Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L. ) varieties of North-West Pakistan / Caracterização bioquímica para determinação de distâncias genéticas entre diferentes variedades indígenas de grão-de-bico (Cicer arietinum L. ) do noroeste do Paquistão

Abstract Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.

Resumo As distâncias genéticas entre as diferentes variedades de grão-de-bico e a avaliação de seus perfis de aminoácidos livres foram determinadas com base na eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). As proteínas solúveis totais foram resolvidas em SDS-PAGE a 10%. Foi observada baixa variabilidade nas variedades testadas. O dendrograma fundamentado em dados eletroforéticos agrupou os genótipos em dois grupos. Os resultados mostraram que o teor médio de proteínas de todas as variedades foi de 26,01%, na faixa de 22,8% para Thal-2006 a 34,06% para Sheenghar-2000 do peso de sementes secas. Com base no conteúdo total de proteínas, Bittal-98, Dasht, Sheen Ghar-2000, Karak-3, CM-98, Paidar-91, Fakhr-e-Thal, C-44, Balaksar e KK-1 apresentaram concentrações semelhantes para o conteúdo de proteínas entre si, mas tiveram variação quanto ao restante das variedades. Diferentes proteínas foram separadas com base nas alterações de seus pesos moleculares por meio de eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). Dasht, CM-98 e Sheen Ghar mostraram 100% de similaridade. Balaksar, Fakhr-e-Thal, KK-2, Chattan e KC-98, KK-1 e Lawaghar foram 100% semelhantes entre si, mas apresentaram variação em relação ao restante dos acessos. O dendrograma geral mostrou alto e baixo nível de variação entre os acessos. A concentração de aminoácidos livres variou entre as 16 variedades de grão-de-bico. Foi encontrada uma diferença significativa entre os aminoácidos essenciais e não essenciais nas cultivares de grão-de-bico. A concentração total de aminoácidos essenciais foi registrada em 40,81 g / 100 g de proteína, enquanto a não essencial foi registrada em 59,18343 g / 100 g de proteína nas cultivares. A maior concentração de aminoácidos essenciais foi encontrada em C-44, seguida de KK-2, KK-1 e Fakhr-e-Thal, enquanto a menor concentração foi registrada em CM-98, Paidar-91 e Sheen Ghar-2000. As cultivares TAL-2006, Chattan e Karak-3 apresentaram concentração máxima de aminoácidos essenciais e endógenos. Em conclusão, para ampliar os pools genéticos em programas de melhoramento ou procurar caracteres exóticos, por exemplo, novos alelos de resistência a doenças, pode ser utilizada a adesão com baixos coeficientes de similaridade (Lawaghar e Battal-98). Além disso, as informações adquiridas neste estudo poderiam ser usadas para criar uma abordagem de criação eficiente, com o objetivo de melhorar a nutrição e ampliar a base genética dessa cultura alimentar essencial do Paquistão.

Amino Acid Profile, Functional Properties and In-vitro Antioxidant Capacity of Cucurbita maxima and Cucurbita mixta Fruit Pulps and Seeds

The study aimed at evaluating the amino acid profile, fatty acid profile, anti-nutritional factors, functional properties and in-vitro antioxidant activities of Cucurbita maxima and Cucurbita mixta fruit pulps and seeds. Freshly harvested Cucurbita mixta and Cucurbita maxima fruit were processed into flour as; Pa: Cucurbita maxima pulp flour, Pi: Cucurbita mixta pulp flour, Sa: Cucurbita maxima seed flour and Si: Cucurbita mixta seed flour and were evaluated for amino acid profile, fatty acid profile, anti-nutritional factors, functional properties and in-vitro antioxidant activities. Triplicate data were analysed and means were separated using New Duncan Multiple Range Test (NDMRT) at p<0.05. The protein content of the fruit pulps and seeds flour samples were 12.77 g/100 g (Pi), 13.22 g/100 g (Pa), 15.37 g/100 g (Sa) and 16.86 g/100 g (Si). Total essential amino acid was 5.33 mg/100 g of protein (Pa) 6.62 mg/100 g of protein (Pi), 9.85 mg/100 g of protein (Sa) and 14.61 mg/100 g of protein (Si). Total essential amino acid of Pi and Si are significantly higher (p>0.05) than Pa and Sa. Statistically, saturated fatty acid and polyunsaturated fatty acid of the Pi and Si were significantly lower (p<0.05) than Pa and Sa respectively. Antioxidant activities against ABTS* (Pi) is significantly higher (p>0.05) than Pa and with no significant differences (p<0.05) between ABTS values of Si and Sa. While there is a significant difference (p<0.05) between Si and Sa as well as Pi and Pa antioxidant activities against DPPH* respectively. In conclusion, Cucurbita seeds and pulps flour contains high protein content, appreciable amount of essential minerals, lower Na/K molar ratio of less than one and they also exhibit a good free radical scavenging abilities against DPPH*, ABTS* and ability to reduce Fe3+ to Fe2+ with high content of total phenol and flavonoid.

