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. Mantel's correlation coefficients between Jaccard's 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.

Isolation and characterisation of legumin promoter sequence from chickpea (Cicer arietinum L.).

Seed specific promoters are useful for expression of foreign genes in the seeds. We have isolated a Cicer arietinum legumin promoter from lambda EMBL genomic library and subcloned in pBluescript II KS (-) in Eco RV and Pst I site. The 2.762 kb Hind II Pst I fragment was sequenced completely by dideoxy chain termination method by creating a set of unidirectional deletions of the inserts in pAKKIII. The insert contains mainly upstream sequence (2240 bps) and only a part of structural gene (522 bps) sequence. The 522 bps of the structural gene shows approximately 80% homology with pea legumin A and this is almost the same as chickpea legumin in its sequence. The amino acid sequence derived from the part of the structural gene was similar to the chickpea 5' part of the legumin structural gene with a few variations. A 21 amino acid signal peptide was also deduced like many other legumes. Transcription start site (CAT) was located at 55 bp upstream of the initiation codon ATG. One codon downstream to ATG codon Hind III site was present. TATA box was observed at -30 position, with a consensus of CCTATAAATAACC. The consensus CATGCAAG, a part of legumin box was noticed at -110 bp position. At -295 to -265 bp upstream AGGA box like sequences were observed and a 56 bp perfect repeat was located between -913 bp and -972 bp. Strong homology with pea promoter sequence near the CAT sequence was noticed which gradually decreased towards the upstream region. Thus the cloned fragment contains a full length promoter which can be utilised for expression of foreign genes in seeds of chickpea.

Isolation and characterization of cDNAs encoding storage proteins of chickpea (Cicer arietinum L.).

A cDNA library was constructed in ;'ZAPll vector from poly(A)+ RNA isolated from developing seeds of chickpea (Cicer arietinum L.). Two cDNA clones encoding legumin protein were obtained by screening the library by plaque hybridization using a heterologous pea legumin cDNA probe. The pBluescript plasmids were excised from the phage clones and two clones designated pCSSK4 and pCSSK5 with inserts of 1.45 kb and 1.82 kb respectively were mapped by restriction and partially sequenced. The partial nucleotide sequence showed that these two cDNAs are not identical and showed sequence homology with the storage protein cDNA clones of other legumes.

Use of polymerase chain reaction in analysing recombinant cDNA clones encoding storage proteins of chickpea Cicer arietinum L.

A simple and reliable method was undertaken for the use of polymerase chain reaction in analyzing cDNA clones. Amplification was done of the inserts from positive legumin clones isolated from a cDNA library constructed from developing chickpea cotyledons in the expression vector, gt11. Amplification was made simple by using oligonucleotide primers which allowed convenient sizing, subcloning and sequencing of inserts by di-deoxy chain termination method. This simple method may provide opportunity to isolate large number of agronomically important genes from gene libraries.

Construction and screening of subgenomic library of chickpea (Cicer arietinum L.) for legumin genes and their analysis.

Legumin storage protein genes of chickpea were identified by hybridizing restricted chickpea genomic DNA transferred on Southern blot with heterologous pea legumin cDNA probes. Southern hybridization of restricted chickpea genomic DNA with pea legumin pDUB6 and pDUB8 cDNA probes indicated presence of pea legumin homologous sequences in chickpea. About 4-5 kb EcoRI fragment was hybridized with both the probes used. DNA corresponding to this size was isolated from the gel and used for construction of subgenomic library in lambda ZAPII vector. After amplification, library was screened for presence of legumin genes in chickpea. Out of clones screened (0.1 x 10(6)), three positive clones were obtained. Restriction analysis and hybridization studies showed presence of complete structural gene in each of the identified positive clones.

Isolation and subcloning of chitinase clone from chickpea genomic library.

Chickpea genomic library constructed earlier in phage lambda (EMBL-3) was screened for the presence of chitinase clone using tobacco chitinase cDNA as a probe. Positive clones obtained by primary screening of plaques (2 x 10(6)) were ascertained by secondary and tertiary screening. Presence of chitinase insert in the positive clones obtained, was further confirmed by restricting phage DNA with Sal I and then doing southern with tobacco chitinase. The insert band was eluted out and subcloned in puc 19 plasmid.

Construction of chickpea genomic library.

For construction of chickpea genomic library, DNA was isolated, purified on CsCl gradient and size fractionated into 15-20 Kb fragments after restriction with Sau 3A. These fragments were ligated to phage lambda (EMBL-3) vector and the recombinant molecules packaged in vitro into viable phage particles. The recombinant phages were obtained as phages on a P2 lysogen of E. coli (Spi- selection) and amplified to establish a permanent library. This is the first report of the construction of chickpea genomic library.