IMP-HRM: an automated pipeline for high throughput SNP marker resource development for molecular breeding in orphan crops

Orphan crops have played a major role in daily diet, health nourishment, economy and livelihood of marginal farmers in under developed and developing world. For various reasons, the majority of these crops including finger millet and tef in cereals; lentil, cowpea and groundnut in legumes; cassava and yam in root crops; have remained neglected and underutilized. With little or no investment these ‘orphan’ crops have lagged far behind in developing genomic resources and are deprived of the application of advanced molecular breeding approaches for their rapid improvement. Single nucleotide polymorphisms, multiple nucleotide polymorphisms, insertions/deletions (indels) and size polymorphisms are important tools for developing rich molecular marker resources. Cataloguing and use of intron based polymorphism have also been demonstrated for applications in molecular breeding. Currently the process of predicting putative introns and designing flanking primers for their amplification requires multiple computer programs and a high level of human intervention. We automated this process by developing intron mapping pipeline (IMP) which allows the generation of large primer sets with minimal human intervention and greater efficiency. Since IMP can take advantage of the highly conserved nature of the introns flanking sequences across a wide range of species, any available model plant genome can be used to predict intron boundaries in related orphan crop species. IMP combined with high resolution melt analysis demonstrated potential for high throughput polymorphism discovery, validation and molecular marker development platform which can generate genomic resources rapidly in orphan species.     

Antimicrobial activity of some medicinal plants from India

The results of a preliminary antimicrobial screening of the methanol extracts of Zingiber officinale, Asteracantha longifolia, Citrus acida, Salacia microsperma and Tinospora cordifolia are reported.     

Exogenous cholesterol prevents cryocapacitation-like changes,membrane fluidity,and enhances in vitro fertility in bubaline spermatozoa

A study was conducted to determine the optimum dosage of the exogenous cholesterol-loaded cyclodextrins (CLC) to get maximum cryoprotection for bubaline spermatozoa. In the present study, 120 × 10⁶ spermatozoa were incubated in 2, 3 and 4 mg of CLC as grouped as Gr II, III and IV, respectively, and sperm progressive motility, intracellular Ca²⁺, capacitation status by protein tyrosine phosphorylation (PTP) assay and zona binding per cent (ZBP) and cleavage rate (CR) of the cryopreserved buffalo spermatozoa by in vitro fertility assay were assessed in comparison with an untreated control group (Gr I). Results revealed that there was a significant (p < .05) linear decrease in percentage of sperm population with higher intracellular Ca²⁺ and percentage of sperm population with medium or high capacitated by PTP in CLC treated from 2 to 3 mg and then increased to 4 mg/120 × 10⁶ spermatozoa whereas sperm progressive motility, percentage of sperm population with low capacitated, ZBP and CR were increased significantly (p < .05) in sperm population treated from 2 to 3 mg CLC and then decreased to 4 mg/120 × 10⁶ spermatozoa. The study has clearly indicated that CLC at 3 mg/120 × 10⁶ spermatozoa has maximum beneficial effects in protection of sperm progressive motility, membrane fluidity (low intracellular Ca²⁺); prevention of cryocapacitation (low capacitation pattern in immunolocalization) and enhancement of in vitro ZBP and CR. Post-thaw motility of the CLC-treated sperm has shown positively significant (p < .05) correlation with sperm population with low intracellular Ca²⁺, low capacitated sperm population, ZBP and CR, whereas it was negatively (p < .05) correlated with sperm population with high intracellular Ca²⁺, medium or high capacitated sperm. The present study has revealed for the first time that incubation of spermatozoa with CLC of higher dose (>3 mg/120 × 10⁶ spermatozoa) had adverse effects on sperm cryopreservation, although incubation of sperm with 3 mg/120 million prior to processing had minimised the freezing–thawing-associated damages in bubaline species.     

Genetic diversity and pathotype determination of Colletotrichum sublineolum isolates causing anthracnose in sorghum

Amplified fragment length polymorphism (AFLP) based genetic diversity was analyzed for 232 Colletotrichum sublineolum isolates collected between 2002 and 2004 from three geographically distinct regions of Texas, and from Arkansas, Georgia, and Puerto Rico. Results revealed significant levels of polymorphism (59%) among the isolates. Even so, genetic similarity between isolates was high, ranging from 0.78 to 1.00. Clustering of similar isolates did not correlate with either geographic origin or year of collection. Pathotypes of 20 of the isolates were determined using 14 sorghum lines previously used in Brazil and the United States and 4 from Sudan. Seventeen new pathotypes were established from the 18 isolates that gave uniform and consistent reactions on all host differentials over 2?years of greenhouse testing. Differentials BTx378 and QL3 were resistant to all isolates while BTx623 and TAM428 were universally susceptible both years. Each of these lines had shown differential responses in prior studies indicating that the pathogen population has sufficient diversity to adapt rapidly to changes in resistant host lines deployed. When the 2-step pathotype classification scheme was used, the 18 isolates examined in this study were placed in four pathotype groups (A, C, D and G), which would further then be separated into ten distinct pathotypes. Common sets of differentials and a standardized nomenclature will allow for comparison to be made among pathotypes of C. sublineolum detected from different regions and also could help direct planting of appropriate sorghum lines and aid in the development of more durable forms of resistance.     

Evaluating grain sorghum hybrids for tolerance to iron chlorosis

Grain sorghum production in calcareous soils is frequently affected by iron (Fe) chlorosis. Greenhouse experiments were conducted to screen sorghum hybrids for their tolerance to iron deficiency chlorosis (IDC) and evaluate the effectiveness of Fe chelate application in alleviating IDC. Treatments in Exp. 1 were a factorial combination of 14 sorghum hybrids and three Fe chelate application rates (0, 3.4 and 6.8?kg product ha^?1) applied in-furrow with the seed at the time of planting. Exp. 2 evaluated two sorghum hybrids (85Y40 and NK5418) and three Fe chelate rates (0 and 3.4?kg product ha^?1) at planting, and a split treatment of 3.4?kg ha^?1. Results showed iron chelate application suppressed IDC and increased leaf chlorophyll content and grain yield in susceptible hybrids. Split application of Fe chelate suppressed IDC and increased grain yield. Our results indicate sorghum hybrids G8G08, 86G32 and 87P06 showed promise for tolerance to IDC.     

Molecular markers and their application in genetic diversity of penaeid shrimp

Penaeidae is a family of shrimp, and it contains many species of economic importance, such as the tiger prawn (Penaeus monodon), white leg shrimp, Atlantic white shrimp and Indian prawn. Identification and population genotype structure of penaeid shrimp have been enhanced by molecular markers that can be classified into three types, namely allozyme, mitochondrial and nuclear markers. The widely used mitochondrial DNA markers are 12S rDNA, 16S rDNA, cytochrome b and control region. Random amplification of polymorphic DNA, amplified fragment length polymorphism, restriction fragment length polymorphism, single-stranded conformational polymorphism and microsatellites are the most commonly used nuclear markers for DNA fingerprinting. Molecular markers play a crucial role in penaeid shrimp to evaluate phenotypic and genetic variation, assess demographic bottleneck, study natural population structure, compare wild and hatchery populations, preserve genetic biodiversity, construct chromosome maps and detect whether genetic tag propagation–assisted rehabilitation programs are effective. Increase in the number of molecular markers, construction of high-density genetic maps and implementation of genomic resources (including genome sequencing) are expected to provide tools for the genetic improvement in these aquaculture species through marker-assisted selection. Molecular markers are versatile tools for the identification of populations with genetic crisis by comparing genetic diversities, which helps to establish management units within these threatened species.     