Nutrient Composition, Amino Acid Profile and Anti-nutritional Factors of Nixtamalized Maize Flour Supplemented with Sprouted Soybean Flour

The high dependence on maize as a staple food in Tropical Africa, coupled with its low nutritive value necessitate investigation on how to improve the nutritional value of maize based foods. The present study was aimed at assessing the effect of supplementing nixtamalized maize flour with sprouted soy bean flour on the proximate composition, mineral content, amino acid profile and anti-nutritional factors in the blends. Nixtamalized maize flour was prepared by cooking maize grains in 1% unripe plantain peel ash solution (pH 10.2), steeped in the cooked solution for 15 h, washed, dried and milled into flour. The flour was supplemented with 0, 10, 20, 30 and 40% sprouted soy flour and analyzed for the aforementioned parameters. The results showed that protein, fat, ash, crude fibre and caloric value increased with increase in soy flour supplementation ranging from 9.26–22.57%, 4.51–10.53%, 1.38–2.06%, 2.14–2.39% and 408.47–434.85 kcal/100 g respectively while carbohydrate decreased from 82.71 – 62.45%. Potassium, calcium, magnesium and zinc contents increased from 267.58 – 286.35mg/100g, 126.93 – 161.03 mg/100 g, 135.71 – 163.81 mg/100 g and 4.52 – 4.85 mg/100 g respectively with soy flour addition. The total amino acids and total essential amino acids increased from 70.55 g/100 g protein and 30.54 g/100 g protein for the control to 87.97 g/100 g protein and 38.98 g/100 g protein for the 40% soy flour supplemented blends respectively. Limiting essential amino acids in both flours were significantly (P = 0.5) improved as a result of soy flour supplementation. Majority of essential amino acid chemical scores were above 100% except for lysine (51.03–66.38%), tryptophan (67.27–95.46%) and threonine (80.88 – 99.12%). Soy flour supplemented blends had higher phytate, trypsin inhibitor and tannin contents than the control sample but their values were low and may not have serious effect on nutrient bioavailability. The study clearly shows that nutritional value of nixtamalized maize flour can be improved by supplementing it with sprouted soy flour.

Biochemical characterization for determination of genetic distances among different indigenous chickpea (Cicer arietinum L.) varieties of North-West Pakistan

Abstract Genetic distances among different chickpea varieties and evaluation of their free amino acid profiles were determined on the basis of Sodium dodecyle sulphate polyacrylamide gels electrophoresis (SDS-PAGE). Total soluble proteins were resolved on 10% SDS Polyacrylamide gel. Low variability in tested varieties was observed. Dendogram based on electrophoretic data clustered the genotypes into 2 groups. The results showed that the average protein content of all the varieties was 26.01% within the range 22.8% for Thal-2006 to 34.06% Sheenghar-2000 of dry seed weight. On the basis of total protein content Bittal-98, Dasht and Sheen Ghar-2000, Karak-3 and CM-98, Paidar -91 and Fakhr-e-Thal, C-44, Balaksar and KK-1showed similar concentrations for protein contents among each other but showed variation from the rest of the varieties. Different proteins were separated on the basis of changes in their molecular weights by means of Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE). Dasht, CM-98, and Sheen Ghar showed 100% similarity. Balaksar and Fakhr-e- Thal, KK-2 and Chattan and KC-98, KK-1 and Lawaghar were 100% similar among each other but showed variation from the rest of the accessions. The overall dendrogram showed high and low level of variation among the accessions. The concentration of free amino acids varied among the 16 chickpea varieties. A significant difference of both essential and non-essential amino acids was found among the chickpea cultivars. The total concentration of essential amino acid was recorded 40.81 g/100 g protein while non-essential was recorded 59.18343 g/100 g protein in the given cultivars. The highest concentration of essential amino acids was found in C-44 followed by KK-2, KK-1 and Fakhr E Tal while the lowest concentration was recorded in Cm-98, Paidar-91 and Sheen Ghar-2000 respectively. Cultivars TAL-2006, Chattan and Karak-3 showed maximum concentration of both essential and endogenous amino acids. In conclusion; for broadening the genetic pools in breeding programs or to search for exotic characters, for instance new disease resistance alleles, accession with low similarity coefficients (Lawaghar and Battal-98) may be utilized. Furthermore the information acquired from this study could be used to device a proficient breeding approach intended at improving nutritional as well as broadening the genetic base of this essential food crop of Pakistan.

