Cytogenetics of F1 hybrids between Cajanus and Atylosia species and its phylogenetic implications

Summary Cytogenetic relationships between two cultivars of Cajanus cajan and six species of Atylosia were investigated. Of the 12 cross combinations obtained, only seven could be studied. Meiotic chromosome pairing, pollen and ovule fertility in parental species and four F1 hybrids were near normal. Some meiotic abnormalities were observed in the Fls: A. lineata x A. scarabaeoides, A. scarabaeoides x A. sericea and C. cajan (UPAS 120) x A. trinervia, indicating varying degrees of chromosomal and genic differences between these species. These observations suggested that A. cajanifolia is the closest wild relative of C. cajan, followed by A. scarabaeoides, A. albicans and A. trinervia. Among the Atylosia species, A. sericea was closer to A. scarabaeoides than to A. lineata.Botanist, Genetic Resources Unit, ICRISAT, Patancheru-502 324, IndiaRegional Plant Production and Protection Officer, FAO, RAPA, Bangkok-2, Thailand     

Genomic relationships among 11 species in the genus Cajanus as revealed by seed protein (albumin and globulin) polymorphisms

SDS-PAGE analysis of seed albumins and globulins from two pigeonpea, Cajanus cajan, cultivars (DSLR-17 and BDN-2) and ten wild species, including C. cajanifolius, C. lineatus, C. sericeus, C. acutifolius, C. lanceolatus, C. reticulates, C. albicans, C. scarabaeoides, C. volubilis and C. platycarpus, resulted in 34 albumin and 27 globulin polypeptides. Proximity matrix analysis based on electrophoretic banding patterns of albumins and globulins jointly revealed C. cajanifolius to be closest to C. cajan having similarity coefficients of 0.595 and 0.676, respectively. Cluster analysis also exhibited the grouping of C. cajanifolius with C. cajan in one cluster. C. platycarpus has been out grouped. The present analysis more or less agreed with the sectional classification of the genus Cajanus and it has been hypothesized that cultivated pigeonpea has evolved through multi-genomic interaction involving C. cajanifolius and that it has experienced minor genomic reorganization during its divergence.     

Genetic variation of trypsin and chymotrypsin inhibitors in pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives

Variation in the trypsin inhibitors (TIs) and the chymotrypsin inhibitors (CIs) among 69 pigeonpea [Cajanus cajan (L.) Millsp.] strains from a wide geographical distribution and among 17 accessions representing seven wild Cajanus species was studied by electrophoretic banding pattern comparisons and by spectrophotometric activity assays. The TI and CI electrophoretic migration patterns among the pigeonpea strains were highly uniform but varied in the inhibitor band intensities. The migration patterns of the inhibitors in the wild Cajanus species were highly species specific. The mean TI activity of pigeonpea strains (2279 units) was significantly higher than that of the wild Cajanus species (1407 units). However, the mean CI activity in the pigeonpea strains (62 units) was much lower than that in the wild species (162 units). Kenya 2 and ICP 9151 were the lowest and the highest, respectively, in both the TI and CI activities among all the pigeonpea strains used in this study. A highly-significant positive correlation was observed between the TI and CI activities. The Bowman-Birk type inhibitors with both TI and CI activities were identified in all the pigeonpea strains and also in the accessions of all the wild species except C. volubilis (Blanco) Blanco. The C. volubilis accession ICPW 169 was found to be null for both CI bands and CI activity. Environment, strain, and environment x strain interaction showed highly-significant effects on both the TI and CI activities. Growing the pigeonpea strains at a different environment from their area of adaptation increased TI and CI activities and also altered the maturity period.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

Characterization of Kenyan Isolates of Fusarium udum from Pigeonpea [Cajanus cajan (L.) Millsp.] by Cultural Characteristics, Aggressiveness and AFLP Analysis

Isolates of Fusarium udum from pigeonpea (Cajanus cajan) plants with wilt symptoms were collected from various districts in Kenya and were characterized using cultural characteristics, aggressiveness and amplified fragment length polymorphism (AFLP). The 56 isolates of F. udum showed a high level of variability in aerial mycelia growth, pigmentation and radial mycelia growth (colony diameter) on potato dextrose agar. The aggressiveness of 17 isolates of F. udum on seven pigeonpea varieties varied and five aggressive groups were observed in the present study. There were no relationships among cultural characteristics and aggressiveness. AFLP analysis of the 56 isolates was tested for genetic variability using seven primer combinations. A total of 326 fragments was generated of which 121 were polymorphic. Ten AFLP groups were identified among the Kenyan isolates and, although they were not genetically distinct, six AFLP subgroups were genetically distinct. AFLP had no relationship with cultural characteristics, aggressiveness and geographical origin of the isolates. This is the first report on the study of genetic variability of F. udum using DNA analysis.     

Nodule haustoria and microbial features of Cajanus and Pongamia parasitized by sandal (sandal wood)

A field observation that roots of sandal wood tree (Santalum album (L.)) formed direct haustorial connections with root nodules of nodulating legumes was confirmed by pot culture studies on interaction between sandal wood plants and pigeonpea (Cajanus cajan (L.) Millsp.) or Pongamia glabra Vent. plants. The number of nodules and nitrogen content of plants decreased in parasitized nodulating species with corresponding increase in the nitrogen content of sandal plants. The root region of sandal had more of nitrogen-fixing bacteria and VAM fungi than those of pigeonpea.     

Broad-based resistance to pigeonpea sterility mosaic disease in wild relatives of pigeonpea (Cajanus: Phaseoleae)

Sterility mosaic disease (SMD), an important biotic constraint on pigeonpea (Cajanus cajan) in the Indian subcontinent, is caused by Pigeonpea sterility mosaic virus (PPSMV) transmitted by the eriophyid mite, Aceria cajani. Distinct PPSMV isolates occur in different geographical regions and broad‐based resistance to all these isolates is scarce in cultivated pigeonpea germplasm. Wild relatives of pigeonpea, which are known to possess resistance to several pests and diseases, were evaluated for broad‐based SMD resistance. One hundred and fifteen wild Cajanus accessions from six species (C. albicans, C. platycarpus, C. cajanifolius, C. lineatus, C. scarabaeoides and C. sericeus) were evaluated against three PPSMV isolates prevailing in peninsular India. Evaluations were done under greenhouse conditions in endemic locations of each isolate through mite‐mediated virus inoculation. Fifteen accessions showed resistance to all three isolates: ICP 15614, 15615, 15626, 15684, 15688, 15700, 15701, 15725, 15734, 15736, 15737, 15740, 15924, 15925 and 15926. Most of the wild accessions did not support mite multiplication. The majority of the accessions resistant to PPSMV following inoculations with viruliferous mites were susceptible by graft inoculation, suggesting that vector resistance is conferring resistance to infection with PPSMV. The 15 accessions identified as being resistant to infection to all three virus isolates tested are cross compatible with pigeonpea by traditional breeding. They are therefore useful for exploitation in breeding programmes to increase both the level of SMD resistance and to diversify its genetic base in the cultivated pigeonpea gene pool.     

Development of simple sequence repeat (SSR) markers for the assessment of gene flow and genetic diversity in pigeonpea (Cajanus cajan)

Pigeonpea (Cajanus cajan) is an important subsistence crop in India where traditional landraces and improved hybrids are grown alongside each other. Gene flow may result in genetic erosion of these landraces and their wild relatives, whilst transgene escape from future genetically engineered varieties is another potential hazard. To assess the impact of these factors gene flow needs to be measured. A set of 10 simple sequence repeat markers have been developed, which exhibit polymorphism across a range of pigeonpea varieties. Use of these markers also offers an efficient system for the assessment of genetic diversity within populations of pigeonpea.     

Partitioning of Carbon and Nitrogen During Growth and Development of Pigeonpea (Cajanus cajan L.)

Budgets for C and N were computed for pigeonpea (Cajanus cajanL.) at 15 d intervals, for the entire life cycle. Maximum Cand N in dry matter was observed at 90 d after sowing. Of theplants total respiratory loss during the vegetative phase, shoots,roots and nodules accounted for 65%, 23% and 12%, respectively.During the reproductive phases, the respiratory burden of theroots increased, while that of shoots and nodules decreased.Total respiratory loss as a proportion of net photosynthateremained more or less constant until ‘flowering and pod-setting’but increased heavily during seed filling, losing nearly 75%of the photosynthate in respiration. The efficiency of nitrogenfixation, in relation to respiratory output of the whole plantand nodulated roots, decreased during the period 60–90d after sowing, while that of nodules decreased from day 45onwards. Photosynthate supply to nodules and nodulated rootsincreased up to 75 d and 90 d after sowing, respectively. During45–90 d, nodules were fixing a constant proportion ofN per unit of C translocated (0.2 mg N mg^–1 C). Nodulatedroots, on an average, fixed 0.07 mg N mg^–1 C translocatedin the vegetative phase and this value decreased considerablyduring the subsequent phases. The crop produced during its lifecycle 50.4 g of glucose equivalents and yielded 3.8 g seed drymatter and 0.8 g seed protein giving an average of 13.2 g g^–1seed dry matter and 62.8 g g^–1 seed protein. Selectioncriteria for the improvement of C, N economy in pigeonpea havebeen suggested. Key words: Cajanus cajan, Carbon, Nitrogen, Dry weight, Plant parts, Growth, Development, Models     

Seedling emergence of chickpea (Cicer arietinum L.), pigeonpea (Cajanus cajan L.) and pearl millet (Pennisetum typhoides L.) Effect of differential soil crusting,as induced by raindrop size,and depth of sowing

The effects of differential soil crusting, as achieved by varying the raindrop size, and depth of sowing on seedling emergence of chickpea (Cicer arietinum L.), pigeonpea (Cajanus cajan L.) and pearl millet (Pennisetum typhoides L.) were investigated. Three drop diameters: 2.75, 1.65 and 0.93 mm and four depths of sowing: 2, 4, 6, and 8 cm were studied. Mean time of emergence increased with increase in raindrop size and depth of sowing. Crops differed in their emergence capacity under adverse soil conditions from delay in the emergence of chickpea to complete failure in the case of pigeonpea. Pearl millet was not significantly affected by drop size but was found to be sensitive to depths beyond 4 cm. Within the chickpea crop, the variety H 208 was observed to have better emergence qualities than Pusa 209. Other parameters like epicotyl diameter, swelling index, and the area of crust broken by the emerging seedling were measured.     

Measurement of N2-fixation in field-grown pigeonpea [Cajanus cajan (L.) Millsp.] using15N-labelled fertilizer

Two experiments were carried out from 1981 to 1983 in Vertisol field at ICRISAT Center, Patancheru, India to measure N₂-fixation of pigeonpea [Cajanus cajan (L.) Millsp.] using the¹⁵N isotope dilution technique. One experiment examined the effect of control of a nodule-eating insect on fixation while another in vestigated the effect of intercroping with cereals on fixation and the residual effect of pigeonpea on a succeeding cereal crop. Although both experiments indicated that at least 88% of the N in pigeonpea was fixed from the atmosphere, one result is considered fortuitous in view of the differential rates of growth of the legume and the control, sorghum [Sorghum bicolor (L.) Moench]. The difference method of calculation in dieated negative fixation and the results emphasized the problem of finding a suitable nonfixing control. In a second experiment, when all plants were confined to a known volume of soil to which¹⁵N fertilizer was added in the field, these problems were overcome, and isotope dilution and difference methods gave similar results of N₂-fixation of about 90%. In intercropped pigeonpea 96% of the total N was derived from the atmosphere. This estimate might be an artifact. There was no evidence of benefit from N fixed by pigeonpea to intercropped sorghum plants. Plant tissue¹⁵N enrichments of cereal crops grown after pigeonpea indicated that the cereal derived some N fixed by the previous pigeonpea. Thus residual benefits to cereals are not only an effect of ‘sparing’ of soil N.     

Insights into Salt Stress-Induced Biochemical,Molecular and Epigenetic Regulation of Spatial Responses in Pigeonpea (Cajanus cajan L.)

Pigeonpea (Cajanus cajan L.) is a rich source of nutritionally good quality proteins, carbohydrates, minerals, and vitamins. However, environmental stresses adversely affect its productivity. Only limited reports are available on biochemical/physiological responses of pigeonpea under salt stress. The objectives of the present study were to screen pigeonpea germplasm accessions for salt stress tolerance, followed by understanding their biochemical, epigenetic and molecular responses. Based on germination, growth, and vigor of seedlings under salt stress, the most contrasting pair of salt-responsive genotypes (ICP1071- most salt-sensitive, and ICP7- most salt-tolerant) were selected. Three-week-old seedlings subjected to 250 mM NaCl stress for 7 days showed a significant increase in proline and reducing sugar contents in the case of ICP7, whereas a considerable increase in cell wall-degrading enzyme activity and protein oxidation was observed in ICP1071. Superoxide dismutase, peroxidase, and glutathione reductase activity increased considerably in shoots of ICP7. We observed the CcCYP gene to be upregulated in root, whereas CcCDR was upregulated in shoots of the salt-tolerant genotype to provide protection against the stress. The extent of DNA hypomethylation in the contrasting pigeonpea genotypes under salt stress was correlated with their salt tolerance level. Bisulfite sequencing of CcCDR revealed that methylation of three cytosine residues in CHH context in shoots of the ICP7 genotype due to salt stress results in 2.6-fold upregulated expression of the gene. With a 6.8% increase in methylation of the coding region of CcCDR, its expression level increased by 22%. To the best of our knowledge, this is the first report on a comprehensive study of salt-induced biochemical, epigenetic and molecular responses of pigeonpea, which might be useful in the development of improved salt-tolerant variety.