Determination of residues of endosulfan in human blood by a negative ion chemical ionization gas chromatographic/mass spectrometric method: impact of long-term aerial spray exposure

A new and sensitive analytical method using negative ion chemical ionization gas chromatography/mass spectrometry in selective ion monitoring (SIM) mode has been developed for the determination of residues of endosulfan in the human blood. The residues of endosulfan are extracted from whole blood samples without separating the serum by the addition of 60% sulfuric acid at 10 degrees C followed by partition with hexane + acetone (9 + 1 by volume). The total endosulfan is quantified as the sum of alpha-endosulfan, beta-endosulfan and endosulfan sulfate in SIM mode. The mass-fragment ions used for this purpose that are monitored for in SIM mode include endosulfan diol: 95, 169, 214, 313, alpha-endosulfan: 99, 242, 270, 406, beta-endosulfan: 99, 242, 270, 406, and endosulfan sulfate: 97, 353, 386. Recovery experiments were conducted at the concentration range 1.0-100 pg ml(-1). Results showed 112-98% recovery of total endosulfan from the whole blood samples. The relative standard deviation was 1.49-2.68%. The method was found to be highly sensitive in quantifying endosulfan residues down to the 0.1 pg ml(-1) level. Conversion of endosulfan to endosulfan diol was found to be less than 0.1% under the conditions used. The results were compared with published data. The applications of the analytical method for the determination of endosulfan residues in real samples was tested by analyzing 106 human blood samples collected from a population living in Padre village, Kasargode District, Kerala, India, where aerial spraying of endosulfan has been a common agricultural practice over the years. The results showed that none of the blood samples contained residues of endosulfan (alpha-endosulfan 4 beta-endosulfan + endosulfan sulfate) or endosulfan diol. The results were confirmed by the detection of the appropriate amounts in a number of these samples which had subsequently been spiked with endosulfan.     

First report of leaf blight of noni caused by <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler

A severe outbreak of leaf blight was observed in noni crop for the first time in Tamil Nadu and Karnataka states of India during 2008–2009. Based on pathogenicity, morphology and ribosomal DNA spacer sequences, the pathogen was identified as Alternaria alternata (Fr.) Keissler. This report is the first of Alternaria leaf blight of noni.     

Does heat accumulation alter crop phenology,fibre yield and fibre properties of sunnhemp (Crotalaria juncea L.) genotypes with changing seasons?

Field experiments were carried out in split plot design during the dry and wet seasons for two years(two seasons each in 2016–2017 and 2017–2018) with two genotypes(SH4 and SUIN053), two plant geometry(30×15 cm and 45×15 cm main plots) and three levels of NPK(20 kg N ha~(–1), 40 kg P ha~(–1) and 40 kg K ha~(–1); 20 kg N ha~(–1), 60 kg P ha~(–1) and 60 kg K ha~(–1); 20 kg N ha~(–1), 80 kg P ha~(–1) and 80 kg K ha~(–1)) with an objective to study the relationship between fibre yield of sunhmep and thermal indices. The results indicated that the thermal units such as cumulative heat unit(CHU), photo thermal unit(PTU) and helio thermal unit(HTU) were the highest during dry seasons, while relative temperature disparity(RTD) was the highest during wet seasons irrespective of the genotypes, plant geometry and fertilizer levels. The combined analysis of variance showed that the suitability of sunnhemp genotypes for obtaining fibre and seed yields varied with season. The results further indicated that sunnhemp grew during dry seasons with longer photoperiod and higher values of growing degree days(GDD), HTU and PTU resulted in a higher fibre yield, while a higher seed yield and relatively longer, finer and stronger fibres were obtained during wet seasons with higher RTD values. Regression analysis indicated that CHU was positively related to fibre yield, while RTD was positively related to seed yield. CHU beyond 2 000 °C d reduced seed yield and favoured fibre production. In contrary to CHU, RTD values were positively related to seed yield and negatively related to fibre yield. Similarly, HTU had an inverse relationship with fibre yield while PTU had a positive relationship with fibre yield. The genotype SH4 produced a seed yield of 1 361 kg ha~(–1) during wet seasons, which was significantly higher than SUIN053, while a fibre yield of 990 kg ha~(–1)(significantly higher than that of SH4) was obtained for SUIN053 that required less CHU to attain the phenological events during dry seasons. The per unit area yields of seed and fibre with the closer spacing(30 cm×15 cm) by virtue of higher plant density were 17.0 and 14.9% higher than those with the spacing of 45 cm×15 cm, respectively. Higher doses of P and K resulted in higher seed and fibre yields.