia retrotransposons were the most abundant classes of repeat elements in jute. The raw data of our study are available in SRA database of NCBI with accession number SRX1506532. The genome sequence has been deposited at DDBJ/EMBL/GenBank under the accession LLWS00000000, and the version described in this paper will be the first version (LLWS01000000).

Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.).

BACKGROUND: Increased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, 'HTM-N22', exhibiting tolerance to a broad spectrum herbicide, 'Imazethapyr', and identified the mutations imparting tolerance to the herbicide. RESULTS: We identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2 plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies. CONCLUSIONS: A novel mutant tolerant to the herbicide "Imazethapyr" was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.

Single-copy gene based 50 K SNP chip for genetic studies and molecular breeding in rice.

Single nucleotide polymorphism (SNP) is the most abundant DNA sequence variation present in plant genomes. Here, we report the design and validation of a unique genic-SNP genotyping chip for genetic and evolutionary studies as well as molecular breeding applications in rice. The chip incorporates 50,051 SNPs from 18,980 different genes spanning 12 rice chromosomes, including 3,710 single-copy (SC) genes conserved between wheat and rice, 14,959 SC genes unique to rice, 194 agronomically important cloned rice genes and 117 multi-copy rice genes. Assays with this chip showed high success rate and reproducibility because of the SC gene based array with no sequence redundancy and cross-hybridisation problems. The usefulness of the chip in genetic diversity and phylogenetic studies of cultivated and wild rice germplasm was demonstrated. Furthermore, its efficacy was validated for analysing background recovery in improved mega rice varieties with submergence tolerance developed through marker-assisted backcross breeding.

Natural allelic diversity in OsDREB1F gene in the Indian wild rice germplasm led to ascertain its association with drought tolerance.

KEY MESSAGE: Three coding SNPs and one haplotype identified in the OsDREB1F gene have potential to be associated with drought tolerance in rice. Drought is a serious constraint to rice production worldwide, that can be addressed by deployment of drought tolerant genes. OsDREB1F, one of the most potent drought tolerance transcription activator genes, was re-sequenced for allele mining and association study in a set of 136 wild rice accessions and four cultivated rice. This analysis led to identify 22 SNPs with eight haplotypes based on allelic variations in the accessions used. The nucleotide variation-based neutrality tests suggested that the OsDREB1F gene has been subjected to purifying selection in the studied set of rice germplasm. Six different OsDREB1F protein variants were identified on the basis of translated amino acid residues amongst the orthologues. Five protein variants were truncated due to deletions in coding region and found susceptible to drought stress. Association study revealed that three coding SNPs of this gene were significantly associated with drought tolerance. One OsDREB1F variant in the activation domain of OsDREB1F gene which led to conversion of aspartate amino acid to glutamate was found to be associated with drought tolerance. Three-dimensional homology modeling assisted to understand the functional significance of this identified potential allele for drought tolerance in rice. The natural allelic variants mined in the OsDREB1F gene can be further used in translational genomics for improving the water use efficiency in rice.

The first draft of the pigeonpea genome sequence.

Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety 'Asha'. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable 'Arhar' simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement.