Mukesh Kumar Rana

Genetic Analysis of Recombinant Inbred Lines for Sorghum bicolor × Sorghum propinquum

We describe a recombinant inbred line (RIL) population of 161 F5 genotypes for the widest euploid cross that can be made to cultivated sorghum (Sorghum bicolor) using conventional techniques, S. bicolor × Sorghum propinquum, that segregates for many traits related to plant architecture, growth and development, reproduction, and life history. The genetic map of the S. bicolor × S. propinquum RILs contains 141 loci on 10 linkage groups collectively spanning 773.1 cM. Although the genetic map has DNA marker density well-suited to quantitative trait loci mapping and samples most of the genome, our previous observations that sorghum pericentromeric heterochromatin is recalcitrant to recombination is highlighted by the finding that the vast majority of recombination in sorghum is concentrated in small regions of euchromatin that are distal to most chromosomes. The advancement of the RIL population in an environment to which the S. bicolor parent was well adapted (indeed bred for) but the S. propinquum parent was not largely eliminated an allele for short-day flowering that confounded many other traits, for example, permitting us to map new quantitative trait loci for flowering that previously eluded detection. Additional recombination that has accrued in the development of this RIL population also may have improved resolution of apices of heterozygote excess, accounting for their greater abundance in the F5 than the F2 generation. The S. bicolor × S. propinquum RIL population offers advantages over early-generation populations that will shed new light on genetic, environmental, and physiological/biochemical factors that regulate plant growth and development.

Multi-locus DNA fingerprinting and genetic diversity in jute (Corchorus spp.) based on sequence-related amplified polymorphism

Sequence-Related Amplified Polymorphism (SRAP) markers were used for genetic diversity assessment and cultivar identification among 31 cultivars of jute belonging to two cultivated species Corchours olitorius L. and C. capsularis L. Forty-three primer-pairs produced a total of 394 bands with an average of 9 bands per primer pair and 89% bands were polymorphic across the genotypes of two species. Average genetic diversity in the cultivars of C. olitorius and C. capsularis was 7.2% (range 2.8–12.3%) and 7.6% (range 2.2–13.1%), respectively. Jute cultivars JRC 698, JRC 7447, TJ 40, S19 and JRO 3690 were more diverse compared to rest of the cultivars. UPGMA cluster analysis grouped all cultivars into two clusters which were representative of C. olitorius and C. capsularis species. All the cultivars could be unequivocally differentiated from one another based on the pooled profile of 43 primer-pairs, however, 24 of 31 cultivars could be identified uniquely. The probability of chance identity of any two cultivars based on SRAP markers was very low and was 6.95 × 10−07 and 2.23 × 10−07 for cultivars of C. capsularis and C. olitorius, respectively. Primer-pairs EM1-ME5, EM4-ME1, EM8-ME1 and EM10-ME1 were found to be useful for genetic diversity and cultivar identification. Our results show that SRAP markers could be effectively used for genetic diversity analyses in jute. For poor genetic diversity and resulting narrow genetic base, these markers will prove to be highly useful for identifying elite germplasm in a jute breeding program.

Comparative analysis of Gossypium and Vitis genomes indicates genome duplication specific to the Gossypium lineage

Genetic mapping studies have suggested that diploid cotton (Gossypium) might be an ancient polyploid. However, further evidence is lacking due to the complexity of the genome and the lack of sequence resources. Here, we used the grape (Vitis vinifera) genome as an out-group in two different approaches to further explore evidence regarding ancient genome duplication (WGD) event(s) in the diploid Gossypium lineage and its (their) effects: a genome-level alignment analysis and a local-level sequence component analysis. Both studies suggest that at least one round of genome duplication occurred in the Gossypium lineage. Also, gene densities in corresponding regions from Gossypium raimondii, V. vinifera, Arabidopsis thaliana and Carica papaya genomes are similar, despite the huge difference in their genome sizes and the different number of WGDs each genome has experienced. These observations fit the model that differences in plant genome sizes are largely explained by transposon insertions into heterochromatic regions.

CAAT box- derived polymorphism (CBDP): a novel promoter -targeted molecular marker for plants

The availability of a simple, reproducible and cost-effective molecular marker is a prerequisite for plant genetic analysis. We have developed a novel promoter-targeted marker, CAAT box- derived polymorphism (CBDP) using the nucleotide sequence of CAAT box of plant promoters. CBDP, like random amplified polymorphic DNA (RAPD), uses single primer in polymerase chain reaction (PCR) for generating markers. However unlike RAPD, the CBDP primers are 18 nucleotides long and consist of a central CCAAT nucleotides core flanked by the filler sequence towards the 5′ end and di- or trinucleotides towards the 3′ end. In this study, a small set of 25 CBDP primer was designed and initially tested in a representative set of eight cultivars of jute for generation of polymorphic markers. Further, to achieve high reproducibility, a touchdown PCR was employed with an annealing temperature of 50ºC. All the CBDP primers generated polymorphic markers in jute cultivars, and an UPGMA dendrogram based on Jaccard’s similarity grouped them into two clusters represented by Corchorus capsularis and C. olitorius, respectively. Interestingly, such grouping of jute cultivars was consistent with genetic relationships established earlier for these cultivars using other DNA markers. Moreover, these CBDP primers also generated polymorphic markers in representative sets of cotton (Gossypium species) and linseed (Linum usitatissimum ) cultivars. Given the high success rate of CBDP primers in generating markers in the tested species and advantages like ease in marker development and assay with reproducible profiles, they could potentially be exploited in other species as well for assessing genetic diversity, cultivar identification, construction of linkage map and marker- assisted selection.

Genetic Analysis of Indian Lentil ( Lens culinaris Medikus) Cultivars and Landraces Using RAPD and STMS Markers

Molecular analysis of 29 lentil (Lens culinaris) cultivars and landraces of Indian origin was carried out using twenty RAPD and ten cross-species STMS primers. A total of 97 markers (72 RAPD and 25 STMS) were amplified of which 42.3% were polymorphic. Genetic similarity among the cultivars and landraces was 89.7%. The observed results indicated low level of genetic diversity in the studied material. UPGMA cluster analysis for the combined data of RAPD and STMS revealed two broad clusters — Cluster I with three landraces and Cluster II containing all remaining landraces and cultivars except Precoz. Germplasm line Precoz was found to be the most distinct in individual as well as combined analyses. All cultivars and landraces except K-75 and L4076 could be discriminated from one another using combined data for the two techniques. Germplasm lines Precoz, L830 and cultivars L4147 and JL3 were quite distinct and could be potential germplasm resource.

Analysis of Indian Sorghum [ Sorghum bicolor (L) Moench] Cultivars and Lines Using RAPD Markers

Random Amplified Polymorphic DNA (RAPD) analysis was performed for the identification and assessment of genetic relationships in Indian sorghum comprising released varieties, hybrids and their parental lines (A, B and R-lines). Twenty-three random decamer primers generated 151 amplicons, of which 87 were found to be polymorphic (57.6%). The primer OPL-03 was the most informative as it could alone discriminate 13 accessions out of the 37 studied. The accessions 27B, C 43, CSV 14R and HC 6 were found to have specific bands. Four of the male sterile (27A, 296A, 2219A and AKMS 14A) lines and their respective maintainer (27B, 296B, 2219B and AKMS 14B) lines could be easily differentiated with thirteen random primers (OPA-09, OPC-02, OPD-07, OPE-04, OPE-16, OPG-09, OPL-01, OPL-03, OPL-05, OPL-15, OPL-18, OPV-01 and OPX-12). Based on Jaccard’s similarity coefficient values, the most distant two accessions were CS 3541 and CSV 14R and the closest were 104A and 104B, with similarity values of 0.64 and 1.00 respectively. The average Jaccard’s similarity coefficient of 0.78 revealed low level of genetic diversity in the investigated material. The dendrogram generated by UPGMA could group all the accessions into distinct clusters consistent with established classification viz. grain and forage sorghum and known pedigree information.

Cloning and in silico analysis of a gene encoding a putative β-carbonic anhydrase from cowpea (Vigna unguiculata L. Walp)

A gene encoding putative β-carbonic anhydrase gene (CA) from cowpea (Vigna unguiculata L. Walp.) was characterized and designated as VuCA1 (GenBank ID: JQ429799). While the genomic sequence of CA was found to be 1470 bp long with 4 introns, the open reading frame was 990 bp in length. The deduced amino acid sequence of CA contained two characteristic conserved domains, i.e. CSDSRV and EYAVLHLKVSNIVVIGHSACG, which showed high degree of homology with other β-CA genes of angiosperms. We have reported three-dimensional model of VuCA1 and quality of the predicted model was analysed with PROCHECK. Molecular docking of modeled VuCA1 revealed similar binding pockets for different substrates and products. The expression of VuCA1 transcripts was detected in different parts of the cowpea plant, with highest level observed in leaves, followed by flower buds, weak expression in stems, and the weakest in roots. The expression of VuCA1 transcripts of leaves was downregulated by zinc deficiency but upregulated by salt and dehydration stress.

Assessment of genetic diversity and DNA profiling of linseed ( Linum usitatissimum subsp. usitatissimum L.) germplasm using SSR markers

Access to genetic diversity is essential for any progress in adapting linseed (Linum usitatissimum subsp. usitatissimum L.) cultivation to changing environmental conditions or to the changing market needs. An attempt has been made in the present study to assess genetic diversity in 96 genotypes of linseed including varieties, landraces and exotic material. A total of 38 SSR primers amplified 153 alleles with 4.0 alleles per marker locus. The number of alleles ranged from 2 to 15 and the observed polymorphism ranged from 50 to 100%. Average genetic dissimilarity ranged from 2 to 50%. In order to analyze the efficiency for unambiguous identification of linseed germplasm, various statistical measures, viz., number of genotyping patterns, polymorphism information content, resolving power, discrimination power, probability of identity and probability of random identity, identified a set comprising of primers LU7, LU27, LU25, LU20 and LU31 (or LU637) for DNA fingerprinting of linseed germplasm. UPGMA cluster analysis showed that all genotypes could be grouped into four main clusters. Cluster 2 was the largest consisting of mainly landraces, whereas, Cluster 4 was the smallest. Cluster 1 consisted of mainly the released cultivars. Cluster 3 and Cluster 4 were smaller clusters and consisted of exotic genotypes. Principal co-ordinate analysis further substantiated the UPGMA clustering patterns of the observed genetic relationship. To explain 70–80% variability, 17–23 PCOs were needed, whereas 70 components were needed to explain the whole variability in the linseed material under study. Analysis of molecular variance indicated that most of the genetic variation is owing to the individuals within single population, whereas grouping of linseed material into varieties, landraces and exotics accounted for nearly 10% of the total genetic variation. The utility of SSR markers in diversity assessment and cultivar identification is discussed.

Erratum to: Genome-wide Analysis of Zinc Transporter Genes of Maize (Zea mays)

Zinc (Zn) is an essential micronutrient for plants and animals. Zinc-regulated transporters and iron-regulated transporter-like proteins (ZIP) are important zinc transporters in plants with the characteristic ZIP domain (Pfam:PF02535). Although individual genes belonging to the ZIP family had been discovered in various plants, genome-wide analysis of the paralouges (ZmZIP) in maize and their relationships with other related genera has so far not been conducted. We performed a genome-wide analysis and identified 12 members of the ZIP gene family in maize. Chromosomal locations, motif organization, and biochemical characterizations of proteins, as well as exon–intron, trans-membrane domains of these ZmZIP genes were determined, which indicated the structural diversity of ZmZIP. Additionally, apart from the identification of the canonical form of the metal binding signature in ZIP domains of the ZmZIP proteins, we also identified a new conserved plant ZIP signature. Further, tissue-specific expressions of those genes were determined by real-time PCR in the flag leaf as well as in 10-day-old-baby kernel among the high and low kernel zinc-containing maize inbreds. We found that overall transcript abundance was higher in the flag leaf than the kernel in both the inbreds for all the members except two, namely ZmZIP5 and ZmZIP11 were expressed more in flag leaf of a high-kernel zinc-containing inbreds than a lowkernel zinc-containing inbreds. Therefore, these results provide a basis for further functional characterization of specific ZmZIP genes in the future.