Genetic relationships of 16 cultivars of pigeonpea (Cajanus cajan (L) Millsp.) and its two wild relatives (C. albicans and C. lineatus) from different parts of the India were analysed using 22 random amplified polymorphic DNAs (RAPDs) primers and 10 inter simple sequence repeats (ISSRs) primers. Twenty two RAPD primers yielded 151 polymorphic markers (71.2%) with an average of 6.8 polymorphic band/primer. Cluster analysis based on these 151 RAPD markers revealed relatively low level (0.434 - 0.714) of genetic diversity among cultivars and high level of diversity between cultivars and wild relatives. Ten ISSR primers produced 100 bands across 16 cultivars and its wild relatives out of which 93 (93%) were polymorphic with an average of 9.3 polymorphic band/primer. Cluster analysis based on these 93 ISSR markers also revealed relatively higher level (0.328 - 0.827) of genetic diversity among cultivars as compared to RAPD markers. The polymorphic markers obtained by both RAPD and ISSR primers were pooled and the genetic diversity analysis based on these 244 markers was analysed. Jaccard’s similarity coefficient obtained by pooled data revealed very narrow range (0.477 - 0.720) among cultivated and high range between cultivated and wild species C. albicans (0.240 - 0.331) and C. lineatus (0.163 - 0.193). In the UPGMA based dendrogram the 16 cultivars were grouped into three distinct clusters. Cluster I contained two genotypes, cluster II had six and cluster III had eight genotypes. Principal components analysis (PCA) also resulted in similar pattern as that of UPGMA based analysis. The first three PCs contributed 56.26%, 5.71% and 4.97% of variation, respectively, with cumulative variation of the first three PCs was 66.96%. Both the markers and the combined data revealed similar pattern with narrow diversity among cultivars and higher diversity between cultivars and wild one, but the genetic diversity range obtained by ISSR markers was relatively higher as compared to RAPD and pooled data. Furthermore, both the markers also correlated the clustering of stress resistant genotypes together. Cultivar Pusa-2002 possessed more diversity with other genotypes in ISSR dendrogram.
Pigeonpea [Cajanus cajan (L.) Millsp.] is an important crop in semi-arid tropical and subtropical farming systems, providing high quality vegetable protein (20% - 22%), animal feed, and firewood. Pigeonpea is a tall, woody, perennial legume with centre of diversity in India [
The productivity of pigeonpea in the last five decades has remained low at about 700 kg/ha (http://faostat.fao.org/site/339/default.aspx). Pigeonpea is an important pulse crop of India but as compared to other grain legumes it has received relatively little research attention. Moreover, genetic improvement in production and productivity of pulse crops has been very slow owing to several constraints. Therefore, many pigeonpea breeding programmes are focused on improving the genetic potential both to increase yield and to provide protection against abiotic and biotic stresses. In order to enhance genetic potential, there must be a comprehensive understanding of the amount and pattern of genetic variation that exists within and between the available cultivated and wild accessions.
A large number of polymorphic markers are required to measure genetic relationships and genetic diversity in a reliable manner [
ISSR marker is based on primers having microsatellite sequences, and detects variation in the size of inter- satellite regions; therefore, a prior knowledge of the template DNA sequence is not required [
The objectives of the present investigation were to study and compare genetic diversity patterns among 16 pigeonpea genotypes and its wild relatives (C. albicans and C. lineatus), using RAPD and ISSR markers, and to evaluate the degree of polymorphism generated by each technique as a pre-requisite for their applicability to breeding programme in pigeonpea.
The plant material comprised of 18 accessions of pigeonpea including 16 cultivars of C. cajan and 2 accessions of its wild progenitor (C. albicans and C. lineatus) (
Pigeonpea seeds were surface sterilized with 0.2% HgCl2 and washed 3 - 4 times and 3grown in pots in sunlight for three weeks. DNA was isolated from young leaves using HiPurA™ Plant genomic DNA Miniprep Purification spin kit (Himedia Laroratories Pvt. Ltd). Quality and quantity of isolated DNA was checked by spectrophotometry as well as by 0.8% agarose gel electrophoresis. The DNA yield obtained was in the range of 1.0 μg to 3.0 μg.
A total of 30 decamer primers (GC content 60% - 90%) were used for RAPD analysis. Out of 30 primers 19 (OPA-19, OPB-14, OPB-17, OPB-19, OPC-05, OPH02, OPH-03, OPH-05, OPH-10, OPH-11, OPH-12, OPM-07, OPP-07, OPP-09, OPAQ-05 OPAQ-18, OPAQ-19, OPAZ-05 and OPAZ-18) belong to Operon series (Operon Technologies USA) and 11 (P-23;GTAGGCGTCG, P-24;GGCTCGTACC, P-25;GACCCCGGCA, P-26; CAGGGGACGA, P-27;CGCCACGTTC, P-28;GCCTCCTACC, P-29;GGCGTCGGGG, P-30;CAGGGCCGCT, P-31;CTCTCCGCCA, P-32;CTCGGCTGGA and P-33;AGGCCCGATG) were selected randomly. The PCR amplification protocol is described in our published paper [
ISSR-PCR was performed by means of 15 primers out of which 11 were 3’-anchored [GAGAGAGAGAGAGA- GAT, GAGAGAGAGAGAGAGAC, GAGAGAGAGAGAGAGAA, ACACACACACACACACCT, AGAGA- GAGAGAGAGAGCT, GTGTGTGTGTGTGTGTA, GTGTGTGTGTGTGTGTCT, CACCACCACGC, CTCT- CTCTCTCTCTCTGC, GAGAGAGAGAGACC, AGAGAGAGAGAGAGAGTG] and four were non-anchored [GACAGACAGACAGACA, GACTGACTGACTGACT, GTGTGTGTGTGTGTGTGT, ACTGACTGACTGA- CTG] primers. Each amplification reaction contained 10 mM Tris-HCl, pH 9.0, 2 mM MgCl2, 50 mM KCl, 0.01% gelatin, 200 µM of each dNTP, 4 µM of primer, 0.6 Units of Taq DNA polymerase (Bangalore Genei, Bangalore, India) and 30 ng of genomic DNA in total volume of 25 µl. The PCR amplification was carried out for 40 cycles in a thermal cycler (PTC-200, Bio-Rad USA). The reaction had initial denaturation step at 94˚C for 5 min, followed by 40 cycles of 94˚C for 1 min, annealing temperature for 1 min, 72˚C for 2 min. The final extension step was at 72˚C for 5 min. Amplified products were separated on 1.8% agarose gels having 0.5 µg・ml−1 of the ethidium bromide at 50 V for 3 h. The gels were observed under a UV light source in a gel documentation system (BIOVIS Gel, Expert Vision Labs Pvt. Ltd, India).
Clearly resolved, unambiguous polymorphic band were scored visually for their presence or absence with each
S. No. | Variety | State | Pedigree | Place of origin | Year of release | Maturity duration (days) | 100 seed wt. (g) | Special features |
---|---|---|---|---|---|---|---|---|
1. | ICPL87 Pragati | M.P, Gujarat, M.S | T21 × JA277 | ICRISAT Patancheru | 1986 | 116 - 126 | 8.6 | Determinate, brown seeded, spreading, resistant to wilt |
2. | AK-101 | Maharashtra | Selection from germplasm | Akola (Maharashtra) | - | 145 - 160 | 7.9 | Tolerant to wilt, semi-spreading, Indeterminate, |
3. | Vamban 1 | Tamil Nadu | (Prabhat × HY3A) × (T21 × 102) | TNAU, Vamban | 1993 | 95 - 100 | 7.5 | Determinate, Suitable for Peanut intercropping |
4. | Jawahar (JKM7) | M.P., Gujarat, Maharashtra | ICP8863 × LRG30 | Jknvv, Khargon | 1996 | 173 - 180 | 9.3 | Tolerant to wilt and Phytophthora blight |
5. | ICPL85063 (Laxmi) | A.P. | BDN1 × (T21 × JA275) | RAS, Lam | 2000 | 160 - 200 | 9.9 | semi-spreading, suitable for rabi planting also |
6. | Azad | UP, Bihar | Bahar × NP (Wr) 15 | CSAUA & T, Kanpur | 1997 | 153 - 210 | 10.1 | Indeterminate, resistant to sterility mosaic semi-spreading |
7. | Pusa 2002 | New Delhi | P945 × Pusa 78 | IARI, New Delhi | 2002 | 130 | 7.5 | Early maturing, indeterminate |
8. | BDN- 2 | Maharashtra | Sel. From local bori11-132-A-1 | ARS, Badnapur | 1978 | 150 - 160 | 8.6 | Indeterminate, Tolerant to wilt |
9. | GT 101 | Gujarat | BWR 24 × Pusa Sweta | S.D. Agri. Univ. S.K. Nagar | 2002 | 133 - 185 | 11.0 | Indeterminate, semi-spreading |
10. | Malviya Vikalp (MA-3) | M.P., Gujarat, Maharashtra | Sel. From land races no MA-2 | BHU, Varansi | 1999 | 178 - 262 | 8.0 | Spreading, Constricted pod, resistant to pod fly |
11. | C11 | M.P, Gujarat, M.S | Sel. from sanga Reddy (A.P.) | A.P. | 1982 | 195 - 200 | 9.0 | Profuse branching, brown seeded, Tolerant to wilt |
12. | Manak | Punjab, Haryana, North Raj, West U.P. | T21 × UPAS120 | HAU, Hisar | 1985 | 120 - 130 | 6.5 | Indeterminate, semi-spreading, small seeded |
13. | Paras (H82-1) | Haryana | EE76 × UPAS120 | HAU, Hisar | 1998 | 133 - 145 | 7.8 | Indeterminate, tolerant to wilt |
14. | Birsa Arhar | Bihar | Local sel. Land races Ranchi | BAU, Kanke (Bihar) | 1992 | 180 - 200 | - | Resistant to wilt under field condition |
15. | Pusa 84 | Punjab, Haryana, North Raj, West U.P | Pusa Ageti × T21 | IARI, New Delhi | 1985 | 140 - 150 | 7.5 | Semi-spreading, determinate, semi tall, brown seeded |
16. | T15x15 | Gujarat | Sel. From land races | GAU, Gujarat | 1985 | 200 - 210 | 9.0 | Indeterminate, White seeded, suitable for vegetable purpose also |
17. | Cajanus albicans (wild) | NKR 185 | A.P. | |||||
18. | Cajanus lineatus (wild) | JM 3366 | Tamil Nadu |
RAPD and ISSR primer. The scores were obtained in the form of a matrix with “1” and “0”, which indicate the presence and absence of bands in each variety respectively. The individual RAPD and ISSR profiles as well as pooled data (RAPD+ISSR) were analysed for genetic diversity analysis. Jaccard’s similarity coefficient [
In RAPD analysis, our earlier results [
ISSR primers produced different numbers of DNA fragments, depending on their simple sequence repeat motifs. Out of the 15 primers tested, 10 produced reproducible and polymorphic patterns. The 10 primers yielded a total of 100 fragments (average 10 bands/primer), of which 93 amplicons (93%) were polymorphic, the number of polymorphic bands per primer ranged from 5 to 15, the average being 10 (
Marker system | Number of primer | Polymorphism (%) | Average number bands per primer | Average number of polymorphic bands per primer |
---|---|---|---|---|
RAPD | 22 | 71.2 | 9.6 | 6.8 |
ISSR | 10 | 93 | 10 | 9.3 |
RAPD + ISSR | 32 | 76.2 | 9.7 | 7.6 |
The genetic diversity among pigeonpea cultivars and wild relatives was analyzed by UPGMA method. In RAPD analysis, 151 polymorphic markers amplified were analyzed for genetic diversity. Jaccard’s similarity coefficients among C. cajan cultivars ranged from 0.434 - 0.714 [
Genetic diversity analysis based on 93 ISSR markers yielded Jaccard’s similarity coefficients range from 0.328 - 0.827 among C. cajan cultivars (
The RAPD and ISSR data were combined for UPGMA cluster analysis. The 244 markers (151 RAPD + 93 ISSR) revealed varying degrees of genetic relatedness among wild and cultivated genotypes. Jaccard’s similarity coefficients among C. cajan cultivars revealed very narrow range from 0.477 - 0.720 and high range between C. cajan cultivars and its wild relatives C. albicans (0.240 - 0.331) and C. lineatus (0.163 - 0.193) (
In the present investigation, two multilocus marker systems viz., RAPD and ISSR were evaluated for their efficiency in revealing the genetic diversity among the pigeonpea genotypes and its wild relatives studied. RAPD markers produced 71.2% polymorphic band with an average 9.63 band per primer. ISSR markers amplified 93% polymorphic band with an average 10 band per primer. Clearly the ISSR markers are more efficient than the RAPD assay, as they revealed more polymorphism than that of RAPD markers. Similar results were obtained in several other studies involving pigeonpea [
Jaccard’s similarity coefficients among 16 C. cajan cultivars used ranged between 0.434 - 0.714 (RAPD), 0.328 - 0.827 (ISSR) and 0.477 - 0.720 (RAPD + ISSR). ISSR marker showed relatively higher range of similarity coefficients as compared to RAPD and RAPD + ISSR data in C. cajan cultivars. Similar results were also found in chickpea [
Cluster analyses revealed three main clusters viz., cluster I, II and III in RAPD + ISSR dendrogram, which has similar pattern to that of ISSR dendrogram whereas, the dendrogram based on RAPD showed some variation in the clustering of genotypes. Sixteen genotypes were grouped in three clusters in ISSR and ISSR + RAPD data; whereas the dendrogram based on RAPD data showed two main clusters. Clustering of genotypes within groups was not similar when RAPD and ISSR derived dendrogram were compared, whereas the pattern of clustering of the genotypes remained more or less the same in ISSR and combined data of RAPD + ISSR. In all the three dendrograms, both the wild species C. lineatus and C. albicans formed out groups as they did not cluster with any of the pigeonpea cultivars. Pusa-2002 showed more diversity from other cultivars in ISSR dendrogram; this pattern was not found in RAPD and RAPD + ISSR dendrograms. On comparing these results it can be concluded that ISSR marker has more differentiating power than RAPD. In ISSR and RAPD + ISSR dendrograms, we obtained the almost same sample distributions while dendrogram from RAPD is little different. The differences in clustering pattern of genotypes using RAPD and ISSR markers may be the different number of PCR products analysed, this confirms the importance of the number of loci and their coverage of the overall genome for producing reliable estimates of genetic relationships among cultivars [
The dendrogram based on RAPD + ISSR markers clearly separated various biotic and abiotic stress tolerant genotypes together (