Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: A bitter melon (Botanical name: Momordica charantia, Family: Cucurbitaceae) medicinal variety, CBM12, with high content of one anticancer phytomedicine, Cucurbitacin-B, and two antidiabetic phytomedicines, Charantin and Plant Insulin, was developed previously by the team from a wild plant of the allied botanical variety, Momordica charantia var. muricata, of the popularly cultivated botanical variety, Momordica charantia var. charantia. Utility application (USSN: 13/179,952) for this variety has been filed on July 11, 2011. Recently we have developed two other medicinal varieties, CBM3 and CBM6, from wild plants of Momordica charantia var. muricata. These two varieties bear larger fruits as compared to CBM12 in addition to high content of the phytomedicines. We observed previously that the charantia varieties produce fruits with superior yield and quality but low content of phytomedicines, while the muricata varieties bear fruits with inferior yield and quality but higher phytomedicine contents. Hence, we developed two dual-purpose hybrids (10C2S2 and 12C1S1) that produce fruits with both desirable fruit traits and phytomedicine contents. Previously, we constructed the first genetic linkage map of bitter melon using amplified fragment length polymorphism (AFLP) markers and positioned genes and quantitative trait loci (QTL) controlling fruit yield and quality traits. However, this map contains long marker intervals in some genomic regions. Therefore, we completed a preliminary sequencing of the two parents, CBM12 and Taiwan White, following a genomic reduction strategy, to verify that the methodology will produce sufficient number of single nucleotide polymorphism (SNP) targets to build a saturated molecular genetic map. This has generated a total 1,285,379 reads (347 Mbp) of sequences, with 439,086 reads derived from CBM12 and 766,235 derived from Taiwan white. Assembly of the sequences with (GSAssembler) Newbler produced 26,614 consensus contigs with an average length of 445 bp and an average read depth of 34. Preliminary SNP discovery identified 3,357 SNPs that fit the minimum parameters for SNP discovery (10 coverage and 95% identity within accession). As expected, transition mutations (A/G or C/T) are the most numerous, outnumbering transversion (A/T, C/A, G/C, G/T) by 2.1-times margin. Extraction from fruits of the F2 mapping population has been done for quantification of the three phytomedicines with a view to positioning the genes governing them in the saturated genetic map. Some of these results were discussed in a keynote presentation by the PD during the second annual conference of the American Council for Medicinally Active Plants held at Huntsville, Alabama during 17-20 July 2011. We demonstrated some of our results to over 20 farmers who visited our laboratories and greenhouses during several phases in 2011. We trained one undergraduate student of Clemson University and two senior scientists sponsored by the Indian Council of Agricultural Research. PARTICIPANTS: Three colleagues from the Department of Genetics and Biochemistry, Clemson University including Ms. Phullara Kole, Dr. Bode A. Olukolu and Prof. Albert G. Abbott; one colleague, Prof. Kenneth R. Marcus, from the Department of Chemistry, Clemson University; and one collaborator, Prof. Jeff Maughan, from Brigham Young University, Provo, UT assisted me in this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The medicinal variety, CBM12, has already been used in the development of a dual-purpose hybrid, 12C1S1; construction of the first genetic linkage map; and discovery of SNP markers. The recently developed two medicinal varieties, CBM3 and CBM6, can be utilized for similar purposes. The dual-purpose hybrid, 10C2S2, along with 12C1S1 can be utilized for breeding of elite varieties with much higher phytomedicine contents through marker-assisted backcross breeding. Our initial SNP discovery effort using CBM12 and Taiwan White, following genome reduction, suggests that SNP development and validation for the proposed mapping population should be highly successful. This strategy samples the genome with a fairly even distribution. Saturating intergenic regions will be important since regulatory regions controlling the expression of genes responsible for the important fruit phenotypes and phytomedicine contents are often located in the noncoding DNA. Development of a saturated genetic linkage map will facilitate identification of molecular markers tightly linked to the genes and QTLs controlling fruit traits and phytomedicine contents. These linked markers can be used in the future for precise molecular breeding for developing superior functional food varieties with superior fruit yield and quality coupled with higher phytomedicine contents. These linked molecular markers can also be useful in positional cloning of QTLs controlling production of the phytomedicines that can be used for genetic transformation in bitter melon and other vegetable and fruit crops. Sequencing of transcriptomes of the two parent varieties is expected to be performed in the near future. This will provide the ability to map most of the genes that harbor polymorphism between the two parents. SNP markers developed from these transcript sequences will facilitate cataloging genes with QTLs, while the down- and up-regulated genes from the transcriptome profile will implicate plausible candidate genes. Candidate genes can also be further validated with reverse transcription (RT)-PCR to confirm their differential expression levels. Ultimately, SNP markers developed from genome reduction and transcriptome sequencing will ensure a more comprehensive saturation of the genome without gaps. The incidence of cancer and diabetes is alarmingly high in the United States as evidenced from the recent estimates of prevalence of cancer (11.7 million) and diabetes (23.6 million). Development of functional food crop varieties bearing fruits with high content of phytomedicines will be useful in combating these two deadly diseases. Potential of employing the concepts and strategies of our project in mapping of the genes controlling the contents of phytomedicines and nutraceuticals in other medicinal plants and nutraceutical crops for improvement of their phytomedicine content through molecular breeding has been comprehensively discussed in the International Workshop on Phytomedimics and Nutriomics and ICPN organized by the PD at San Diego, CA on 17 January, 2011 (www.intlpag.org). Financial support for the investigations was obtained from USDA-AFRI through a NRI-CGP grant.
