Progress 10/01/11 to 09/30/15
Outputs
Target Audience: The target audience include research scientists mainly plant breeders, nematologists and agronomists. Ultimate beneficiaries are farmers in the cotton belt. Changes/Problems: The tragic and untimely loss of our original PI Dr. Ramesh Kantety on August 20, 2012 after an extended illness. The co-PI Dr. Govind C. Sharma is currently serving as a PI. What opportunities for training and professional development has the project provided? The project has graduated one PhD student and it is currently training one additional PhD student, one MS students and 2 undergraduate and 1 high school students. A post-doctoral associate is also under training for the part of the year. The facilities and the resources of the lab are utilized by several part time undergraduate students who wish to learn more about biotechnology. How have the results been disseminated to communities of interest? The results are regularly shared with the user community through oral and poster presentations in fora within the state and in cotton growing belt, The results and the findings are also shared via International Cotton Genome initiative (ICGI) annual meeting at Plant and Genome Conference, as well as ICGI biennial meetings that rotate between global cotton producing areas. Publications in the journals and reposition of genomic data via NCBI are additiona and important method of sharing data and findings.In addition to the publications listed above several oral and poser presentations are also being utilized. A select list of such presentations is included below: Sripathi, V.R., Nyaku, S.T., Sripathi, P. Genome-wide computational identification of microRNAs and their targets in the Rotylenchulus reniformis. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville Botanical Garden, February 24th-25th, 2012. (Poster presentation). Nyaku, S.T., Sripathi, V.R. Next Generation Sequencing and Analysis of the Reniform Nematode Transcriptome. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville Botanical Garden, February 24th-25th, 2012. (Oral presentation). Sripathi, V.R., Nyaku, S.T. Genome-wide computational identification of microRNAs and their targets in the Rotylenchulus reniformis. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville Botanical Garden, February 24th-25th, 2012. (Poster presentation). Williams, A., V R. Sripathi, and Ramesh Kantety. Identification of Tissue Specific Small RNAs in Upland Cotton, Gossypium hirsutum. In: 5th Annual STEM Day. Conquering 21st century challenges through science and technology, 20 April 2011, Alabama A&M University, Normal, USA. Sripathi, V R ,and R.Buyyarapu. Isolating Cotton Chromosomes Using Laser Capture Microdissection. In: 5th Annual STEM Day, 20 April 2011, Alabama A&M University, Normal, USA. What do you plan to do during the next reporting period to accomplish the goals? Our focus for the coming year will be on finalizing genomic shotgun sequence data for reniform genome and publishing it to be available to the cotton research community.
Impacts
What was accomplished under these goals?
1. The reniform nematode, Rotylenchulus reniformis, infests over 300 plant species worldwide and over the last two decades it has emerged as a major cotton pest in the southeastern United States. Nine locations in Alabama and one location in Mississippi were selected for study of R. reniformis. Thirteen morphometric measurements were made on 20 male and 20 female nematodes from each population. The sex and location interaction was significant (P < 0.005) for all traits except total body length (P = 0.29) and the derived ratio trait a (body length / maximum body width, P = 0.06). Canonical discriminant analysis effectively separated the 10 sampling locations into three distinct groups; among them, Group 1 and 3 were distinct with an intermediate group (Group 2) differentiating in the middle. Furthermore, both female and male R. reniformis based on the morphometrics measured here adhered to this metrics-based grouping. Belle Mina (Limestone County, AL), Huxford (Escambia County, AL), and Mississippi State University, MSU (Oktibbeha County, MS) locations were separated from the remaining seven locations based on the large positive CAN1 centroid means. Eight out of 13 traits had high phenotypic correlations (r > 0.80) with CAN 1 for both sexes. Anal width and length of the hyaline portion of the tail measurement accounted for a significant amount (r > 80%) of the variation in total and sex-based canonical structure. Occurrence of the three non-overlapping morphometric groups in cotton-growing fields in close proximity (250 mile radius) suggests a greater biological variation in this species than expected. Cotton cultivars with differential resistance and soil types are among the major factors to be tested for further delineating the causes of morphometric variation in R. reniformis. 