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White grub species, Melolontha cuprescens, Melolontha furcicauda, Melolontha indica and Polyphylla fullo are serious pests of different crops in north-western Himalaya. None of these species are, however, characterized at molecular level including DNA barcodes. Here we provide DNA barcodes for these species. The adults of these species were collected from different locations of Himachal Pradesh and described by molecular characterization. The mitochondrial Cytochrome c oxidase 1 (COX1) and 28S ribosomal RNA (28S rRNA) genes of these species were amplified and sequenced for molecular identification and phylogenetic analysis. The sequences of these genes from other species of these genera and other related genera were also retrieved from DNA databases and included in the analysis. The COX1 sequences of M. cuprescens were 99.88% similar to P. fullo, whereas those of M. furcicauda were 84.91% similar to M. indica. Among retrieved COX1 sequences, Melolontha hippocastani (HM120757 & KX087316) were closer to M. furcicauda (86.14–86.50%) and M. indica (85.74–86.04%) as compared to M. cuprescens (81.44–81.47%) and P. fullo (81.32–81.47%). Based on 28S rRNA gene sequences, M. indica showed 99.42% similarity to Melolontha melolontha (EU084231) as compared to M. cuprescens and M. furcicauda which were distantly related. The COX1 was a better DNA barcode for the identification of Melolontha and Polyphylla species. The 28S rRNA has a limited utility in DNA barcoding of Melolontha species.

Treatment with EMS (0.05%, 0.10% and 0.15%), gamma-rays (150 Gy, 200 Gy, 300 Gy) and their all possible combinations were utilised to generate genetic variability in chickpea variety HPG-17. The potential of each treatment was analysed on the basis of variability generated which included range of viable and non-viable mutants. Treatment of 0.10% EMS produced maximum viable macro-mutants. Low doses of both EMS and gamma rays individually, as well as in combination failed to produce chlorophyll mutants. Highest frequency of chlorophyll mutants was observed in treatment 300 Gy + 0.15% EMS. Wide range of mutants for plant type, leaf colour, leaf shape, seed colour, seed texture and sterility was also observed.

Ficus roxburghii, “elephant ear” or wild fig is valued for its edible fruits, medicinal uses, fodder for cattle and other purposes like use of leaves as plates for serving food. However, the population of this species is declining at an alarming rate in Himachal Pradesh as revealed by the survey conducted recently, possibly due to low germination, changing climate and loss of habitat. Many elite genotypes have disappeared over time in this process. Hence, a study was carried out to raise F. roxburghii through alternative propagation technique to conserve the elite genotypes and produce true to type nursery stock of selected genotypes for further plantation in field.

Low temperature (LT) causes significant yield losses in chickpea (

Low temperature (LT) causes significant yield losses in chickpea (Cicer arietinum L.). The sucrose starch metabolism is associated with abiotic-stress tolerance or sensitivity in plants. The changes in sugars and starch contents under LT in chickpea have already been studied, however, no information is available on LT-induced alterations in transcription of carbohydrate metabolic pathway genes in chickpea. To understand the differences in the regulation of sucrose and starch metabolism under LT, the expression of sucrose and starch metabolism genes was studied in leaves of cold-sensitive (GPF2) and cold-tolerant (ICC 16349) chickpea genotypes. The mRNA sequences of chickpea genes were retrieved from the public databases followed by confirmation of identity and characterization. All the genes were functional in chickpea. Between the two paralogues of cell wall invertase, cell wall invertase 3×2 (CWINx2) was the truncated version of cell wall invertase 3×1 (CWINx1) with the loss of 241 bases in the mRNA and 67 amino acids at N terminal of the protein. Comparison of expression of the genes between control (22°C day / 16°C night) and LT treated (4°C; 72 h) plants revealed that granule bound starch synthase 2 (GBSS2) and β-amylase 3 (BAM3) were upregulated in ICC 16349 whereas sucrose phosphate synthase 2 (SPS2), CWINx1, CWINx2 and β-amylase 1 (BAM1) were downregulated. In contrast to this, SPS2, CWINx1, CWINx2 and BAM1 were upregulated and GBSS2 downregulated in GPF2 under LT. The gene expression data suggested that UGPase, CWINs, GBSS2 and BAM3 are important components of cold-tolerance machinery of chickpea.

Chickpea has a narrow genetic base, and consequently many DNA-based markers were unsuccessful in revealing polymorphism. The problem was overcome by the development of simple sequence repeat and single nucleotide polymorphism markers that revealed adequate polymorphism and these became markers of choice in chickpea. In addition, transcription factor gene–derived microsatellite markers, intron targeted primers, expressed sequence tag polymorphisms, diversity arrays technology, and insertion-deletion markers have also been developed for chickpea. Genotyping by sequencing is also gaining popularity. Saturated linkage maps of chickpea with adequate marker density on each linkage group have also been constructed; however, several of the recently developed markers are still to be placed on linkage maps. The markers have subsequently been used to map genes or identify quantitative trait loci (QTLs) for biotic stress resistance, abiotic stress tolerance, and agro-morphological traits. Using reference genomes of chickpea and QTL intervals, candidate genes in QTL regions for several of these characters have been identified. Notably, genes involved in flowering in chickpea have been identified. Same QTL was also found to govern more than one trait, e.g., drought tolerance and plant vigor, salinity tolerance and higher plant vigor, salinity tolerance and higher reproductive success. The markers linked closely to genes/QTLs of interest have also been exploited to introgress wilt and blight resistance, and double podding character into agronomically superior cultivars.

Productivity of cultivated chickpea is hampered by various abiotic and biotic stresses and this offers limited genetic improvements in domesticated crop varieties. Wild Cicer species harbor several important traits, which are not present adequately in the cultigen, e.g., resistance to cold, drought, pod borer, and ascochyta blight, and hence these species could be a wonderful resource for their introgression into the cultigen to broaden its genetic base. The cytogenetic studies have been used to karyotype the chromosomes and characterize hybrids cytologically, and introgression of traits also can be monitored using marker-assisted selection and fluorescence in situ hybridization approaches. Marker-assisted selection also offers added advantages of introgression of multiple traits or traits that are difficult to assess through phenotyping. A majority of the chickpea cultivars released so far have been developed using selection and intraspecific hybridization involving common parents, which has resulted into the narrow genetic base of released cultivars. To attain further breakthrough for enhancing yield and resistance against prevailing stresses, new gene sources need to be identified in wild species and introgressed for widening the genetic base of cultivated gene pool.

Identification of the fungus Fusarium oxysporum f. sp. pisi (Fop), the causal organism of wilt disease of pea, is a time consuming and arduous task. Diagnosis of Fop by traditional means requires more than 2 months and involves two steps, identification of species using morphological characters and formae specialis ‘pisi’ using pathogenicity assays. The ambiguous morphological differences between F. solani and F. oxysporum further complicate the diagnosis of F. oxysporum. A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) based method was developed to detect Fop from India. A PCR–RFLP marker, HPACAPS1380, generated after restriction of 28S rDNA region with enzyme MvaI, detected accurately the Fop among several other fungi with detection sensitivity of 5 fg of Fop genomic DNA. In a mixture of Fop and pea DNA, the sensitivity was 500 pg of Fop DNA in 50 ng of pea DNA. The assay was further refined to detect the Fop from infected tissues and infested soil. The current assay can detect Fop from culture, plant tissues and soil in a considerably shorter period of time compared to traditional methods.

Chickpea (Cicer arietinum L.) is the third most important food legume globally and is an important source of high-quality proteins to both humans and animals. It is highly sensitive to low temperature (LT) resulting in large scale yield losses. LT induces flower abortion in chickpea by disruption of gamete development and induction of pollen sterility. However, molecular mechanisms governing LT induced pollen sterility in chickpea are unknown. Disruption of carbohydrate metabolism has been implicated in LT induced damage to crops whereas proline and antioxidants protect plants from LT stress. In this study, the impact of LT on carbohydrates, proline and enzymatic antioxidants and expression of genes of these metabolite pathways were investigated in anthers at vacuolated pollen stage. The study included two genotypes, a cold-sensitive (GPF2) and a cold-tolerant (ICC 16349) that differed for pollen sterility under LT. Metabolite profiling revealed a reduction in starch and proline content in GPF2 under LT, whereas these parameters did not change but non-reducing sugars increased in ICC 16349. At the transcriptional level, cold initially lowered the transcription of all genes (19) under study in GPF2 and 14 genes out of 19 in ICC 16349. Initial down-regulation was followed by differential reactivation of transcription machinery in GPF2 and ICC 16349 at 30 min. Under LT, expression of starch synthesis, starch degradation, sucrose synthesis and sucrose transport genes showed a contrasting response in the two genotypes, where GPF2 showed downregulation of these genes while ICC 16,349 showed upregulation. The proline transport gene (proline transporter 1) showed higher downregulation in GPF2 than ICC 16349. Overall, pollen viability under LT in ICC 16349 was linked to the maintenance of starch, reducing sugars and proline levels and over-expression of UDP glucose pyrophosphorylase and cell wall invertase, while these processes were compromised in the GPF2 possibly leading to pollen sterility.

Chickpea is one of the most economically important food legumes, and a significant source of proteins. It is cultivated in more than 50 countries across Asia, Africa, Europe, Australia, North America, and South America. Chickpea production is limited by various abiotic stresses (cold, heat, drought, salt, etc.). Being a winter-season crop in northern south Asia and some parts of the Australia, chickpea faces low-temperature stress (0–15°C) during the reproductive stage that causes substantial loss of flowers, and thus pods, to inhibit its yield potential by 30–40%. The winter-sown chickpea in the Mediterranean, however, faces cold stress at vegetative stage. In late-sown environments, chickpea faces high-temperature stress during reproductive and pod filling stages, causing considerable yield losses. Both the low and the high temperatures reduce pollen viability, pollen germination on the stigma, and pollen tube growth resulting in poor pod set. Chickpea also experiences drought stress at various growth stages; terminal drought, along with heat stress at flowering and seed filling can reduce yields by 40–45%. In southern Australia and northern regions of south Asia, lack of chilling tolerance in cultivars delays flowering and pod set, and the crop is usually exposed to terminal drought. The incidences of temperature extremes (cold and heat) as well as inconsistent rainfall patterns are expected to increase in near future owing to climate change thereby necessitating the development of stress-tolerant and climate-resilient chickpea cultivars having region specific traits, which perform well under drought, heat, and/or low-temperature stress. Different approaches, such as genetic variability, genomic selection, molecular markers involving quantitative trait loci (QTLs), whole genome sequencing, and transcriptomics analysis have been exploited to improve chickpea production in extreme environments. Biotechnological tools have broadened our understanding of genetic basis as well as plants' responses to abiotic stresses in chickpea, and have opened opportunities to develop stress tolerant chickpea.

Sharma, K. D., Chen, W., and Muehlbauer, F. J. 2005. Genetics of chickpea resistance to five races of Fusarium wilt and a concise set of race differentials for Fusarium oxysporum f. sp. ciceris. Plant Dis. 89:385-390. Genetics of resistance in chickpea accession WR-315 to Fusarium wilt was investigated, and a concise set of differentials was developed to identify races of Fusarium oxysporum f. sp. ciceris. A population of 100 F7 recombinant inbred lines (RILs) from a cross of WR-315 (resistant) and C-104 (susceptible) was used to study genetics of resistance to races 1A, 2, 3, 4, and 5 of F. oxysporum f. sp. ciceris, and a population of 26 F2 plants from a cross between the same two parents was used to study inheritance of resistance to race 2. Segregations of the RILs for resistance to each of the five races suggest that single genes in WR-315 govern resistance to each of the five races. A 1:3 resistant to susceptible ratio in the F2 population indicated that resistance in WR-315 to race 2 was governed by a single recessive gene. A race-specific slow disease progress reaction was observed in chickpea line FLIP84-92C(3) to infection by race 2, a phenomenon termed as slow wilting, that is different from previously reported late wilting with respect to latent period, disease progress rate, and final disease rating. Twenty-nine germ plasm lines (27 Cicer arietinum and two C. reticulatum) including previously used differentials were evaluated for their reactions to infection by the five races. Only eight of the 29 germ plasm lines differentiated at least one of the five races based on either resistant or susceptible reactions, whereas the remaining germ plasm lines were either susceptible or resistant to all five races or differentiated them by intermediate reactions. A concise set of eight chickpea lines comprised of four genotypes and four F7 RILs with vertical resistance was developed as differentials for race identification in F. oxysporum f. sp. ciceris. These differential lines were characterized by early appearance of wilt symptoms, and clear and consistent disease phenotypes based on no wilt or 100% wilt incidence, which offers important improvements over previously available differential sets and provides more precise and unambiguous identification of the races.

Chickpea, a vital food legume, shows high sensitivity to cold stress at reproductive stage, and temperatures below 20/10 °C, (as day/night temperature) at this stage result in disruption in developmental and functional aspects of reproductive components, causing floral abortion, poor pods and significant yield losses. In the present study, chickpea seeds (a cold sensitive genotype GPF2) were cold primed at 5 °C ± 1 °C in the dark for 30 days, followed by gradual air-drying at 15 °C to the original moisture content (11–13%). The crop was raised in the month of November from non-primed and primed seeds in the outdoor environment (temperature profile: 11.7 °C; mean day/night temperature; 1300–1500 μmol m−2 s−1 light intensity, 65–70% relative humidity), until the onset of budding and flowering (45 days after sowing). Subsequently, the plants were separated into different sets to impose following treatments in controlled environment a) control (at optimum temperature; a set of plants were grown at optimum temperature (25/18 °C) until maturity, b) cold-stressed; a set of the plants were grown at low temperature (15/8 °C) until maturity, c) raised from cold-primed seeds but grown at optimum temperature (25/18 °C) until maturity, d) raised from cold-primed seeds and exposed to low temperature (15/8 °C), until maturity. The plants were tested for various traits in the leaves at 0–25 days after exposure to stress. Cold stress resulted in damage to membranes, photosynthetic ability (as loss of chlorophyll, photosystem II function and chlorophyll fluorescence, stomatal conductance), photo-assimilation capacity (as sucrose metabolism), hydration status (as leaf water content), production of osmolytes (as disruption in metabolism of proline, trehalose, glycine betaine, and GABA production) and decreased the redox status of the cells (as decrease in activity and expression of various enzymatic and non-enzymatic antioxidants. Reproductive function was markedly decreased, as was evident from reduction in pollen viability, pollen germination, stigma receptivity and ovule viability). Pod number and seed weight plant−1 showed 65.5 and 68.4% reduction because of cold stress. The plants raised from cold-primed seeds were remarkable benefitted at reproductive stage, which showed significantly improved pollen and stigmatic function, resulting in considerable recovery of the pod number and seed weight plant-1. The benefits of the cold priming were possibly related to improved leaf function (hydration status, photosynthetic and carbon fixation ability), which increased the sucrose concentration in the leaves to support the reproductive function, along with enhanced anti-oxidative capacity and osmo-protectants’ production, which were significantly more in cold-stressed plants, grown from cold-primed seeds, compared to the controls. Our findings in this regard are novel and suggest an effective strategy to enhance cold tolerance in chickpea.

Chickpea (Cicer arietinum L.) is susceptible to low temperature (LT) at reproductive stage. LT causes flower abortion and delays pod set in chickpea until terminal drought becomes an issue, thereby decreasing yield potential. In chickpea, flower and anther/pollen development as well as LT-induced abnormalities on anther and pollen development are described inadequately. In the present manuscript, we report flower development stages, anther development stages, and aberrations in male gamete formation in chickpea under LT. Flower length was linearly correlated to flower and anther stages and can be used to predict these stages in chickpea. LT affected male gamete development in a flower/anther age-dependent manner where outcome ranged from no pollen formation to pollen sterility or no anther dehiscence to delayed dehiscence. In anthers, LT inhibited microsporogenesis, microgametogenesis, tapetum degeneration, breakage of septum and stomium, and induced pollen sterility. Whereas disruption of male function was the prime cause of abortion in flowers below vacuolated pollen stage, flower abortion was due to a combination of male and female reproductive functions in flowers with mature pollen. The study will help in elucidating mechanisms governing flower development, anther and pollen development, and tolerance/susceptibility to LT.

The internal transcribed spacer (ITS) region was used to discriminate 18 Saccharomyces cerevisiae strains isolated from fermented foods of the North-Western Himalayas. ITS sequences were compared with other known lineages available in literature. A phylogenetic tree grouped these strains into two clusters, the first comprising Malaysian, North American, Sake, West African and wine/European strains and the second comprising all Indian strains along with certain other strains from different countries. In both clusters, mixed grouping was observed, with the exception of a few strains with a region-based grouping pattern. The baking and brewing abilities of these strains were studied to expose functional diversity. Four strains, viz. Sc06, Sc11, Sc19 and Sc20, were found promising for baking and three strains, viz. Sc04, Sc05 and Sc24, were found promising for brewing, thereby indicating the existence of functional diversity. At a genetic level, mining of the ADH1 gene of ten randomly selected yeast strains, showed very little variation in the gene sequences irrespective of their alcohol production ability. In contrast, mining of the ATF1 gene showed a lot more variation in the gene sequences, revealing the ATF1 gene to be an appropriate marker to reveal differences in ester formation among these indigenous yeast strains.

Of the various environmental stresses that a plant can experience, temperature has the widest and most far-reaching effects on legumes. Temperature extremes, both high (heat stress) and low (cold stress), are injurious to plants at all stages of development, resulting in severe loss of productivity. In response to unfavorable temperatures, plant biomolecules such as stress proteins, enzymatic and non-enzymatic antioxidants, organic osmolytes and phytohormones come into play, usually, as a part of the plant defense mechanisms. The accumulation of these molecules, which may be useful as metabolic indicators of stress tolerance, depend on the plant species exposed to the temperature stress, its intensity and duration. Some of these molecules such as osmolytes, non-enzymatic antioxidants and phytohormones may be supplied exogenously to improve temperature stress tolerance. Legumes show varying degrees of sensitivity to high and low-temperature stresses, which reduces their potential performance at various developmental stages. To address the ever-fluctuating temperature extremes that various legumes are being constantly exposed, efforts are being made to develop tolerant plant varieties via conventional breeding methods as well as more recent molecular breeding techniques. In this review, we describe the progress made towards the adverse effects of abnormal temperatures on various growth stages in legumes and propose appropriate strategies to resolve these effects.

Dactylorhiza hatagirea (D. Don) Soo, family Orchidaceae is an endangered medicinal herb inhabiting higher altitudes of western Himalayas. Due to reckless exploitation for its roots, it is of high conservation concern. In the present study fifteen microsatellites were developed and characterized across twenty collections of D. hatagirea. The total numbers of alleles amplified by these microsatellites were 64 with an average of 4.2 alleles per marker. Average observed and expected heterozygosity values for polymorphic loci were 0.623 and 0.631, respectively. Mean polymorphism information content value of the polymorphic markers was 0.532. Of the fourteen polymorphic microsatellites, 7 deviated from Hardy–Weinberg equilibrium. Microsatellites reported here can be utilized to address questions related to genetic characteristics in this species.

Chickpea is sensitive to chilling stress, especially at its reproductive stage and experiences abortion of flowers and poor pod set at temperatures below 10 °C. The metabolic controls governing chilling-sensitivity in chickpea, particularly involving proline are not known. Hence, in the present study we explored the role of proline in this regard. A set of chickpea plants (cv. GPF2) growing under warm conditions of the glass house (temperature – 28/14 °C as average maximum and minimum till early flowering stage) was exposed to low temperature conditions of the field (8.3–9.6/2.8–5.3 °C; average maximum and minimum temperature, respectively) during the onset of reproductive phase while another set of plants continued to grow under warm conditions. In case of chilling-stressed plants, one set of the plants was treated with 10 μM proline while the other set not treated with proline served as control under low temperature conditions. In untreated chilling-stressed plants, the endogenous proline increased to 230 μmol g −1 dry weight (DW) on 4th day of stress and decreased thereafter to reach 28 μmol on 7th day. In plants treated with 10 μM proline, its endogenous content reached 310 μmol g −1 DW on 4th day and stayed significantly higher than untreated chilling-stressed plants. The proline-treated plants showed significant improvement in retention of flowers and pods leading to better seed yield compared to the untreated ones. The proline-applied plants also had greater pollen viability, pollen germination, pollen tube growth and ovule viability. The stress injury measured as oxidative stress, electrolyte leakage, loss of chlorophyll and decrease in leaf water content was mitigated significantly in proline-treated plants. Additionally, proline application increased the level of sucrose and trehalose (cryoprotectants) in chilling-stressed plants. The studies revealed that proline application was significantly effective in reducing the impact of chilling injury on reproductive growth in chickpea.

