Cloning of the beet cyst nematode resistance gene Hs4 from the wild beet Patellifolia procumbens

Plant parasitic nematodes are important pests in crop production worldwide. Therefore, breeding nematode resistant crops is an important aim nowadays. The beet cyst nematode Heterodera schachtii causes severe losses in sugar beet production. The only source of resistance are sugar beet wild relatives from the genus Patellifolia. Sugar beet lines carrying a translocation stemming from chromosome 1 of P. procumbens are resistant to beet cyst nematode. The aim of this study was to clone the beet cyst nematode resistance gene Hs4. In this study, I characterized the translocation segment in the resistant translocation line TR520. I then identified the translocation breakpoint to the precision of a single base pair. Finally, I identified a putative resistance gene. I functionally characterized my candidate gene by two approaches, CRISPR-Cas mediated knock-out and overexpression studies. To characterize the translocation segment, I first generated a de-novo assembly of the resistant translocation line TR520. Then, using whole genome sequencing reads from P. procumbens, I identified the translocation specific scaffolds. Finally, two super scaffolds for the whole translocation segment, super scaffold 1 and super scaffold 2 were generated which are 1.109 and 2.121 Mb in size, respectively. I identified the translocation breakpoint in the following way. I inspected mate-pair read libraries for TR520 and found a set of reads joining super scaffold 2 with a scaffold from chromosome 9 of the sugar beet genome. Therefore, I concluded that the breakpoint lies between these two scaffolds. I amplified the breakpoint by PCR, followed by Sanger sequencing, and I could identify the exact nucleotide position at which the sequence of chromosome 9 from sugar beet ended and the translocation sequence started. To find the resistance gene, I used whole genome sequencing data from two resistant translocation lines, TR520 and TR363, and one γ-irradiated susceptible translocation line TR659. I then identified the translocation regions that are present in the resistant lines but absent in the susceptible line. I found three regions, encompassing a total of 229 kb, and referred to them as “critical regions”. The next step was to identify the putative resistance gene. Using transcriptome data from the roots of the resistant plant after infection, I could narrow down the search to 19 gene-models within the critical regions. None of the genes in the critical regions was annotated as resistance gene analogue. Considering the role of proteases in plant-defence mechanisms, I chose a rhomboid-like protease encoding gene as a putative Hs4, which is predicted to be bound to the endoplasmic reticulum. To functionally characterize my candidate gene, I performed CRISPR-Cas mediated knockout in hairy roots of a resistant translocation variety and overexpression study in hairy roots of a susceptible sugar beet line. Knock-out of my candidate gene resulted in complete loss of resistance, while overexpression led to complete resistance. This study has revealed the Hs4 gene, encoding a rhomboid-like protease and conferring complete resistance to beet cyst nematodes. This gene is the first protease which alone causes resistance to a pest. Thus, it constitutes a previously unknown mechanism of plants to fight plant parasitic nematodes

Cost Benefit of Poultry Farming in Kashmir Valley: Case Study in Anantnag District

Poultry farming from last decade earn a good name in terms of income and employment generation in Kashmir valley. Taking this point as our concern we go through the cost benefit of poultry farming in Kashmir valley. As our study area is district Anantnag and we take one medium size poultry farm as our sample. The study shows that the income from poultry farming is so reasonable and normal profits are earned with less effort. So government should have to think about this industry and support them financially and give them new information’s and training regarding poultry farming through which the production and productivity increase.

SCREENING AND GROWTH KINETIC STUDIES OF WILD CHLOROPHYCEAN FRESH WATER MICROALGAL SPECIES FOR BIOMASS AND BIOFUEL APPLICATIONS

Objective: Microalgae are studied for decades for various products such as, protein rich animal/fish feed, lipids, pigments, neutra ceuticals, therapeutic agents, primary products and biomass. Lipid content was prime target in most of the research programs for production of biodiesel as an alternate to fossil fuel. Chlorophycean microalgae has the potential to meet all these requirements. The objective of this study was to collect and identify chlorophycean microalgae from various water bodies of Jharkhand State of India and to estimate their total lipid content.Methods: Wild cholorophycean fresh water species from Jharkhand were collected and studied for biomass, total lipid, carotenoids and chlorophyll content. Fourier transform infrared spectroscopy (FTIR) data were obtained for further verification of lipid estimation in all the species. Light microscopy as well as Scanning Electron Microscopy (SEM) was performed to identify the species.Results: The observation revealed two groups of micro algae, among these Scenedesmus sp and Chlorella sp. Showed highest lipid accumulation of 45.1 and 41.5 % respectively, while Legerhemia sp. Showed highest biomass production (21.2 g/l). Productivity/day for an 80K L pond system was calculated by extrapolation of results; that changed the choice of organism to Desmodesmus sp.Conclusion: The microalgae collected from highly polluted sites were efficient enough to yield high lipid (AKS-1/AKS-8) and biomass (AKS-6). The laboratory scale study was extrapolated with mass scale culture data and the choice of organism changed to AKS-16 from AKS-1/AKS-8 (for high lipid content) or AKS-6 (for high biomass).Â

Molekularna analiza i istraživanje polimorfizma egzona 10 gena za receptore folikulostimulirajućeg hormona (FSHR) u indijskih pasmina goveda

FSH receptors are important binding sites of the follicle stimulating hormone (FSH) in ovaries and are coded by the FSHR gene which has 10 exons and 9 introns. Exon 10 is the largest (>1200 bp) of all the exons. In the present study, exon 10 of the FSHR gene partial coding sequence (CDS) was cloned and characterized in Sahiwal and Hariana cattle breeds, and DNA polymorphism was investigated using AluI/PCR-RFLP assay. The partial CDS of the Sahiwal and Hariana FSHR exon 10 was 99.3% to 100% similar to exotic cattle breeds at the nucleotide and amino acid level. A missense mutation was found in Sahiwal and Hariana at position 1118 (C→G) that caused an amino acid change at 373 (Thr→Ser) and two nonsense mutations were found at position 729 (G→A), 1180 (C→T). Phylogenetic analysis clearly showed that Sahiwal and Hariana cattle are more closely related to yak and Bos taurus. The 306 bp region of exon 10 on digestion with AluI restriction enzyme revealed three types of genotypes, namely: CC (243 bp and 63 bp), GG (193 bp, 63 bp and 50 bp) and CG (243 bp, 193 bp, 63 bp and 50 bp), where the CG genotype was more frequent (45.0%) than CC (13.5%) and GG (41.5%) genotypes, and the frequency of the G allele was higher (0.64) than the C allele (0.36) in all the screened animals. Chi square (χ2) analysis revealed that the screened animal population was in Hardy-Weinberg equilibrium. An association study revealed a significant (P1200 bp) od svih egzona. U ovom istraživanju, egzon 10 kloniran je iz djelomično kodiranog slijeda (CDS) FSHR gena. Kao materijal za istraživanje poslužila su goveda pasmina sahival i hariana u kojih je DNK polimorfizam analiziran pomoću AluI/PCR-RFLP testa. Na razini nukleotida i aminokiselina, djelomično kodirani slijed (CDS) egzona 10 FSHR gena u goveda sahival odnosno hariana pasmina bio je 99,3% do 100% sličan onom u egzotičnih pasminama goveda. U navedene dvije pasmine na položaju 1118 (C → G) pronađena je pogrešna mutacija koja je uzrokovala promjenu aminokiseline na 373 (THR → SER), te dvije besmislene mutacije koje su nađene na položajima 729 (G → A) i 1180 (C → T). Filogenetska analiza jasno je pokazala da su goveda pasmina sahival i hariana u većoj mjeri povezana s jakom i domaćim govedom (Bos taurus). Područje egzona 10, koje sadrži 306 parova baza pokazalo je nakon cijepanja s AluI restrikcijskim enzimom 3 genotipa: CC (243 bp i 63 bp), GG (193 bp, 63 bp i 50 bp) i CG (243 bp, 193 bp, 63 bp i 50 bp). Genotip CG bio je učestaliji (45,0%) od genotipova CC (13,5%) i GG (41,5%). Uzevši u obzir sve pretražene životinje, učestalost G alela bila je veća (0,64) od C alela (0,36). Hi-kvadrat (χ 2) analizom je utvrđeno da je pretražena populacija goveda bila u Hardy-Weinberg ravnoteži. Statistička analiza povezanosti pokazala je znakovite (P<0,05) razliku između genotipova za obilježja ukupnog prinosa mlijeka i trajanja laktacije, pri čemu se istaknuo genotip CC s većim prinosom mlijeka u odnosu na druge genotipove

A Study of Female Figure in Bapsi Sidhwa’s ‘The Pakistani Bride’ and Tehmina Durrani’s ‘My Feudal Lord’

The Feminist Movement, of the twentieth century that metamorphosed the attitude and perception of the world. It provided a better understanding of women’s issues and rights and endeavored to bring them forward because they had been marginalized for so long. While the privileged women of the continent got into limelight by means of their writings, women belonging to third world still had their voice unheard. There is also a rich tradition of Women writing in English being carried out efficiently with some remarkable writers, who have not only gained recognition at internationally acclaimed. The novels strongly comments about the influence of cultural conflicts and its impact on the social order. Bapsi Sidhwa's novel The Pakistani Bride deals with the repression of women in the patriarchal Pakistani society. The novel is based on a true story narrated to Sidhwa when with her family; she stayed at an army camping the remotest regions of the Karakoram Mountains. My Feudal Lord is one of the extraordinary autobiographies that can ever be written. Durrani uses it as a means of exposing the hypocrisy of ruling elites in Pakistan generally and the cruel nature of her husband specifically. In this book she talks about social ethos of Pakistani marital life by citing her own marriage as an example.Keywords: privileged, milieu, promise, experience, autobiography, high.Introductio

Molekularna karakterizacija cjelovite kodirajuće sekvencije gena MBL1 u indijskog vodenog bivola (Bubalus bubalis)

Mannose-binding lectin (MBL) is a member of the collectin protein family that binds a broad range of microorganisms and activates the lectin-complement pathway of innate immunity. A number of mutations have been found in both the coding as well as the non-coding regions of the MBL1 gene in various species, of which several variations affected the assembly of MBL1, thus leading to a low level of plasmic MBL and innate immune dysfunctions. In the present study, we have reported molecular cloning and characterization of the complete coding sequence of the MBL1 gene in the Indian buffalo breed Murrah. A 951 bp fragment of the MBL1 gene was amplified, cloned and sequenced. Multiple sequence alignment with other buffalo and cattle breeds revealed that the Murrah buffalo MBL1 CDS was 98.1-99.6% homologous to other buffalo breeds, and 98.3-98.5% similar to cattle breeds at nucleotide level. It was 96.8-98.8% homologous to buffalo breeds and 96.8-97.2% similar to cattle breeds at amino acid level. The amino acid sequence of the Murrah buffalo MBL1 contained two non-synonymous amino acid substitutions (L204P and S180P). Further, PCR-RFLP was performed to screen 50 Murrah buffalo for the presence of SNPs, g.855G>A in intron I and g.2686T>C, as well as g.2651G>A in exon 2 region of the MBL1 gene. The ApaI/intron I PCR-RFLP assay revealed a polymorphic pattern with three genotypes viz., AG (90%), GG (8%) and AA (2%), with allelic frequencies 0.94 for G and 0.06 for A. HaeIII/exon 2 PCR-RFLP assay exhibited the presence of three genotypes, namely, TC (66%), TT (32%) and CC (2%) with allelic frequencies 0.15 for T and 0.85 for C. StyI/exon 2. PCR-RFLP assay showed a monomorphic pattern for g.2651G>A with GG genotype only. We further examined the association of these SNPs with milk production traits and somatic cell score (SCS), and found no significant difference for any of the traits. Since the present study has formulated the results on the basis of a relatively small sample size, further studies with a larger sample size are required to validate the effects of polymorphisms.Lektin koji veže manozu (MBL), član je porodice proteina kolektina koja veže veliki broj mikroorganizama i aktivira lektinski put komplementa prirođene imunosti. U različitim vrstama pronađene su brojne mutacije u kodirajućim i nekodirajućim regijama gena MBL1. Za nekoliko je mutacije utvrđeno da utječu na strukturu gena MBL1, što dovodi do snižavanja razine MBL-a i prirođenog poremećaja imunosnog sustava. U ovom je istraživanju provedeno molekularno kloniranje i karakterizacija cjelovitog slijeda gena MBL1 u indijskog vodenog bivola pasmine Murrah. Fragment s ukupno je 951 bp gena MBL1 umnožen je kloniran i sekvenciran. Višestruko poravnanje sekvencija s drugim pasminama bivola i goveda otkrilo je da je kodirajuća sekvencija gena MBL1 bila 98,1 – 99,6 % podudarna s drugim pasminama bivola i 98,3 – 98,5 % podudarna s drugim pasminama goveda na nukleotidnoj razini. Na razini aminokiselina ustanovljena je podudarnost od 96,8 do 98,8 % s pasminama bivola i 96,8 – 97,2 % s pasminama goveda. Sekvencija aminokiselina MBL1 u vodenih bivola pasmine Murrah sadržavala je dvije nepodudarne supstitucije aminokiselina (L204P i S180P). Osim toga proveden je PCR-RFLP kako bi se 50 bivola pasmine Murrah analiziralo na prisutnost jednonukleotidnih polimorfizama (SNP), g. 855G > A u intronu I i g. 2686T > C, kao i g. 2651G > A, u eksonu 2 regije gena MBL1. Test ApaI/intron I PCR-RFLP otkrio je polimorfni obrazac s tri genotipa: AG (90 %), GG (8 %) i AA (2 %) s učestalošću alela od 0,94 za G i 0,06 za A. Test HaeIII/exon 2 PCR-RFLP pokazao je prisutnost triju genotipova: TC (66 %), TT (32 %) i CC (2 %) s učestalošću alela od 0,15 za T i 0,85 za C. Test StyI/exon 2 PCR-RFLP pokazao je monomorfni obrazac za g. 2651G > A, samo s genotipom GG. Osim navedenog istražena je i povezanost SNP-ova s proizvodnim svojstvima mlijeka i omjerom somatskih stanica (SCS). Nije pronađena znakovita razlika ni za jedno svojstvo. S obzirom na to da se ovo istraživanje temelji na malom broju uzoraka, potrebna su istraživanja koja će uključiti veći broj životinja kako bi se provjerili učinci polimorfizama