2. Upland cotton, Gossypium hirsutum L., is highly susceptible to infection by reniform nematode (Rotylenchulus reniformis), which can cause over 10% reduction of cotton yields in Alabama. Detection of reniform nematode (RN) and analysis for molecular variation within its population is important for understanding its interactions with cotton and other host plant species. Restriction analysis of PCR products of ITS1 regions was achieved using four restriction enzymes, HaeIII, HhaI, MspI, and RsaI. These showed similar banding patterns for both male and female populations. However, MspI digestion of ITS1 amplification products showed variants within the combined sex and location effects primarily attributed to a 500 bp fragment that was absent in other restriction digestions. Intra-nematodal variations in 18S and ITS1 rDNA were studied in detail by sequencing a minimum of ten clones in each individual male and female RN isolates in both directions. Multiple sequence alignment of the 18S rDNA sequences showed two major types of sequences within this gene for both male and female RN clones, which could be distinguished at 27 specific sites. Two distinct ITS1 fragments of lengths (550 bp and 720 bp) were observed; referred to as ITS1S and ITS1L respectively. Neighbor-joining analysis was used in revealing the relationships and grouping characteristics between male and female RN clones,with clones grouping together irrespective of sex and isolate. Sequencing of one-third of the 18S and ITS1 rDNA regions provided clear evidence of intra-and inter-nematode variability, in addition to gene conversion events in the 18S rDNA of individual male and female RN clones. 3. The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1(RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene. 4. The reniform nematode (RN), Rotylenchulus reniformis, is an agriculturally important pest with a broad host range that results in a large economic impact in tropical, subtropical and in warm temperate zones. In an initial effort to understand the transcriptome and gene expression in RN, we present EST results that reveal numerous putative parasitism-related genes some of which play roles in plant cell wall modification. The characterized contigs included 8,362 (40.6%) matches to unique proteins. Coding contigs predicted were 10,656 (51.7%) or 3079 (14.9%), that was similar to those identified in Brugia malayi and Caenorhabditis elegans as reference organisms respectively. Specific transcripts studied in more detail include putative plant parasitism genes, prominent among them were several plant cell wall modification genes. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes included expansins, hexosa-minidase, glycosyl hydrolases family, 14-3-3 protein, xylanases, glutathione peroxidase, pectate lyase, β-1,4-endoglu-canase, major sperm protein, aminopeptidase, c-type lectin, chitin synthase, FMR famide-like peptide, and calreticulin. These genes function in suppression of host defenses and development of feeding sites.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2011
Citation:
Ramesh Buyyarapu, Venkateswara Rao Sripathi, Sarah Beth Cseke, Ramesh V. Kantety (2011). Leland Cseke, Peter Kaufman, Gopi K. Podila, and Chung-Jui Tsai. Laser Capture Microdissection and Whole Genome Amplification. Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edi. CRC Press. Boca Raton, FL. 695.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nyaku, S. T, R. V. Kantety, K. S. Lawrence, E. van Santen and G. C. Sharma. 2013. Canonical discriminant analysis of
Rotylenchulus reniformis in Alabama. Nematropica 43:171-181.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
McEwan,Robert E. Ramesh Kantety, Seloame T. Nyaku, Kathy Lawrence, Edzard van Santen and Govind C. Sharma 2014. Relative Response of Four Tomato Species to Rotylenchulus reniformis Infestation. American Journal of Plant Sciences 5(1):55-62.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nyaku, Seloame T., Ramesh V. Kantety, Yonathan Tilahun, Kathy S. Lawrence, Khairy M. Soliman, Ernst Cebert, and Govind C. Sharma. 2013. 18S and ITS1 Genomic Sequence Variations in Rotylenchulus reniformis
Isolates from Alabama. The Journal of Cotton Science 17:184194.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nyaku ST, Sripathi VR, Kantety RV, Gu YQ, Lawrence K, and Govind C. Sharma. 2013. Characterization of the Two Intra- Individual Sequence Variants in the 18S rRNA Gene in the Plant Parasitic Nematode, Rotylenchulus reniformis. PLoS ONE 8(4): e60891. doi:10.1371/journal.pone.0060891.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nyaku, Seloame T, Venkateswara R. Sripathi, Graham Wiley, Fares Z. Najar, Leland J. Cseke, Govind C. Sharma, Bruce A. Roe, Sarah Beth Cseke, Elica Moss, Ramesh V. Kantety.2013. The expressed parasitism genes in the reniform nematode (Rotylenchulus reniformis) American Journal of Plant Sciences 4:780-791.