Publications
- Kole C, Olukolu BA, Kole P, Abbott AG (2010) Association mapping of fruit traits and phytomedicine contents in a structured population of bitter melon (Momordica charantia L.). In: Thies JA, Kousik S, Levi A (eds) Cucurbitaceae 2010 Proceedings. American Society of Horticultural Science, Alexandria, VA, pp 42-45 (reprint provided in 2011)
- Kole C (2011) Molecular breeding in medicinally active plants: bitter melon as a model. Key Note presentation. Second Annual Conference of the American Council for Medicinally Active Plants. Huntsville, AL, 17-20 January, pp 35-36
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: A bitter melon (Momordica charantia) F2 population consisting of 146 plants was evaluated with regard to segregation at molecular level and fruit yield and quality characters. This population was derived earlier from hybridization of a horticultural variety, Taiwan White, possessing superior fruit yield and quality traits but containing low concentrations of three phytomedicines cucurbitacin-B, charantin and polypeptide-p also known as plant insulin with a medicinal variety, CBM12 developed by us (US Provisional Patent Application Serial No. 61/364,938), having high content of cucurbitacin-B, charantin and plant insulin but exhibiting poor fruit yield and quality traits. The population exhibited Mendelian segregation for color, luster, surface structure and shape of fruits and color of seed and stigma. Six characters including average length, average maximum diameter, average weight, number and yield of fruits and seed number exhibited quantitative variation. One hundred thirty-one (131) amplified fragment length polymorphism markers were generated by using DNA samples extracted from leaf tissues of the F2 plants. These markers and genotypes of the five qualitative traits were used for linkage analysis leading to the construction of a genetic linkage map that included 108 amplified fragment length polymorphism markers and five qualitative trait loci. This genetic map included 11 linkage groups equaling to the haploid chromosome number of bitter melon and spanned 3060.7 cM with mean marker interval of 27.09 cM. The genes controlling color, luster and surface structure of fruits, stigma color and seed color mapped to three linkage groups. Fifteen quantitative trait loci controlling average length (2), average maximum diameter (1), average weight (1), number (4) and yield (4) of fruits and seed number (3) were detected on five linkage groups. Extraction of cucurbitacin-B, charantin and plant insulin from the fruits of the F2 plants and their quantification are in progress. Some of these results were presented in the XVIII International Conference on the Status of Plant and Animal Genome Research held at San Diego, CA, January 9-13, 2010 and in the XI Biennial International Cucurbit Conference at Charleston, SC, November 14-18, 2010. We demonstrated our results to over 70 organic farmers who visited our laboratories and greenhouses during several phases in 2010. We trained two students through this project. Four senior scientists from India sponsored by the Indian Council of Agricultural Research through its National Agricultural Innovation Project were trained on marker-assisted breeding through this project. PARTICIPANTS: Three colleagues from the Department of Genetic and Biochemistry, Clemson University including Ms. Phullara Kole, Dr. Bode A. Olukolu and Prof. Albert G. Abbott; and one colleague, Prof. Kenneth R. Marcus, from the Department of Chemistry, Clemson University assisted me in this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The long-term goal of this project is to develop vegetable and fruit varieties for consumption as functional foods and/or dietary supplements to combat several chronic diseases, particularly cancer and diabetes. The varieties with superior fruit yield and quality traits coupled with higher content of phytomedicines should be developed for popularization of their cultivation. The first genetic linkage map of a cucurbit vegetable bitter melon we developed based on amplified fragment length polymorphism markers has already facilitated mapping of the quantitative trait loci controlling six quantitative traits underlying yield and its component characters and genes governing five qualitative traits including three fruit quality characteristics, viz. color, luster and surface structure. This genetic map will be used in the near future in mapping of the quantitative trait loci controlling the contents of one anticancer bioactive, cucurbitacin-B, and two antidiabetic phytomedicines, charantin and polypeptide-p also known as plant insulin. Identification of amplification fragment length polymorphism markers linked to the genes and quantitative trait loci will be helpful in using them in molecular breeding for developing varieties with superior fruit yield and quality traits coupled with high content of anticancer and antidiabetic phytomedicines. In our recent investigations we found the two parental varieties of the mapping population, Taiwan White and CBM12 to have contrasting contents of other anticancer phytomedicines including momordin, lycopene and beta-carotene. The molecular map we have developed can be used in the future to position the genes controlling the contents of these three phytomedicines and their future introgression into popularly cultivated bitter melon varieties. The genes controlling the phytomedicine contents can be cloned in the future and used in intransgenic breeding of other vegetable crops including popular cucurbits. The incidence of cancer and diabetes is increasing at an alarming rate in the United States. Recent estimates of prevalence of cancer and diabetes are 11.7 and 23.6 million, respectively, and annual deaths due to cancer and diabetes are 0.57 and 0.234 million, respectively. Development of vegetable and fruit varieties bearing fruits with high content of phytomedicines will be useful in combating these two deadly diseases. Importance of using phytomedicines and nutraceuticals to combat fatal diseases particularly cancer and diabetes has recently been discussed in the International Workshop on Phytomedimics and Nutriomics organized by the PD (http://www.intl-pag.org/19/19-pag.html). The concepts and strategies of this project can be employed in other medicinal plants and nutraceutical crops for mapping of the genes controlling the contents of phytomedicines and nutraceuticals for further improvement of their contents through molecular breeding. Financial support for the investigations was obtained from the Clemson University, South Carolina Department of Agriculture, and USDA-AFRI through a NRI-CGP grant.
Publications
- Kole C, Olukolu B, Kole P, Abbott AG (2010a) Towards phytomedomics with bitter melon (Momordica charantia L.) as a model. In: International Conference on the Status of Plant & Animal Genome Research, January 9-13, 2010, San Diego, CA, USA, P164
- Kole C, