Molecular mapping of QTLs for resistance to Fusarium wilt (race 1) and Ascochyta blight in chickpea (Cicer arietinum L.)

Fusarium wilt (FW) and Ascochyta blight (AB) are two important diseases of chickpea which cause 100 % yield losses under favorable conditions. With an objective to validate and/or to identify novel quantitative trait loci (QTLs) for resistance to race 1 of FW caused by Fusarium oxysporum f. sp. ciceris and AB caused by Ascochyta rabiei in chickpea, two new mapping populations (F_2:3) namely ‘C 214’ (FW susceptible) × ‘WR 315’ (FW resistant) and ‘C 214’ (AB susceptible) × ‘ILC 3279’ (AB resistant) were developed. After screening 371 SSR markers on parental lines and genotyping the mapping populations with polymorphic markers, two new genetic maps comprising 57 (C 214 × WR 315) and 58 (C 214 × ILC 3279) loci were developed. Analysis of genotyping data together with phenotyping data collected on mapping population for resistance to FW in field conditions identified two novel QTLs which explained 10.4–18.8 % of phenotypic variation. Similarly, analysis of phenotyping data for resistance to seedling resistance and adult plant resistance for AB under controlled and field conditions together with genotyping data identified a total of 6 QTLs explaining up to 31.9 % of phenotypic variation. One major QTL, explaining 31.9 % phenotypic variation for AB resistance was identified in both field and controlled conditions and was also reported from different resistant lines in many earlier studies. This major QTL for AB resistance and two novel QTLs identified for FW resistance are the most promising QTLs for molecular breeding separately or pyramiding for resistance to FW and AB for chickpea improvement.     

In vitro direct plant regeneration and Agrobacterium‐mediated transformation of lucerne (Medicago sativa L.)

In vitro direct plant regeneration of lucerne was achieved by simultaneous application of thidiazuron (TDZ) and 6‐benzyladenine (BA) in Murashige and Skoog (MS) medium. Seedlings were germinated and grown for 6 d on growth regulator–containing MS medium. The shoot tip, consisting of the apical meristem along with parts of the cotyledonary leaves and hypocotyl, was then cultured on a medium containing the growth regulator(s). Adventitious budding of the shoot tip was promoted synergistically by treatment with TDZ and BA, and a maximum of thirty‐five shoots per explant was obtained on a medium supplemented with 2 mg L^?1 TDZ and 1 mg L^?1 BA. Plant regeneration frequency varied from 67 to 93%, and five Indian lucerne cultivars responded well to the regeneration protocol. The Agrobacterium‐mediated transformation frequency from co‐cultivated explants was 13% following multiple shoot induction. Southern analysis of the T₀ plants and T₁ progenies confirmed stable inheritance of the hpt marker gene. Agrobacterium infection of the explant caused a significant reduction in the plant regeneration frequency (23%) and the number of shoots induced (11) when compared with uninfected explants. A single shoot tip provided sufficient material to regenerate and establish twenty‐seven lucerne plants, whereas only nine plants could be regenerated from an Agrobacterium co‐cultivated explant. This transformation protocol could represent a valuable improvement over existing ones for lucerne.     

A major QTL for gluten strength in durum wheat (Triticum turgidum L. var. durum)

Gluten strength is an important characteristic, determining the end product quality of durum wheat semolina. To identify the genetic basis of gluten strength in North Dakota durum cultivars, a doubled haploid population was developed from the cross of a weak gluten cultivar ‘Rugby’ and a strong gluten cultivar ‘Maier’. A framework linkage map consisting of 228 markers was constructed and used with phenotypic data on gluten strength (measured by sedimentation volume) to conduct single- and two-locus QTL analyses. Only one consistent QTL (QGs.ndsu-1B) contributing up to 90% of the phenotypic or 93% of the genotypic variation was detected on 1BS. No QTL × QTL or QTL × environment interactions were observed. The QGs.ndsu-1B was flanked by two DArT markers which were converted to STS markers and used along with SSR and EST-SSRs to develop a map of 1BS. QTL analysis delineated QGs.ndsu-1B in a 7.3 cM region flanked by an STS marker (STS-wPt2395) and a SSR marker (wmc85). The adapted background of this material and availability of PCR-based markers closely associated with this locus represent invaluable resources for marker-assisted introgression of gluten strength into other durum wheat varieties. A single QTL segregating in this population also makes it an ideal target for map-based cloning.     