     

Improved growth of Cajanus cajan (L.) Millsp. in an unsterile tropical soil by three mycorrhizal fungi

Under glasshouse conditions Cajanus cajan plants grown in a dark red latosol were fertilized with soluble simple superphosphate and hardly soluble rock phosphate and inoculated with three VA mycorrhizal fungi (M₁, Gigaspora margarita; M₂, Scutellospora verrucosa; M₃, Acaulospora rehmii) from the Cerrado ecosystem, Brazil. Only with rock phosphate plant growth was significantly increased by all fungi. Enhanced P uptake corresponded with higher yields and proved to be a characteristic of the VA myccorhizae. A definite relationship between infection intensity and efficiency of VA mycorrhizae was not detected. Spore production was generally more pronounced in the treatment with rock phosphate, especially with M₁ and M₂. Nodulation of Cajanus cajan was greatly improved by all fungi in the treatment with rock phosphate. It is suggested that the increased plant development and nodulation was due to improved uptake of P by mycorrhiza.     

Occurrence of Mosaic Mottle, a Viroid Disease of Pigeonpea (Cajanus cajan) in India

During investigations of viroid diseases of food crops in Delhi, India, an infectious low-molecular-weight RNA (M_r 1.3 × 10⁵) was isolated from pigeonpea (Cajanus cajan) plants showing mosaic mottling and reduced size of leaves, stunting and no flowering. This RNA infected mechanically inoculated C. cajan, Chenopodium amaranticolor, Nicotiana glutinosa, and N. clevelandii. The causal agent, for which the name pigeonpea mosaic mottle viroid (PMMVd) is proposed, differed from N. glutinosa stunt viroid (NgSVd), the only viroid reported to infect legumes, in size and symptom expression on the original host species.     

Evaluation and Validation of Housekeeping Genes as Reference for Gene Expression Studies in Pigeonpea (Cajanus cajan) Under Drought Stress Conditions

Gene expression analysis using quantitative real-time PCR (qRT-PCR) is a very sensitive technique and its sensitivity depends on the stable performance of reference gene(s) used in the study. A number of housekeeping genes have been used in various expression studies in many crops however, their expression were found to be inconsistent under different stress conditions. As a result, species specific housekeeping genes have been recommended for different expression studies in several crop species. However, such specific housekeeping genes have not been reported in the case of pigeonpea (Cajanus cajan) despite the fact that genome sequence has become available for the crop. To identify the stable housekeeping genes in pigeonpea for expression analysis under drought stress conditions, the relative expression variations of 10 commonly used housekeeping genes (EF1α, UBQ10, GAPDH, 18SrRNA, 25SrRNA, TUB6, ACT1, IF4α, UBC and HSP90) were studied on root, stem and leaves tissues of Asha (ICPL 87119). Three statistical algorithms geNorm, NormFinder and BestKeeper were used to define the stability of candidate genes. geNorm analysis identified IF4α and TUB6 as the most stable housekeeping genes however, NormFinder analysis determined IF4α and HSP90 as the most stable housekeeping genes under drought stress conditions. Subsequently validation of the identified candidate genes was undertaken in qRT-PCR based gene expression analysis of uspA gene which plays an important role for drought stress conditions in pigeonpea. The relative quantification of the uspA gene varied according to the internal controls (stable and least stable genes), thus highlighting the importance of the choice of as well as validation of internal controls in such experiments. The identified stable and validated housekeeping genes will facilitate gene expression studies in pigeonpea especially under drought stress conditions.     

Effect of phenolic compounds on symbiotic nitrogen fixation in pigeonpea(Cajanus cajan (L.) Millsp.)

The effect of different phenolic compounds:p-hydroxybenzoic acid, resorcinol and chlorogenic acid (mono-, di- and polyphenol) was studied on nodulation and related metabolic processes in pigeonpea (Cajanus cajan L. cv. Al-15). Nitrogenease activity, leghaemoglobin and ascorbic acid content of the nodules increased with the application of phenols. Phenols increase the contents of amino acids, proteins and total soluble carbohydrates in the nodules as reserve food materials.     

Karyotypic analysis of Cajanus,Atylosia, and Rhynchosia species

Summary Somatic chromosomes of two cultivais of Cajanus cajan, eight species of Atylosia (A. albicans, A. cajanifolia, A. lineata, A. platycarpa, A. scarabaeoides, A. serica, A. trinervia and A. volubilis), and of Rhynchosia rothii were analysed. All species had 2n=22. Eight of the 10 species studied had two pairs of satchromosomes while A. scarabaeoides and A. sericea had only one sat-chromosome pair. Based on relative chromosome length (L%), arm ratio (pa-value) and presence or absence of secondary constriction, a karyotype formula for each species was formulated. Based on these parameters the chromosome pairs could also be assigned to groups ranging from 8 to 10 in different species. Except for the asymmetrical karyotype of A. albicans, the other species had rather moderately symmetrical karyotypes.     

Pod surface exudates of wild relatives of pigeonpea influence the feeding preference of the pod borer, Helicoverpa armigera

Wild relatives of crops are an important source of resistance genes against insect pests. However, it is important to identify the accessions of wild relatives of crops with different mechanisms of resistance to broaden the basis and increase the levels of resistance to insect pests. Therefore, we studied the feeding behavior of pod borer, Helicoverpa armigera, which is the most damaging pest of pigeonpea, in relation to biochemical characteristics of the pod surface exudates in a diverse array of germplasm accessions belonging to 12 species of pigeonpea wild relatives. Feeding by H. armigera larvae was significantly lower on the unwashed or water-, methanol-, or hexane-washed pods of Canajus sericeus, C. scarabaeoides, Flemingia bracteata, F. stricta, and Rhynchosia aurea than those of C. acutifolius, C. albicans, C. cajanifolius, C. lineatus, D. ferruginea, P. scariosa, R. bracteata, and the cultivated pigeonpea, C. cajan genotypes, ICPL 87, and ICPL 332, although there were a few exceptions. The methanol-washed pods of wild relatives were less preferred for feeding by the H. armigera larvae than the unwashed pods, but the hexane-washed pods were preferred more than the unwashed pods. The results suggested that methanol extracted the phagostimulants from the pod surface, while hexane removed the antifeedants. The high-performance liquid chromatography (HPLC) finger printing of methanol and hexane pod surface extracts showed qualitative and quantitative differences in compounds present on the pod surface of different wild relatives of pigeonpea. Some of the peaks in HPLC profiles were associated with feeding preference of the third-instar larvae of H. armigera. There was considerable diversity in wild relatives of pigeonpea as revealed by principal component analysis based on HPLC fingerprints of pod surface extracts in methanol and hexane, and H. armigera feeding on the pods. Wild pigeonpea accessions with low amounts of phagostimulants and high amounts of antifeedants may be used for introgression of resistance genes into the cultivated pigeonpea to develop varieties with broad-based resistance to H. armigera. There is considerable diversity among the wild relatives of pigeonpea, and the accessions with resistance to pod borer. These can be used to broaden the basis and increase the levels of resistance to H. armigera.     

Genetic patterns of domestication in pigeonpea (Cajanus cajan (L.) Millsp.) and wild Cajanus relatives

Pigeonpea (Cajanus cajan) is an annual or short-lived perennial food legume of acute regional importance, providing significant protein to the human diet in less developed regions of Asia and Africa. Due to its narrow genetic base, pigeonpea improvement is increasingly reliant on introgression of valuable traits from wild forms, a practice that would benefit from knowledge of its domestication history and relationships to wild species. Here we use 752 single nucleotide polymorphisms (SNPs) derived from 670 low copy orthologous genes to clarify the evolutionary history of pigeonpea (79 accessions) and its wild relatives (31 accessions). We identified three well-supported lineages that are geographically clustered and congruent with previous nuclear and plastid sequence-based phylogenies. Among all species analyzed Cajanus cajanifolius is the most probable progenitor of cultivated pigeonpea. Multiple lines of evidence suggest recent gene flow between cultivated and non-cultivated forms, as well as historical gene flow between diverged but sympatric species. Evidence supports that primary domestication occurred in India, with a second and more recent nested population bottleneck focused in tropical regions that is the likely consequence of pigeonpea breeding. We find abundant allelic variation and genetic diversity among the wild relatives, with the exception of wild species from Australia for which we report a third bottleneck unrelated to domestication within India. Domesticated C. cajan possess 75% less allelic diversity than the progenitor clade of wild Indian species, indicating a severe "domestication bottleneck" during pigeonpea domestication.