|
Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The audience of this work include plant biologists and plant nematologists. Producers of cotton and Students interested in training to become agricultural professionals. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project is currently training one PhD student, one MS student and one undergraduate student. A postdoctoral associate is also under training for the part of the year. The facilities and the resources of the lab are utilized by several part time undergraduate students who wish to learn more about biotechnology. Periodic summer workshop has been held for science teachers. How have the results been disseminated to communities of interest? The results are regularly shared with the user community through oral and poster presentations in fora within the state and in cotton growing belt, The results and the findings are also shared via International Cotton Genome initiative (ICGI) annual meeting at Plant and Genome Conference, as well as ICGI biennial meetings that rotate between global cotton producing areas. Publications in the journals and reposition of genomic data via NCBI are additiona and important method of sharing data and findings. What do you plan to do during the next reporting period to accomplish the goals? Our focus for the coming year will be on finalizing genomic shotgun sequence data for reniform genome and publishing it to be available to the cotton research community.
Impacts
What was accomplished under these goals?
1. The reniform nematode, Rotylenchulus reniformis, infests over 300 plant species worldwide and over the last two decades it has emerged as a major cotton pest in the southeastern United States. Nine locations in Alabama and one location in Mississippi were selected for study of R. reniformis. Thirteen morphometric measurements were made on 20 male and 20 female nematodes from each population. The sex and location interaction was significant (P < 0.005) for all traits except total body length (P = 0.29) and the derived ratio trait a (body length / maximum body width, P = 0.06). Canonical discriminant analysis effectively separated the 10 sampling locations into three distinct groups; among them, Group 1 and 3 were distinct with an intermediate group (Group 2) differentiating in the middle. Furthermore, both female and male R. reniformis based on the morphometrics measured here adhered to this metrics-based grouping. Belle Mina (Limestone County, AL), Huxford (Escambia County, AL), and Mississippi State University, MSU (Oktibbeha County, MS) locations were separated from the remaining seven locations based on the large positive CAN1 centroid means. Eight out of 13 traits had high phenotypic correlations (r > 0.80) with CAN 1 for both sexes. Anal width and length of the hyaline portion of the tail measurement accounted for a significant amount (r > 80%) of the variation in total and sex-based canonical structure. Occurrence of the three non-overlapping morphometric groups in cotton-growing fields in close proximity (250 mile radius) suggests a greater biological variation in this species than expected. Cotton cultivars with differential resistance and soil types are among the major factors to be tested for further delineating the causes of morphometric variation in R. reniformis. 2. Upland cotton, Gossypium hirsutum L., is highly susceptible to infection by reniform nematode (Rotylenchulus reniformis), which can cause over 10% reduction of cotton yields in Alabama. Detection of reniform nematode (RN) and analysis for molecular variation within its population is important for understanding its interactions with cotton and other host plant species. Restriction analysis of PCR products of ITS1 regions was achieved using four restriction enzymes, HaeIII, HhaI, MspI, and RsaI. These showed similar banding patterns for both male and female populations. However, MspI digestion of ITS1 amplification products showed variants within the combined sex and location effects primarily attributed to a 500 bp fragment that was absent in other restriction digestions. Intra-nematodal variations in 18S and ITS1 rDNA were studied in detail by sequencing a minimum of ten clones in each individual male and female RN isolates in both directions. Multiple sequence alignment of the 18S rDNA sequences showed two major types of sequences within this gene for both male and female RN clones, which could be distinguished at 27 specific sites. Two distinct ITS1 fragments of lengths (550 bp and 720 bp) were observed; referred to as ITS1S and ITS1L respectively. Neighbor-joining analysis was used in revealing the relationships and grouping characteristics between male and female RN clones,with clones grouping together irrespective of sex and isolate. Sequencing of one-third of the 18S and ITS1 rDNA regions provided clear evidence of intra-and inter-nematode variability, in addition to gene conversion events in the 18S rDNA of individual male and female RN clones. 3. The 18S rRNA gene is fundamental to cellular and organismal protein synthesis and because of its stable persistence through generations it is also used in phylogenetic analysis among taxa. Sequence variation in this gene within a single species is rare, but it has been observed in few metazoan organisms. More frequently it has mostly been reported in the non-transcribed spacer region. Here, we have identified two sequence variants within the near full coding region of 18S rRNA gene from a single reniform nematode (RN) Rotylenchulus reniformis labeled as reniform nematode variant 1(RN_VAR1) and variant 2 (RN_VAR2). All sequences from three of the four isolates had both RN variants in their sequences; however, isolate 13B had only RN variant 2 sequence. Specific variable base sites (96 or 5.5%) were found within the 18S rRNA gene that can clearly distinguish the two 18S rDNA variants of RN, in 11 (25.0%) and 33 (75.0%) of the 44 RN clones, for RN_VAR1 and RN_VAR2, respectively. Neighbor-joining trees show that the RN_VAR1 is very similar to the previously existing R. reniformis sequence in GenBank, while the RN_VAR2 sequence is more divergent. This is the first report of the identification of two major variants of the 18S rRNA gene in the same single RN, and documents the specific base variation between the two variants, and hypothesizes on simultaneous co-existence of these two variants for this gene. 4. The reniform nematode (RN), Rotylenchulus reniformis, is an agriculturally important pest with a broad host range that results in a large economic impact in tropical, subtropical and in warm temperate zones. In an initial effort to understand the transcriptome and gene expression in RN, we present EST results that reveal numerous putative parasitism-related genes some of which play roles in plant cell wall modification. The characterized contigs included 8,362 (40.6%) matches to unique proteins. Coding contigs predicted were 10,656 (51.7%) or 3079 (14.9%), that was similar to those identified in Brugia malayi and Caenorhabditis elegans as reference organisms respectively. Specific transcripts studied in more detail include putative plant parasitism genes, prominent among them were several plant cell wall modification genes. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes included expansins, hexosa-minidase, glycosyl hydrolases family, 14-3-3 protein, xylanases, glutathione peroxidase, pectate lyase, β-1,4-endoglu-canase, major sperm protein, aminopeptidase, c-type lectin, chitin synthase, FMR famide-like peptide, and calreticulin. These genes function in suppression of host defenses and development of feeding sites.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Buyyarapu, R., R. V. Kantety J. Z. Yu, S. Saha and G. C. Sharma. 2011. Development of new candidate gene and ESTbased
molecular markers for Gossypium species. Int. J. of Plant Genomics 2011:1-9 (#894598).
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nyaku, Seloame T, Venkateswara R. Sripathi, Graham Wiley, Fares Z. Najar, Leland J. Cseke, Govind C. Sharma, Bruce A. Roe, Sarah Beth Cseke, Elica Moss, Ramesh V. Kantety.2013. The expressed parasitism genes in the reniform nematode (Rotylenchulus reniformis) American Journal of Plant Sciences 4:780-791.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Nyaku, S. T, R. V. Kantety, K. S. Lawrence, E. van Santen and G. C. Sharma. 2013. Canonical discriminant analysis of
Rotylenchulus reniformis in Alabama. Nematropica 43:171-181.
|
Progress 10/01/12 to 09/30/13
Outputs
Target Audience: The target audiences are research scientists working on reniform resistance and its genomic and transcriptomic characterization and also nematologists. The additional users of the information include.include the farmers in the Cotton Belt in US but Alabama and the southeast in particular. The work done here also serves as a hands-on training platform for undergraduate, graduate and post-doctoral students. Changes/Problems: The tragic and untimely loss of our original PI Dr. Ramesh Kantety on August 20, 2012 after an extended illness. The co-PI Dr. Govind C. Sharma is currently serving as a PI. What opportunities for training and professional development has the project provided? The project is currently training one PhD student, one MS students and 4 undergraduate and 1 high school student. A post-doctoral associate is also under training for the part of the year. The facilities and the resources of the lab are also utilized by several part time undergraduate students who wish to learn more about biotechnology and genomics. How have the results been disseminated to communities of interest? This dissemination is occurring by presentation of papers in professional society meetings and by publication of full length papers in scientific journals. What do you plan to do during the next reporting period to accomplish the goals? 1. We will be studying the expressed Parasitism Genes of the Reniform Nematode. 2. We will also pursue18S and ITS1 genomic sequence variations in Rotylenchulus reniformis. 3. Work on small RNA will be initiated.
Impacts
What was accomplished under these goals?
