K. R. Koundal

Single-copy genes define a conserved order between rice and wheat for understanding differences caused by duplication, deletion, and transposition of genes

The high-quality rice genome sequence is serving as a reference for comparative genome analysis in crop plants, especially cereals. However, early comparisons with bread wheat showed complex patterns of conserved synteny (gene content) and colinearity (gene order). Here, we show the presence of ancient duplicated segments in the progenitor of wheat, which were first identified in the rice genome. We also show that single-copy (SC) rice genes, those representing unique matches with wheat expressed sequence tag (EST) unigene contigs in the whole rice genome, show more than twice the proportion of genes mapping to syntenic wheat chromosome as compared to the multicopy (MC) or duplicated rice genes. While 58.7% of the 1,244 mapped SC rice genes were located in single syntenic wheat chromosome groups, the remaining 41.3% were distributed randomly to the other six non-syntenic wheat groups. This could only be explained by a background dispersal of genes in the genome through transposition or other unknown mechanism. The breakdown of rice–wheat synteny due to such transpositions was much greater near the wheat centromeres. Furthermore, the SC rice genes revealed a conserved primordial gene order that gives clues to the origin of rice and wheat chromosomes from a common ancestor through polyploidy, aneuploidy, centromeric fusions, and translocations. Apart from the bin-mapped wheat EST contigs, we also compared 56,298 predicted rice genes with 39,813 wheat EST contigs assembled from 409,765 EST sequences and identified 7,241 SC rice gene homologs of wheat. Based on the conserved colinearity of 1,063 mapped SC rice genes across the bins of individual wheat chromosomes, we predicted the wheat bin location of 6,178 unmapped SC rice gene homologs and validated the location of 213 of these in the telomeric bins of 21 wheat chromosomes with 35.4% initial success. This opens up the possibility of directed mapping of a large number of conserved SC rice gene homologs in wheat. Overall, only 46.4% of these SC genes code for proteins with known functional domains; the remaining 53.6% have unknown function, and hence, represent an important, but yet, under explored category of genes.

AFLP Analysis of the Phenetic Organization and Genetic Diversity in the Sugarcane Complex, Saccharum and Erianthus

Amplified fragment length polymorphism (AFLP) markers were evaluated for determining the phylogenetic relationships, and the diversity in the Saccharum complex using 30 clones belonging to S. officinarum, S. robustum, S. spontaneum, S. barberi, S. sinense and the related genus Erianthus. The phenetic tree of the species clones based on AFLP data was consistent with the known taxonomical relationships. AFLP gave higher resolution of closely related species into discrete groups than that by RAPD and RFLP markers, reported earlier. The levels of diversity within the various Saccharum species were also found to be higher than those obtained previously with the same set of clones using RAPD markers. The intraspecies similarity in S. barberi and S. sinense was much higher than interspecies similarity suggesting a clear separation of the two, which are considered ‘horticultural species’. The genetic similarity matrix derived from a single primer combination highly correlated (r = 0.980) with that obtained from all the 12 primer combination used in the study, thus highlighting the efficiency of a single primer combination in delineating species relationships. All the primer combinations could identify markers that are specific to each of the species and the genus Erianthus. Among the species, specific markers were highest in S. spontaneum followed by S. robustum, S. barberi, S. officinarum and S. sinense. Erianthus had a distinct profile with 30% of the total amplified fragments being specific to it. This offers great scope for identifying intergeneric hybrids, which has been very difficult using morphological traits and RAPD markers. High degree of correspondence between the results from the cluster analysis based on Jaccards similarity index, Neighbour Joining tree based on Sokal and Michener distance matrix and AFTD (Analyses Factorielle on Table of Distances) analysis clearly demonstrated that AFLP markers would be an appropriate tool in providing better information about the relationships among the species, estimation of diversity, and in revealing species and genus specific markers that could be directly applied in sugarcane breeding programmes.

A comparison of physiological and yield characters in old and new wheat varieties

The Indian subcontinent has witnessed a spectacular improvement in yield of wheat during the past decade (Rao, 1978). This is reflected in the improvement of the average national yields as well as of those regions where wheat is grown as an irrigated crop (Sinha & Aggarwal, 1981). However, after the release of the double dwarf variety Kalyansona, only marginal improvement in yield has occurred in recent years. Despite this, the semi-dwarf character continues to be considered a major factor for improvement of wheat. Asana & Chattopadhyay (1970), Konar & Asana (1975), and Wattal & Asana (1976) observed no significant difference in yield between tall and semi-dwarf (medium tall) varieties in pot culture experiments where lodging was prevented and competition was partly reduced. They ascribed prevention from lodging and improvement in the ratio of grain to total above-ground dry matter as major advantages in the modern varieties. Somewhat similar conclusions have recently been drawn by Austin et al. (1980). However, a detailed comparison of various physiological and biochemical characters lias not been made to determine whether any advance has occurred in basic processes such as photosynthesis and nitrogen assimilation. The present study was an effort in this direction.

Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera

Protease inhibitors in legumes are one of the most promising weapons that confer resistance against insects by inhibiting proteases present in the gut of insect larvae. In the present study, trypsin inhibitor activity was detected in the seed flour extracts of 10 selected varieties of chickpea. The presence of inhibitor was confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor has been purified to near homogeneity to 60.46 fold and 29.20% recovery from chickpea seeds using heat denaturation, ammonium sulphate fractionation, DEAE-Sephadex A-25 and Sephadex G-75. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 30,000 Da. The purified inhibitor was active over a wide pH range although it retained maximum activity between pH 6 and 10. The inhibitor protein was stable up to 80°C but retained only 40% of activity when heated at 100°C for 20 min. The inhibitor lost its activity completely at 121°C. The chickpea trypsin inhibitor exhibited inhibitory activity against Helicoverpa armigera both in vitro and in vivo. In insect bioassay, a progressive decline in larval weight, growth and survival as well as temporal extension of larval growth was observed after feeding H. armigera larvae on diet supplemented with increasing concentrations of chickpea trypsin inhibitor. The adult emergence was also adversely affected by the inhibitor. It may be concluded that chickpea trypsin inhibitor has insecticidal potential against H. armigera.

SSR and AFLP based genetic diversity of soybean germplasm differing in photoperiod sensitivity

Forty-four soybean genotypes with different photoperiod response were selected after screening of 1000 soybean accessions under artificial condition and were profiled using 40 SSR and 5 AFLP primer pairs. The average polymorphism information content (PIC) for SSR and AFLP marker systems was 0.507 and 0.120, respectively. Clustering of genotypes was done using UPGMA method for SSR and AFLP and correlation was 0.337 and 0.504, respectively. Mantels correlation coefficients between Jaccards similarity coefficient and the cophenetic values were fairly high in both the marker systems (SSR = 0.924; AFLP = 0.958) indicating very good fit for the clustering pattern. UPGMA based cluster analysis classified soybean genotypes into four major groups with fairly moderate bootstrap support. These major clusters corresponded with the photoperiod response and place of origin. The results indicate that the photoperiod insensitive genotypes, 11/2/1939 (EC 325097) and MACS 330 would be better choice for broadening the genetic base of soybean for this trait.

Oxidation of 2-mercaptoethanol in the presence of tris buffer

2-Mercaptoethanol is oxidized by molecular oxygen, especially at high pH. This reaction is markedly increased by primary amines such as asparagine, glutamine and their corresponding amino acids and by the buffer tris(hydroxymethyl)aminomethane. Thus, the use of 2-mercaptoethanol in Tris buffer may present several complications: an uptake of oxygen which could be confused with metabolic activity; destruction of the protective mercaptoethanol; and by inference, quite possibly an enhanced oxidation of the protein sulphydryls in need of protection.

Assessment of Genetic Diversity in Ziziphus mauritiana Using Inter-Simple Sequence Repeat Markers

Genetic diversity among 47 ber accessions belonging to cultivated species (Ziziphus mauritiana Lam) and one wild accession of Ziziphus nummularia (Burm F) Willed was investigated using Inter-Simple Sequence Repeat (ISSR) markers. A total of 167 amplification products were detected with 18 ISSR primers of which 152 (89.96%) were polymorphic. Most of the primers that produced distinct bands (14 primers out of 18) contained dinucleotide repeats. Primers based on (AC)n and (AG)n repeats produced more polymorphic bands. Genetic similarity ranging from 43.07% to 90.30% suggested that the 48 Ziziphus genotypes used in the study were divergent. Cluster analysis based on UPGMA method and Bootstrap analysis separated all the 48 genotypes in four distinct clusters. The present study has successfully distinguished morphologically similar genotypes that emphasize the use of molecular markers to the taxonomists. Morphologically similar but genetically distinct genotypes, identified using ISSR markers could be potential sources for genotype identification and to resolve controversies over misnomination of ber genotypes. Present study is the first report on the exploitation of ISSR markers in ber for genetic diversity analysis.

Evaluation of Microsatellite Markers for Genome Mapping in Indian Mustard (Brassica juncea L)

Microsatellites are robust markers for genome mapping, gene tagging and marker assisted selection. The genus Brassica, having a large and complex genome, requires such type of markers for various applications in genetics and breeding. A set of 202 microsatellite markers were used to screen two parental genotypes of Indian mustard (Brassica juncea) namely, ‘Varuna’, an indigenous cultivar and BEC144, an exotic collection from Poland, of which 36 (17.8%) were informative and usable for segregation analysis. The polymorphic markers detected heterozygosity in advanced generation recombinant inbred lines (RILs) developed earlier from the cross Varuna × BEC144 with a varying frequency that ranged from 0% to 23.5%. Normal Mendelian segregation for majority of microsatellite markers was observed. Eleven markers showed significant deviation from the expected 1:1 segregation ratio. Twelve markers were assigned to six different linkage groups of Indian mustard genome map. The level of polymorphism between the parents and the percentage of useful informative markers as observed in this study, suggested that many more markers are needed to achieve a reasonable coverage of mustard genome. This is the first report on the evaluation of microsatellite markers for genome mapping in B. juncea.

Construction of Cowpea (Vigna unguiculata L) cDNA Library and Characterization of an Isolated Lectin Gene

The cDNA library of cowpea was constructed in λ ZAP II vector using poly (A+) RNA from developing seeds of cowpea. Initially, to demonstrate the presence of lectin genes in the cowpea genome, cowpea total genomic DNA was isolated, Southern blotted and hybridized with the heterologous pea lectin cDNA probe. It is observed that there exists a strong homology between lectin genes of cowpea and pea. Hence, cowpea cDNA library was screened for the presence of lectin genes using pea lectin cDNA probe. After primary, secondary and tertiary screening, eight positive clones were obtained. When subcloned into pBluescript, out of these eight clones, only four showed presence of larger cDNA inserts. When the DNA of these positive clones were Southern blotted and hybridized, they showed strong hybridization with the probe DNA. One of the clones was sequenced completely and the nucleotide sequence also showed strong homology with other legume lectin genes.

Cloning and Characterization of Legumin Storage Protein Gene of Chickpea (Cicer arietinum L)

Genomic DNA clones coding for legumin storage protein were isolated from chickpea (Cicer arietinum L.) subgenomic library using pea legumin cDNAs as a probe. The complete nucleotide sequence of the legumin gene (leg3) was determined. Sequence analysis indicated that leg3 contains 2022 bp long structural gene interrupted by three small introns. The gene encodes a polypeptide of 496 amino acids including a signal peptide of 21 amino acid residues and a-b subunit cleavage site. Leg3 polypeptide is rich in amide amino acids and contains 5 methionine and 6 cysteine amino acids. DNA sequence comparison showed about 70–80% identity with legumin genes from pea, soybean and broad bean. The 5’ flanking region contains regulatory elements such as TATA and legumin box and 3’ region contains a stop codon and two polyadenylation signals. Southern hybridisation indicated the presence of multiple copies of /eg3in chickpea and estimated about 10–12 copies per haploid genome. Northern blot hybridisation showed that leg3 expresses in developing seed and not in other tissues of chickpea indicating temporal and tissue-specific expression of this gene.