Resumo As distâncias genéticas entre as diferentes variedades de grão-de-bico e a avaliação de seus perfis de aminoácidos livres foram determinadas com base na eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). As proteínas solúveis totais foram resolvidas em SDS-PAGE a 10%. Foi observada baixa variabilidade nas variedades testadas. O dendrograma fundamentado em dados eletroforéticos agrupou os genótipos em dois grupos. Os resultados mostraram que o teor médio de proteínas de todas as variedades foi de 26,01%, na faixa de 22,8% para Thal-2006 a 34,06% para Sheenghar-2000 do peso de sementes secas. Com base no conteúdo total de proteínas, Bittal-98, Dasht, Sheen Ghar-2000, Karak-3, CM-98, Paidar-91, Fakhr-e-Thal, C-44, Balaksar e KK-1 apresentaram concentrações semelhantes para o conteúdo de proteínas entre si, mas tiveram variação quanto ao restante das variedades. Diferentes proteínas foram separadas com base nas alterações de seus pesos moleculares por meio de eletroforese em gel de poliacrilamida com dodecil sulfato de sódio (SDS-PAGE). Dasht, CM-98 e Sheen Ghar mostraram 100% de similaridade. Balaksar, Fakhr-e-Thal, KK-2, Chattan e KC-98, KK-1 e Lawaghar foram 100% semelhantes entre si, mas apresentaram variação em relação ao restante dos acessos. O dendrograma geral mostrou alto e baixo nível de variação entre os acessos. A concentração de aminoácidos livres variou entre as 16 variedades de grão-de-bico. Foi encontrada uma diferença significativa entre os aminoácidos essenciais e não essenciais nas cultivares de grão-de-bico. A concentração total de aminoácidos essenciais foi registrada em 40,81 g / 100 g de proteína, enquanto a não essencial foi registrada em 59,18343 g / 100 g de proteína nas cultivares. A maior concentração de aminoácidos essenciais foi encontrada em C-44, seguida de KK-2, KK-1 e Fakhr-e-Thal, enquanto a menor concentração foi registrada em CM-98, Paidar-91 e Sheen Ghar-2000. As cultivares TAL-2006, Chattan e Karak-3 apresentaram concentração máxima de aminoácidos essenciais e endógenos. Em conclusão, para ampliar os pools genéticos em programas de melhoramento ou procurar caracteres exóticos, por exemplo, novos alelos de resistência a doenças, pode ser utilizada a adesão com baixos coeficientes de similaridade (Lawaghar e Battal-98). Além disso, as informações adquiridas neste estudo poderiam ser usadas para criar uma abordagem de criação eficiente, com o objetivo de melhorar a nutrição e ampliar a base genética dessa cultura alimentar essencial do Paquistão.

Characterization of New Quality Protein Maize (QPM) Varieties from a Breeding Program: Analysis of Amino Acid Profiles and Development of a Variety- Diagnostic Molecular Marker.

Aims of the Study: This study was to determine the amino acid profile of two newly developed quality protein maize varieties and to develop variety-diagnostic molecular markers. Methodology: Two new maize varieties, named MUDISHI 1 and MUDISHI 3 have been developed by breeders and farmers using the participatory breeding approach. Total protein and amino acid profiles of the two new lines were compared to their respective parental population and a locally released genetically improved normal maize variety. Maize accessions from the DR-Congo breeding program were analyzed using ISSR primers. Variety-diagnostic markers were identified and characterized. Results: Protein analysis data revealed that MUDISHI 1 and MUDISHI 3 are QPM varieties that are distinct from their original population, Longe 5 QPM from NARI- Unganda and DMR-ESR-W-QPM from the International Institute for Tropical Agriculture (ITTA, Ibadan), respectively. Lysine content in MUDISHI 1, and MUDISHI 3 were 3.5 g and 3.6 g of lysine / 100 g, respectively, which represents a significant increase of 20% and 23% over the genetically improved normal maize variety (Salongo 2) that is currently released. There was a significant increase of 25% of tryptophan and 33% of methionine in MUDISHI 3 compared to its parental variety while the amount of lysine was similar for the two varieties. There were 10% and 15% decrease of lysine and tryptophan, respectively in MUDISHI 1 compared to its original parent Longe 5 QPM. Genomic DNA was extracted from different maize varieties. One ISSR diagnostic-marker of 480 bp that was identified was unique to the QPM variety MUDISHI 3. This sequence was converted to a sequence characterized amplified region (SCAR) marker using a pair of designed primers. This SCAR sequence was not specific to MUDISHI 3 as it was present in all the varieties tested. Conclusion: The newly developed varieties are typical QPM lines. The development of an ISSR diagnostic marker indicates that it is possible to develop a molecular breeding program involving QPM and normal varieties.