1. Reniform nematode populations in Alabama and Mississippi are being studied for presence of biotypes or other differences in 18S and ITS1 region of the genes. 2. RN transcriptomeis being utilized to identifycandidate parasitism genes and their functions and generate expressed sequence tags (ESTs) through high-throughput sequencing of cDNA libraries prepared from mixed RN stages (eggs to L4). 3. sRNA analyses are being initiated to elucidate post transcriptional gene alterations.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2012
Citation:
Williams, A., Nyaku, S.T. Development of a formula to compare qPCR Concentration to Actual Rotylenchulus reniformis Counts. Sixth annual Science, Technology, Engineering, and Mathematics (STEM) day held at Alabama Agricultural and Mechanical University (AAMU) on April 12th, 2012. (Poster presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2012
Citation:
Nyaku, S.T., Sripathi, V.R. Next Generation Sequencing and Analysis of the Reniform Nematode Transcriptome. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville botanical gardens, February 24th-25th, 2012. (Oral presentation)
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2012
Citation:
Nyaku, S.T., Sripathi, V., Sripathi, P. Reniform Nematode Genome Sequencing, Characterization, and Analysis. Proceedings from Plant and Animal Genome XX Conference, San Diego, California. January 14-18, 2012. (Poster presentation)
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Buyyarapu, R., R. V. Kantety J. Z. Yu, S. Saha and G. C. Sharma. 2011. Development of new candidate gene and EST-based molecular markers for Gossypium species. Int. J. of Plant Genomics 2011:1-9 (#894598).
- Type:
Book Chapters
Status:
Published
Year Published:
2011
Citation:
Ramesh Buyyarapu, Venkateswara Rao Sripathi, Sarah Beth Cseke, Ramesh V. Kantety 2011. Laser Capture Microdissection and Whole Genome Amplification. In Leland Cseke, Peter Kaufman, Gopi K. Podila, and Chung-Jui Tsai, editors, Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edi. CRC Press. Boca Raton, FL. 695.
|
Progress 10/01/11 to 09/30/12
Outputs
Target Audience: The primary target audience was scientists and students interested in plant molecular biology and those studying plant: nematode interaction. The project also assisted undergraduate and high school students to learn more about hands-on work in molecular biology.The secondary target audience includes the farmers in the Cotton Belt but in Alabama and the southeast in particular. Changes/Problems: The tragic and untimely loss of our original PI Dr. Ramesh Kantety on August 20, 2012 after an extended illness. What opportunities for training and professional development has the project provided? The project is currently training two PhD students, one MS students and 4 undergraduate and 1 high school students. A post-doctoral associate is also under training for the part of the year. How have the results been disseminated to communities of interest? Sripathi, V.R., Nyaku, S.T., Sripathi, P. Genome-wide computational identification of microRNAs and their targets in the Rotylenchulus reniformis. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville Botanical Garden, February 24th-25th, 2012. (Poster presentation). Nyaku, S.T., Sripathi, V.R. Next Generation Sequencing and Analysis of the Reniform Nematode Transcriptome. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville Botanical Garden, February 24th-25th, 2012. (Oral presentation). Sripathi, V.R., Nyaku, S.T. Genome-wide computational identification of microRNAs and their targets in the Rotylenchulus reniformis. Partnership for Biotechnology Research 13th Annual Bio Retreat, Huntsville Botanical Garden, February 24th-25th, 2012. (Poster presentation). Williams, A., V R. Sripathi, and Ramesh Kantety. Identification of Tissue Specific Small RNAs in Upland Cotton, Gossypium hirsutum. In: 5th Annual STEM Day. Conquering 21st century challenges through science and technology, 20 April 2011, Alabama A&M University, Normal, USA. 7) Sripathi, V R ,and R.Buyyarapu. Isolating Cotton Chromosomes Using Laser Capture Microdissection. In: 5th Annual STEM Day, 20 April 2011, Alabama A&M University, Normal, USA. What do you plan to do during the next reporting period to accomplish the goals? 1. We will be studying the expressed Parasitism Genes of the Reniform Nematode. 2. We will also pursue18S and ITS1 genomic sequence variations in Rotylenchulus reniformis.
Impacts
What was accomplished under these goals?
1. Reniform nematode populations in Alabama and Mississippi are being studied for presence of biotypes or other differences in 18S and ITS1 region of the genes. 2. RN transcriptomeis being utilized to identifycandidate parasitism genes and their functions and generate expressed sequence tags (ESTs) through high-throughput sequencing of cDNA libraries prepared from mixed RN stages (eggs to L4). 3. sRNA analysis are being initiated to elucidate post transcriptional alterations.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Ramesh Buyyarapu, Ramesh V. Kantety, John Z. Yu, Sukumar Saha, and Govind C. Sharma (2011). Development of New Candidate Gene and EST-BasedMolecular Markers for Gossypium Species. International Journal of Plant Genomics, 2011 Article ID 894598, 9 pages, doi:10.1155/2011/894598.
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