Comparative Compositional Analysis and Pesticidal Efficacy of Essential Oils from Leaves of Skimmia Aanquetilia N.P. Taylor and Airy Shaw

The objective of the current study was to re-examine the chemical components of the essential oil (EO) from the aerial parts of Skimmia anquetilia N.P. Taylor &amp; Airy Shaw in two different seasons designated as Skimmia anquetilia rainy season essential oil (SKREO) and Skimmia anquetilia winter season essential oil (SKWEO). The GC-MS analysis of SKREO and SKWEO resulted in the identification of 42 and 48 constituents, comprising of 95.3 % and 95.4 % of the total composition respectively. Both SKREO and SKWEO varied in their chemical composition in terms of quantity viz: linalyl acetate (15.8% - 17.6%), linalool (13.2% - 13.9%), geijerene (11.6% - 11.7%), α-thujene (11.3% - 11.1%), α-terpineol (6.1% - 6.1%), geranyl acetate (5.0% - 5.1%), α-terpinyl acetate (3.3% - 3.1%), myrcene (3.0% - 3.1%), geraniol (2.6% - 1.9%), α-pinene (2.1% - 2.2%), trans-β-ocimene (2.1% - 2.3%), cis-β-ocimene (2.0% - 2.2%) and neryl acetate (2.3% - 2.4%). Besides qualitative differences SKREO and SKWEO, both were studied for their pesticidal activities. The study exhibited potent antifeedant activity against Spodoptera litura and nematicidal activity against Meloidogyne incognita. Based on the present observations, it was found that besides its academic importance, shrub Skimmia anquetilia can be a good source of phytochemicals like linalyl acetate, linalool, geijerene, thujene and can be used for the development of herbal source for antifeedant and nematicidal activity after proper clinical trials

Variation of antioxidant activity and Phenolic content of some common home remedies with storage time

The variation of antioxidant activity and total phenolic content of some plants commonly used as home remedies were screened on the basis of time factor such as methonlolic extracts of short term and long term air dried plant Ocimum sanctum (leaf), Cucumis sativus (Seed), and ethanolic extracts of Capsicum frutescens (Seed)and Coriandrum sativum(Seed). Antioxidant activities and total phenolic content values were estimated using DPPH reagent and Folin Ciocalteu reagent methods respectively. The antioxidant activity in short term air dried Ocimum sanctum plant methanol extract (IC50=0.03±0.01 mg/ l) stoods at the highest side than other plant extracts. On other side Coriandrum sativum plant ethanol extract represent higher phenolic content (70.24±3.4/66.57±4.1 mg/g) than other plant extracts. It was found that there is no correlation between antioxidant and phenolic content of these plant extracts and long term air dried samples had shown slight variation of antioxidant activities and total phenol contents from short term dried samples. Methanol extract of Ocimum sanctum. plant had shown statistical significance in antioxidant activity (short term IC50 0.03±0.01mg/dl, long term IC500.05±0,01mg/dl) than other study extracts These findings have high significance in the ageing of food chemistry