Chilling stress (

Chickpea wilt caused by Fusarium oxysporum f. sp. ciceris is one of the major yield limiting factors in chickpea. The disease causes 10–90% yield losses annually in chickpea. Eight physiological races of the pathogen (0, 1A, 1B/C, 2, 3, 4, 5 and 6) are reported so far whereas additional races are suspected from India. The distribution pattern of these races in different parts of the world indicates regional specificity for their occurrence leading to the perception that F. oxysporum f. sp. ciceris evolved independently in different regions. Pathogen isolates also exhibit differences in disease symptoms. Races 0 and 1B/C cause yellowing syndrome whereas 1A, 2, 3, 4, 5 and 6 lead to wilting syndrome. Genetics of resistance to two races (1B/C and 6) is yet to be determined, however, for other races resistance is governed either by monogenes or oligogenes. The individual genes of oligogenic resistance mechanism delay onset of disease symptoms, a phenomenon called as late wilting. Slow wilting, i.e., slow development of disease after onset of disease symptoms also occurs in reaction to pathogen; however, its genetics are not known. Mapping of wilt resistance genes in chickpea is difficult because of minimal polymorphism; however, it has been facilitated to great extent by the development of sequence tagged microsatellite site (STMS) markers that have revealed significant interspecific and intraspecific polymorphism. Markers linked to six genes governing resistance to six races (0, 1A, 2, 3, 4 and 5) of the pathogen have been identified and their position on chickpea linkage maps elucidated. These genes lie in two separate clusters on two different chickpea linkage groups. While the gene for resistance to race 0 is situated on LG 5 of Winter et al. (Theoretical and Applied Genetics 101:1155–1163, 2000) those governing resistance to races 1A, 2, 3, 4 and 5 spanned a region of 8.2 cM on LG 2. The cluster of five resistance genes was further subdivided into two sub clusters of 2.8 cM and 2.0 cM, respectively. Map-based cloning can be used to isolate the six genes mapped so far; however, the region containing these genes needs additional markers to facilitate their isolation. Cloning of wilt resistance genes is desirable to study their evolution, mechanisms of resistance and their exploitation in wilt resistance breeding and wilt management.

Sequence-tagged microsatellite site (STMS) and sequence-tagged site (STS) markers linked closely to Fusarium oxysporum f. sp. ciceris race 3 resistance gene in chickpea were identified, and linkage between three wilt resistance genes was elucidated. The resistance to race 3 in chickpea germplasm accession WR-315 was inherited as a single gene, designated foc-3, in 100 F(7) recombinant inbred lines derived from the cross of WR-315 (resistant) x C-104 (susceptible). The foc-3 gene was mapped 0.6 cM from STMS markers TA96 and TA27 and STS marker CS27A. Another STMS marker, TA194, at 14.3 cM, flanked the gene on the other side. Linkage between foc-3 and two other chickpea wilt resistance genes, foc-1 (syn. h(1)) and foc-4, was established. foc-3 was mapped 9.8 cM from foc-1 and 8.7 cM from foc-4, whereas foc-1 and foc-4 are closely linked at 1.1 cM. The identification of closely linked markers to resistance genes will facilitate marker-assisted selection for introgression of the race 3 resistance gene to susceptible chickpea lines.

In the present study, the β-carotene, calcium, iron and zinc content in the leaves of 46 accessions of three Chenopodium species viz. Chenopodium album, C. album ssp. amaranticolor and Chenopodium quinoa was investigated. A wide range of variability, inter-specific as well as varietal, for the β-carotene [0.19-5.91 mg 100 g(- 1) fresh weight (FW)], calcium (358.35-960.10 mg 100 g(- 1) FW), iron (0.56-7.90 mg 100 g(- 1) FW) and zinc content (0.07-4.26 mg 100 g(- 1) FW) was observed. The C. album ssp. amaranticolor accessions IC341710 and IC469275 had a high content of all the four nutrients and were ideal food to alleviate nutritional deficiencies in humans. Nutritionally rich IC341710 and IC469275 were further studied to estimate micronutrient retention in cooked leaves. Between the two methods used to cook leaves, stir-frying showed better retention of micronutrients than pressure cooking.