Progress 01/01/10 to 09/30/14

Outputs
Target Audience: Plant genetics/ molecular biology researchers and plant breeders. Changes/Problems: Extraction of RNA from individual pollen grains was attempted to facilitate gene expression profiling. However extremely limited amount of nucleic material within isolated pollen grain rendered this endeavor unsuccessful. What opportunities for training and professional development has the project provided? This project provided pre-college, undergraduate and graduate students’ research trainings for Department of Agricultural & Environmental Sciences. Through micromanipulation, AFLP and sequencing-gel procedures described above, students’ as well as that of a USDA Borlaug fellow trainings in molecular techniques were provided. The trainings of undergraduate and graduate students as well as that of a USDA Borlaug fellow focused on research applications. Four pre-college summer internship students were exposed to aspects of cotton genetics at their academic levels. The summer interns presented their work to other interns, college faculty and their family members. The undergraduate student has continued training in molecular research techniques till graduation. The training of a graduate student focused on molecular research application in cotton genetics and thus helped to graduate while completing a M.S. thesis. How have the results been disseminated to communities of interest? During this period presentations of the procedures developed and research results were made available to stakeholders. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Upland cotton substitution lines with most of the genome from G. hirsutum line TM-1 while substituting chromosomes numbers 01, 02, 04, 05sh, 06, 07, 11sh, 12sh, 14sh, 15sh, 16, 17, 18, 22lo, 22sh, 25 and 26lo (sh/lo= short/long arm) from G. barbadense double haploid line 3-79 were obtained from Dr. Saha (USDA/ARS Genetics and Precision Agriculture Research Unit). Growing of the cotton plants, sample collections and DNA sample preparations were conducted while training college students in agricultural techniques. DNA from parental leaf samples were extracted using DNeasy Plant Mini Kit (Qiagen, Santa Clara, CA) and grinding matrix along with Bio Fast Prep System (Q. Biogene, Irvine, CA). Presences of DNAs were verified in a 1% agarose gel and DNA concentrations were quantified using a spectrophotometer (Eppendorf, Hamburg, Germany). Pollen grains from selected cotton lines were collected and stored in the -20oC freezer. Pollen germination media were tested on all these line, however, in vitro germination of cotton pollen grains required specific germination media [25%(w/v) sucrose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM MgSO4 and 1μM gibberellic acid, pH 7.6] to develop pollen tubes. The individual pollen grains from CS-B17 cotton lines were isolated (via micromanipulator) and then suspended in 5 µl germination media for at-least two hours to release their DNAs before storing sample in the -20oC freezer. The pollen DNA was amplified via MasterAmp™ Extra-Long PCR kit (EPICENTRE®, Madison, WI) with an array of continuous random 15-mer primer (Operon Technologies, Alameda, CA) using primer extension pre-amplification (PEP) protocol. The amplification of pollen DNA by this procedure using specific EPICENTRE®enhancers resulted in many genomic copies distributed in fragments of varied lengths. Pollen PEP-DNA products were visualized in 1% agarose gel after staining with ethidium-bromide. Genetic analyses on the parental CS-B17 lines as well as its pollen progeny and chromosomes17 specific recombinant inbred lines (RILs) were completed using molecular markers. After digestion of the genomic DNA of germinated pollen grains from CS-B17 cotton line with Mse-I and EcoR I enzymes generating cohesive ends on restriction fragments, T4 DNA Ligase was used to ligate these fragments with oligonucleotide adapters consisting of core and enzyme-specific sequences. The resulting DNA products were then subjected to pre-selective PCR amplification of amplified fragment length polymorphism (AFLP) reactions where primers were specific to adapters’ sequence while having a single selective nucleotide for plant genome. Later set of 35 pre-amplified pollen samples’ DNA along with that of parent were subjected to amplification of AFLP markers using 14 IRDye fluorescent-labeled (IRD-800 and IRD-700) primers. Thus AFLP analyses were concluded on the DNAs extracted from leaves of parent plants (TM-1, 3-79 and CS-B17) and 47 RILs as well that amplified from pollen grains of CS-B17 cotton line. For the comparative marker analyses of the parental and its pollen progeny as well as cotton RILs, the AFLP profiles were run through dual-dye (IRD-800 and IRD-700) automated analyses system (Li-Cor 4300).

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: A. Naseer Aziz. 2014. Isolation and DNA-Marker Based Genotyping of Individual Pollen Grains. Pure and Applied Biology. 3(3): 107-114. http://thepab.org/index.php/17-abstracts/72-isolation-and-dna-marker-based-genotyping-of-individual-pollen-grains

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Tennessee State Univerity faculty, students and studens's parents. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project provided pre-college, undergraduate and graduate students’ research trainings for Department of Agricultural & Environmental Sciences. Through AFLP and linkage mapping procedures described above, students’ trainings in molecular techniques as well as that of a USDA Borlaug fellow were provided. The trainings of undergraduate and graduate students as well as that of a USDA Borlaug fellow focused on research applications. Two pre-college students were exposed to aspects of cotton genetics at their academic levels. How have the results been disseminated to communities of interest? During this period presentations of the procedures developed and research results were made to stakeholder as described below; A. Naseer Aziz, J.N. Jenkins, Jack C. McCarty, D.M. Stelly and S. Saha. Pollen Genotyping in Cotton for Genetic-Linkage Analysis. Poster presentation at 35th Annual University-Wide Research Symposium of Tennessee State University (Nashville, TN), April 1-5, 2013. [Selected by Association of Pre-Professional Life Scientists for 2013 Poster Award on the Innovation and Feasibility Evaluations]. Jaron Hayes and Jasmine Smith. Cotton Genetics (profiling via AFLP, Infrared/ RAMAN Micro Spectroscopy). College of Agriculture, Human and Natural Sciences: 2013-Summer Apprenticeship Program Presentations. Tennessee State University; June 28, 2013. What do you plan to do during the next reporting period to accomplish the goals? Linkage distances derived from AFLP profiles of the parental and its pollen progeny as well as cotton RILs after comparative analyzing through dual-dye (IRD-800 and IRD-700) sequencing electrophoresis and JoinMap3.0 will be refined using previous data. The resulting genetic maps will then be disseminated through manuscript and presentation reporting channels. The bioinformatics tools used for the analytical procedures described above will also contribute in graduate research training at TSU Department of Agricultural & Environmental Sciences.

Impacts
What was accomplished under these goals? During this reporting period comparative analyses on Upland cotton substitution (CS) line that has most of the genome from G. hirsutum line TM-1 while substituting chromosomes number 17 (B17) from G. barbadense double haploid line 3-79 as well as its pollen progeny and chromosomes17 specific recombinant inbred lines (RILs) were completed using molecular markers. After digestion of the genomic DNA of germinated pollen grains from CS-B17 cotton line with Mse-I and EcoR I enzymes generating cohesive ends on restriction fragments, T4 DNA Ligase was used to ligate these fragments with oligonucleotide adapters consisting of core and enzyme-specific sequences. The resulting DNA products were then subjected to pre-selective PCR amplification of amplified fragment length polymorphism (AFLP) reactions where primers were specific to adapters’ sequence while having a single selective nucleotide for plant genome. Later set of 35 pre-amplified pollen samples’ DNA along with that of parent were subjected to amplification of AFLP markers using 14 IRDye fluorescent-labeled (IRD-800 and IRD-700) primers. Thus AFLP analyses were concluded on the DNAs extracted from leaves of parent plants (TM-1, 3-79 and CS-B17) and 47 RILs as well that amplified from pollen grains of CS-B17 cotton line. For the comparative analyses the parental and its pollen progeny as well as cotton RILs, the AFLP profiles were run through dual-dye (IRD-800 and IRD-700) automated analyses system (Li-Cor 4300). Later these AFLPs were then analyzed through JoinMap3.0 for linkage distance estimations.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: A. Naseer Aziz, J.N. Jenkins, Jack C. McCarty, D.M. Stelly and S. Saha. Pollen Genotyping in Cotton for Genetic-Linkage Analysis. Poster presentation at 35th Annual University-Wide Research Symposium of Tennessee State University (Nashville, TN), April 1-5, 2013. [Selected by Association of Pre-Professional Life Scientists for 2013 Poster Award on the Innovation and Feasibility Evaluations]
• Type: Other Status: Other Year Published: 2013 Citation: Jaron Hayes and Jasmine Smith. Cotton Genetics (profiling via AFLP, Infrared/ RAMAN Micro Spectroscopy). College of Agriculture, Human and Natural Sciences: 2013-Summer Apprenticeship Program Presentations. Tennessee State University; June 28, 2013.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: During this reporting period molecular-markers based analyses on individual cotton pollen DNA were conducted. Individually isolated pollen grains from Upland cotton (G. hirsutum line TM-1) substitution line with chromosomes number 17 replaced by that from G. barbadense (double haploid line 3-79) were germinated in five micro-liter germination media. The released pollen DNAs were then amplified via MasterAmp Extra-Long PCR kit (EPICENTRE, Madison, WI) with an array of continuous random 15-mer primer (Operon Technologies, Alameda, CA) using primer extension pre-amplification (PEP) protocol. Amplified fragment length polymorphism (AFLP) analyses were conducted on the genomic DNA of germinated pollen grains from CS-B17 cotton line. Since the pollen genomic DNA was amplified in fragments of varied lengths as confirmed on agarose gel, the subsequent AFLP analyses were challenging especially the restriction digestion step through Mse-I and EcoR I enzymes. Using T4 DNA Ligase these digested fragments were ligated with oligonucleotide adapters to undergo pre-selective PCR amplification with primers specific to adapters' sequence while having a single selective nucleotide. In this study we found that 25% of our samples showed success in the AFLP reactions indicating adequately sized genomic DNA fragments. Currently selective AFLP amplifications using fluorescent-labeled primers consisting of a core sequence, enzyme specific sequence and a selective extension are being conducted on the pollen samples. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Extraction of RNA from individual pollen grains was attempted to facilitate gene expression profiling. However extremely limited amount of nucleic material within isolated pollen grain rendered this endeavor unsuccessful.

Impacts
This project has strengthened interdisciplinary internal and external collaboration with USDA/ARS Genetics and Precision Agriculture Research Unit (Mississippi State) which has enhanced the TSU thrust in molecular genetics research on the most important textile crop. An interdisciplinary team composed of plant scientists, geneticists, poultry nutritionists and chemists has been formed to explore cotton plants that produce both high quality oils and suitable cottonseed meal for poultry feed. Geneticists and plant breeders value this research due to methods developed for genetic linkage and marker assisted breeding studies in cotton and other crops. One Department of Agriculture & Environ Sciences student had been involved during protocols development for this research project. Student was excited to work on cotton plants using micromanipulation and pollen germination procedures for this research project. Since individual gametes based linkage maps can be produced without requiring large farm operations and controlled pollinations, the procedures developed from this ongoing research would be of a great value to develop suitable varieties cost-effectively.

Publications

• Jackson, R., Aziz, A. N., Jenkins, J.N., McCarty, J.C., Stelly, D.M. and Saha, S. 2012. DNA Amplification of Individual Cotton Pollen Grains. 34th Annual University-Wide Research Symposium (Sustaining the Legacy of Excellence through Research) of TSU, Poster Presentation. Tennessee State University; March 26-30, 2012.
• Aziz, A. N., Jenkins, J.N., McCarty, J.C., Stelly, D.M. and Saha, S. 2013. Submitted: Pollen Genotyping in Cotton for Genetic-Linkage Analysis. For 35th Annual University-Wide Research Symposium of Tennessee State University (Nashville, TN) to be held on April 1-5, 2013.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The main focus during this reporting period was to isolate and germinate cotton pollen grains for subsequent genetic analyses. Pollen grains from Upland cotton substitution lines with most of the genome from G. hirsutum line TM-1 while substituting chromosomes numbers 01, 02, 04, 05sh, 06, 07, 11sh, 12sh, 14sh, 15sh, 16, 17, 18, 22lo, 22sh, 25 and 26lo (sh/lo= short/long arm) from G. barbadense double haploid line 3-79 (obtained from Dr. Saha, USDA/ARS Genetics and Precision Agriculture Research Unit) were collected and stored in the -20oC freezer. However, the pollen grains did not germinate in the pollen germination media [0.29 M sucrose, 1.62 mM H3BO3 and 1.27mM Ca(NO3)2, (pH 5.7)] tested. Therefore, fresh pollen were collected from CS-B17 cotton line with the micromanipulator and placed in a different media [25%(w/v) sucrose, 0.52 mM KNO3, 3.06 mM MnSO4, 1.66 mM H3BO3, 0.42 mM MgSO4 and 1μM gibberellic acid, (pH 7.6)] for in vitro germination of cotton pollen grains. The pollen grains were suspended in 5 microliter germination media for at-least two hours and stored in the -20oC freezer. The pollen DNA was amplified via MasterAmp Extra-Long PCR kit (EPICENTRE, Madison, WI) with an array of continuous random 15-mer primer (Operon Technologies, Alameda, CA) using primer extension pre-amplification (PEP) protocol. The amplification of pollen DNA by this procedure using specific EPICENTRE enhancers resulted in many genomic copies distributed in fragments of varied lengths. Pollen PEP-DNA products were visualized in 2% agarose gel after staining with ethidium-bromide. Currently released DNAs from CS-B17 individual pollen grains are being amplified for subsequent marker based genotyping of single gametes. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
While enhancing the TSU thrust in molecular genetics research on the most important textile crop, this project has facilitated interdisciplinary internal and external collaboration with USDA/ARS Genetics and Precision Agriculture Research Unit (Mississippi State), Mississippi State University to explore cotton plants that produce both high quality fibers and suitable cottonseed meal for poultry feed. Two pre-college summer interns and one Department of Agriculture & Environ Sciences student had been involved during protocols development for this research project. The summer interns presented their work to other interns, college faculty and their family members. One undergraduate student of Department of Agricultural & Environmental Sciences who is excited to work on cotton plants that can lead to combining adapatabilty and fiber quality, had been involved for micromanipulation and pollen germination procedures for this research project. Since individual gametes based linkage maps can be produced without requiring large farm operations and controlled pollinations, the procedures developed from this ongoing research would be of a great value to develop suitable varieties cost-effectively.

Publications

• Sahithi. Kommireddy, V. L. 2011. Comparative Molecular Markers Based Analyses of Cotton Lines. Submitted to Graduate School of Tennessee State University in Partial Fulfillment of the Requirements for the Degree of Master of Science. (M.S. Thesis)
• Aziz, A.N., Sahithi Kommireddy, V.L., Jenkins, J.N., McCarty, J.C., Stelly, D.M. and Saha, S. 2011. Enrichment of Chromosome 17 Specific Molecular Markers of Pima Cotton Substituted in Upland Cotton Lines. Program and Abstracts: Association of Research Directors 16th Biennial Symposium, p 124. (Abstract)

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The goal of this project is to genetically characterized breeding lines of Upland cotton (Gossypium hirsutum) that have been created by deleting some Upland genetic material and replacing it from Pima cotton (G. barbadense). Upland cotton is cultivated widely for textile fibers, food, as well as ruminant feed and prized for high yields while Pima cotton is very important to American textile industry due to the exceptional fiber length, strength, fineness, and thus provides a significant price to farmers. Thus these chromosomal substitutions lines are created combining superior fiber quality and diverse adaptability of these two kinds of cotton plants. Upland cotton substitution lines with most of the genome from G. hirsutum line TM-1 while substituting chromosomes numbers 01, 02, 04, 05sh, 06, 07, 11sh, 12sh, 14sh, 15sh, 16, 17, 18, 22lo, 22sh, 25 and 26lo (sh/lo= short/long arm) from G. barbadense double haploid line 3-79 were obtained from Dr. Saha (USDA/ARS Genetics and Precision Agriculture Research Unit). Several representatives young leaf samples from each the cotton line grown from seeds in the greenhouse are collected. The necessary supplies needed for the molecular markers based analyses of these plants are also being acquired. DNA from leaf samples were extracted using DNeasy Plant Mini Kit (Qiagen, Santa Clara, CA) and grinding matrix along with Bio Fast Prep System (Q. Biogene, Irvine, CA). Presences of DNAs were verified in a 1% agarose gel and DNA concentrations were quantified using a spectrophotometer (Eppendorf, Hamburg, Germany). Growing of the cotton plants, sample collections and DNA sample preparations were conducted while training college students in agricultural techniques. From these and other appropriate cotton lines individual pollen grains will be collected from dry pollen spread and isolated individually in PCR tubes using a micromanipulator for molecular markers' based analyses. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Cotton is the most important fiber crop and is vital for U.S. textile industry. Tennessee State University (TSU) did not have any cotton research program and this project has thus initiated this important program at TSU's School of Agriculture and Consumer Science (SACS). New collaboration with cotton geneticist at USDA/ARS Genetics and Precision Agriculture Research Unit, Mississippi State University is also established. Since in this project molecular markers are being used to understand the genetics of desirable characters of cotton, this would strengthen SACS's mission of spearheading biotechnology and bio-security research for general service to stakeholders. This research is genetically characterizing the chromosomal substitutions lines that are created to combining superior fiber quality from Pima cotton with diverse adaptability of Upland cotton plants. American cotton and textile industries will benefit from this research. This project has standardized the molecular procedures for Upland cotton chromosomal substitutions lines. The project undertaken has also trained three of students including two pre-college summer interns. The students learned to grown plants in greenhouse, prepare leaf samples in requisite sizes for cryogenic storage, as well as prepare DNA samples and quantify them. Students were trained in the use of DNeasy Plant Mini Kit (Qiagen, Santa Clara, CA), Bio Fast Prep System (Q. Biogene, Irvine, CA), agarose gel analyses and Eppendorf spectrophotometer (Hamburg, Germany).

Publications

• No publications reported this period