Muhammad Abubakkar Azmat

Extraction of DNA suitable for PCR applications from mature leaves of Mangifera indica L.

Good quality deoxyribonucleic acid (DNA) is the pre-requisite for its downstream applications. The presence of high concentrations of polysaccharides, polyphenols, proteins, and other secondary metabolites in mango leaves poses problem in getting good quality DNA fit for polymerase chain reaction (PCR) applications. The problem is exacerbated when DNA is extracted from mature mango leaves. A reliable and modified protocol based on the cetyltrimethylammonium bromide (CTAB) method for DNA extraction from mature mango leaves is described here. High concentrations of inert salt were used to remove polysaccharides; Polyvinylpyrrolidone (PVP) and β-mercaptoethanol were employed to manage phenolic compounds. Extended chloroform-isoamyl alcohol treatment followed by RNase treatment yielded 950–1050 μg of good quality DNA, free of protein and RNA. The problems of DNA degradation, contamination, and low yield due to irreversible binding of phenolic compounds and coprecipitation of polysaccharides with DNA were avoided by this method. The DNA isolated by the modified method showed good PCR amplification using simple sequence repeat (SSR) primers. This modified protocol can also be used to extract DNA from other woody plants having similar problems.

Comprehensive screening and selection of okra (Abelmoschus esculentus) germplasm for salinity tolerance at the seedling stage and during plant ontogeny

The okra germplasm was screened for salinity tolerance at the seedling stage and during plant ontogeny. Substantial variation existed in okra for salinity tolerance at the seedling stage. An 80 mmol/L NaCl concentration was suitable for discriminating tolerant and non-tolerant okra genotypes. The pooled ranking of the genotypes, based on individual rankings for each trait (root and shoot length, germination percentage, and relative Na+ and K+) in individual NaCl concentrations, was effective for selecting tolerant genotypes. Genotypes selected at the seedling stage maintained their tolerance to NaCl during plant ontogeny, suggesting that screening of the germplasm entries and advanced breeding materials for salt tolerance at the seedling stage is effective. Among 39 okra genotypes, five were identified as the most tolerant genotypes and showed potential for use in breeding programs that focus on the development of salt-tolerant, high-yield okra cultivars.

Single recessive gene controls powdery mildew resistance in pea.

Powdery mildew caused by Erysiphe polygoni DC causes severe reductions in the green pod yield of pea (Pisum sativum L). The objective of this study was to reveal the genetics of powdery mildew resistance in six Resistant × Susceptible crosses and their generations (F1, F2, BCs, and BCr) against three isolates of E. polygoni. Cautious selection of the parental material and evaluation of 873 individual F2 plants from six crosses gave a perfect 3 susceptible:1 resistant segregation and 1 resistant:1 susceptible in BCr. These results led to the conclusion that resistance was conferred by the single recessive gene (er).

Screening Pea Germplasm Against Erysiphe polygoni for Disease Severity and Latent Period

Powdery mildew disease caused by Erysiphe polygoni DC significantly reduces yield quantity and quality of edible seed in pea (Pisum sativum L.). Development of powdery mildew–resistant varieties is an economical and environmentally friendly approach to managing the disease. One hundred forty-six pea accessions, collected from different countries, were screened against powdery mildew. It was determined that accessions 9057, 9370, 9375, 10609, 10612, 18293, 18412, 19598, 19611, 19616, 19727, 19750, 19782, 20126, 20152, 20171, It-96, No. 267, and No. 380 were resistant; accessions It-96 and No. 267 were highly resistant. The correlation between disease score and latent period was negative, suggesting that these parameters should be used simultaneously for more precise screening against powdery mildew. The resistant accessions selected might be used as powdery mildew–resistant parent(s) in breeding programs depending upon the nature and number of gene(s) controlling for resistance.

Role of epicuticular waxes in the susceptibility of cotton leaf curl virus (CLCuV)

Cotton leaf curl virus (CLCuV) is the causal agent of the damaging disease of cotton that is caused by number of begomaviruses and vectored by silver leaf whitefly. In the present study, an attempt was made by infecting Gossypium arboreum variety 786, its wax mutant GaWM3 along with Gossypium hirsutum MNH-93 with viruliferous whiteflies. The presence of symptoms on leaves and amplification by PCR for virus in G. hirsutum MNH-93 and wax mutant GaWM3 but not in G. arboreum variety 786 clearly determined the presence of virus in G. hirsutum MNH-93 and wax mutant GaWM3 but not in G. arboreum variety 786. The results indicate that wax may act as physical barrier and provide hindrance in transfer of virus by whitefly.

MORPHOLOGICAL CHARACTERIZATION AND SSR BASED DNA FINGERPRINTING OF ELITE COMMERCIAL MANGO CULTIVARS

Thirteen premium quality commercial mango cultivars grown in Pakistan were assayed with seven SSR primer series viz., mMiCIR, MiSHRS, MIAC, MITGIT, LMMA, UBC and MiIIHR. Among the 120 primers used, 101 produced bands and revealed a narrow genetic base ranging from 0.62-0.8 with maximum similarity among the cvs. ‘Anwar Ratole’ and ‘Sensation’ while the maximum divergence was between cvs. ‘Sindhri’ and ‘Sensation’. Chaunsa was genetically more similar (74.2%) and ‘Kala Chaunsa’ was more dissimilar (31.2%) respectively when compared with all the other cultivars. Of 101 SSR primers, we have identified 30 (29.7%) highly informative primers (PIC value ≥0.6), which could be useful in the molecular characterization of mango germplasm. The primer series MIAC, UBC and MiIIHR had the maximum percentage of highly informative primers in descending order. The UPGMA based Euclidian dendrogram constructed with similarity indices placed all the cultivars according to regions of their origin and magnitude of diversity among them. The morphological characterization of the commercial cultivars was also done using 11 quantitative traits; however, none of the traits except fruit weight was informative for the assessment of genetic diversity. SSR based molecular characterization appeared to be reliable, reproducible and cost effective.

Characterization of Pea Germplasm

There is a benefit to continued development of improved lines and cultivars of edible plants by effectively using the available gene pool. Pea is a staple food throughout the world (Pisum sativum L.). A field experiment was conducted for two consecutive seasons to assess the genetic diversity of 146 pea accessions. The characterization was based on quantitative and qualitative morphological characters. The pea accessions were collected from different agro-climatic zones of the world. A high degree of diversity was found among the pea germplasms both for qualitative and quantitative traits. Principal component analysis showed that days to 50% flowering, 100-seed weight (fresh and dry), plant height, and number of pods per plant are among the most important descriptors which accounted for more than 90% of the variation. Such a high magnitude of variation suggests the use of all these traits for efficient characterization of pea germplasm. Correlation studies revealed positive and significant correlations among yield, seeds per pod, 100-seed weight (fresh and dry) pod length, and width. Cluster analysis assigned all of the accessions to nine distinct clusters based on Euclidean distances and Unweighted Pair Group Method with Arithmetic Mean (UPGMA), which suggests that there is no parallelism between genetic and geographical diversity.

Pathogenicity and Characterization of Geographically Distributed Isolates of Erysiphe polygoni

The incidence of powdery mildew disease on pea (Pisum sativum L.) is a major limiting factor to maximizing yields in different ecological and geographical zones due to environmental factors and the presence of pathogenic variation in Erysiphe polygoni DC. In order to develop a widely adopted powdery mildew–resistant cultivar it is necessary to challenge genotypes with all available pathogenic variants. In order to examine pathogenic variation, 23 naturally occurring single colony isolates of E. polygoni, collected from geographically diverse locations, were subjected to pathogenicity testing and characterization using a detached leaf assay. Isolates demonstrated small differences in pathogenicity against pea. Isolates MUZ-1 and MUZ-2 were the most virulent and produced disease symptoms even on the resistant cultivars. Isolates TTS-2 and TTS-6 were less virulent. Significant variation occurred among isolates for quantitative and qualitative parameters regardless of the degree of pathogenicity. There was variation for conidium length, conidium width, cleistothecia number, number of hyphae, ascus number, ascus length, ascospore number, and ascospore width. An unweighted pair group method with arithmetic mean phenogram, constructed on the basis of quantitative parameters, indicated that isolates were morphologically different but pathogenicity was not controlled by morphological features of the isolates, though morphological features can be used to distinguish between isolates.

Premium Quality Mango Genotypes for Extended Harvest Season

Mango (Mangifera indica L.) is delicious and nutritious fruit grown in the tropical and subtropical regions of the world. The IndoPak subcontinent–Myanmar region has been reported as the consensus center of diversity for mango (Yamanaka et al., 2006). Mango in Pakistan is a major fruit crop. Due to appealing organoleptic quality, taste, pleasant aroma, and excellent texture, the demand for Pakistani mangoes is increasing in the international market. Most of the mangoes grown in Pakistan are midseason maturing cultivars (Rajwana et al., 2008). Since all the elite and exportable mango cultivars grown in Pakistan mature during mid-July to midAugust (mid harvest season); leaving the early and late harvest season export windows unattended. In spite of high production, the mango exports from Pakistan are meagre and stagnant. Mango production in Pakistan has been affected due to insect–pest infestation (mainly caused by mango leafhopper, mango mealybug, and fruit fly) and different physiological disorders.Mango quick wilt disease (MQWD) (Ceratocystis manginecans) is now widespread in the commercial fruit producing areas of Pakistan which is a serious threat to the production and cultivation of quality mangoes (van Wyk et al., 2007). Most commercial mango cultivars grown in Pakistan typically propagated by means of seedling selection from the natural populations of indigenous cultivars, which are more prone to MQWD (van Wyk et al., 2007). Therefore, together with limited genetic base of commercial mango cultivars and the growing threat of MQWD, the situation has become more critical. Pakistan is located at the western periphery of the natural range of monoembryonic mangoes. Pakistan’s commercial mango cultivars are mostly selected landrace clones and chance seedlings being propagated by amateur gardeners and nurserymen (Rajwana et al., 2008). Selection of chance seedlings arising fromcommercial cultivars has been the preferred method of cultivar development (Chaikiattiytos et al., 2000; Khan et al., 2015; Rajwana et al., 2011). Keeping in view the importance of the identification of alternative mango genotypes in relation to disease progression in the mango growing areas with the same legacy of taste and aroma of Pakistani mangoes, a research project was funded by Punjab Agricultural Research Board. An extensive survey was conducted for 4 consecutive years (2010–14) throughout the mango growing areas of Punjab and Azad Jammu and Kashmir. More than 500 diversified seedling trees were tagged and evaluated for different morphological, physicochemical, and molecular traits. At the completion of this project, nine mango genotypes were identified as potential and alternative genotypes for commercial cultivation in mango growing areas with extended harvesting season.