Effect of feeding of calcium hydroxide-treated or vitamin E-supplemented cottonseed meal on plasma gossypol levels, blood parameters, and performance of Bikaneri lambs

To study the effect of feeding calcium hydroxide-treated or vitamin E-supplemented cottonseed meal (CSM) incorporated diets on plasma gossypol, blood parameters and animal performance, 24 male Bikaneri lambs of 6–7 months of age and of uniform body weight were divided into four groups of six animals each in a completely randomized design and respectively fed isonitrogenous and isocaloric concentrate mixtures containing 20 % soybean meal (CON) or 40 % raw CSM (RCSM), 40 % raw CSM supplemented with 500 IU of vitamin E per head per day (ERCSM), and 40 %, 1.5 % calcium hydroxide-treated CSM (CaCSM) along with ad libitum wheat straw throughout 510 days of experimental feeding. The lambs on all the diets grew linearly throughout the experimental period. The total weight gain, in turn the average daily gain (ADG), was not affected by dietary variations. The daily intake of dry matter, crude protein (CP), digestible crude protein (DCP) and total digestible nutrients (TDN) were found comparable among lambs of all the groups. Though total gossypol intake was similar in RCSM, ECSM and CaCSM groups, however, free gossypol intake was significantly higher (P?<?0.01) in RCSM, ECSM groups as compared to CaCSM group. Serum iron and blood hemoglobin levels were significantly (P?<?0.05) lower in RCSM group as compared to CaCSM and CON groups, and ALT activity was significantly (P?<?0.05) higher on RCSM group as compared to other groups. Plasma gossypol and osmotic fragility of erythrocytes were significantly (P?<?0.05) increased in RCSM group as compared to CaCSM and ERCSM groups. However, there was no significant difference in the concentration of other blood/serum biochemical constituents among the lambs on different groups. Based on the results, it may be concluded that feeding of 40 % CSM in the concentrate mixture of the diet in Bikaneri lambs did not have any major adverse effect in blood parameters and animal performance. Either calcium hydroxide treatment or vitamin E supplementation did not produce any major additional benefits.     

Rapid detection of Corynebacterium pseudotuberculosis in clinical samples from sheep

Corynebacterium pseudotuberculosis, a Gram-positive bacterium is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep, goats and other warm blooded animals. In the present study, a total of 1,080 sheep reared under semi-intensive system on organized farms situated in the semi arid tropical region of Rajasthan, India, was clinically examined. Pus samples from superficial lymph nodes of 25 (2.31 %) adult sheep showing clinical lesions similar to CLA were collected for laboratory analyses. On the basis of morphological, cultural and biochemical characteristics 12 (48 %) bacterial isolates from pus identified it as C. pseudotuberculosis. A polymerase chain reaction (PCR) assay targeting Putative oligopeptide/dipeptide ABC transporter, nicotinamide adenine dinucleotide phosphate (NADP) oxidoreductase coenzyme F420-dependent and proline iminopeptidase (PIP) genes of C. pseudotuberculosis was developed that showed 14 pus samples as positive. All C. pseudotuberculosis isolates were also found positive for these genes in the PCR. The specificity of the PCR products was confirmed by sequencing of the amplified products that showed 98–100 % homology with published sequences available in the NCBI database. The present study shows the incidence of CLA as 2.31 %, 1.1 % and 1.29 % based on clinical, bacterial culture and direct pus PCR assay, respectively. The PCR assay was rapid, specific and as significant as bacterial culture in detecting bacteria directly in the clinical pus samples. The PCR assay developed in the study can be applied for the diagnosis and control of CLA. Furthermore, the assay can also be applied to detect C. pseudotuberculosis in various clinical samples.     

Immunization with a multisubunit vaccine considerably reduces establishment of infective larvae in a rodent model of Brugia malayi

Although recombinant vaccines have several advantages over conventional vaccines, protection induced by single antigen vaccines is often inadequate for a multicellular helminth parasite. Therefore, immunoprophylactic efficacy of cocktail antigen vaccines comprised of several combinations of three Brugia malayi recombinant proteins BmAF-Myo, Bm-iPGM and Bm-TPP were evaluated. Myosin + TPP and iPGM + TPP provided the best protection upon B. malayi infective larval challenge with ∼70% reduction in adult worm establishment over non-vaccinated animals that was significantly higher than the protection achieved by any single antigen vaccine. Myosin + iPGM, in contrast did not provide any enhance protection over the single recombinant protein vaccines. Specific IgG, IgM level, IgG antibody subclasses levels (IgG1, IgG2a, IgG2b, IgG3), lymphocyte proliferation, reactive oxygen species level and cytokines level were also determined to elucidate the characteristics of the protective immune responses. Thus the study undertaken provided more insight into the cocktail vaccination approach to combat LF.     

Effect of yeast supplementation on the growth performance of Malpura lambs

Thirty-six Malpura lambs (age?=?58 days; 8.9 kg BW) were equally divided into three groups (N?=?12; six males and six females) to assess the effect of probiotics supplementation on growth, digestibility, rumen fermentation and carcass attributes. The lambs of the control group (CON) were not supplemented with probiotics, while test groups received either Saccharomyces cerevisiae (SC) or combination of S. cerevisiae and Lactobacillus sporogenes (SCLS) at 1.5 % of concentrate mixture. The lambs were fed ad libitum concentrate mixture and bajra (Pennisetum typhoides) straw in a cafeteria system until 180 days of age. Daily feed intake and weekly live weight changes were recorded. A metabolism trial was conducted on six lambs at 90 days. Rumen fermentation study was conducted at 105 days. At 6 months, all male lambs were slaughtered and carcass characteristics were recorded. Body weight and average daily gain (ADG) were similar among the three groups. The digestibilities of all the nutrients were also similar among the groups, except acid detergent fiber (ADF) digestibility, which was higher (P?=?0.032) in SC and SCLS than the CON. The rumen fluid pH was higher (P?=?0.04) in CON and SC group than SCLS at 0 h while NH₃-N at 8 h sample was higher (P?=?0.031) in SC and SCLS group than the control. Pre-slaughter weight, hot carcass weight and dressing yields were similar. ADF digestibility and rumen fermentation was improved in lambs by probiotic supplementation. However, carcass traits remained unchanged due to probiotics supplementation.     

Acellular Dermal Matrix for Surgical Repair of Ventral Hernia in Horses

The aim of the present study was to evaluate acellular dermal matrix (ADM) of rat origin for the repair of ventral hernia in horses. The skin from rats, to be used as a graft, was de-epithelialized using hypertonic solution and further decellularized with 0.5% sodium dodecyl sulfate and 0.25% tri-(n-butyl)-phosphate. Under general anesthesia, the hernial ring was exposed and repaired with the ADM graft using inlay graft technique. Blood samples were collected at postimplantation days 0, 15, and 30 and were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis to assess the serum protein concentration of the animals, as well as for gelatin zymography for the identification of matrix metalloproteinases. All animals had an uneventful recovery without clinical signs of wound dehiscence, infection, or recurrence of hernias during 6-month follow-up period. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of concentration of the serum proteins revealed that this was increased at day 15 and had decreased again at day 30. Gelatin zymography showed only one major band of 92 kDa in the serum of all the horses with the implant, but the relative amount of 92 kDa was higher at day 15 as compared with day 0 and day 30. It may be concluded that ADM of rat origin can be used safely in horses for repair of ventral hernia.     

Development and validation of a sensitive enzyme immunoassay (EIA) for blood plasma cortisol in female cattle,buffaloes, and goats

A highly sensitive enzyme immunoassay (EIA) that used the second antibody coating technique and the cortisol-horseradish peroxidase conjugate as a label for determination of free and total cortisol in blood plasma of dairy animals (cows, buffaloes, and goats) was developed. For biological validation of the EIA, blood samples were collected from the animals at 48 and 24 h before and 0, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, and 132 h after dexamethasone administration. The EIA was performed directly with 20 μL of fresh plasma (for free cortisol) and also with 20 μL of heat-treated plasma (for total cortisol) after 1:5 dilutions with PBS. Cortisol standards ranging from 0.39 to 200 pg/well/20 μL were used, and the sensitivity of the EIA procedure was found to be 0.39 pg/well/20 μL, which corresponded to 0.02 ng/mL. In comparison with RIA the EIA was at least 4 times more sensitive and required 5 times less cortisol antiserum. In female cattle, buffaloes, and goats, the total, free, and bound plasma cortisol before dexamethasone administration was significantly (P < 0.05) higher than the total, free, and bound cortisol after dexamethasone administration. It can be concluded from these studies that the direct, sensitive EIA validated for estimating the free and total cortisol concentrations was sufficiently reliable and quick for studying the dynamics of cortisol distribution in blood plasma of dairy animals.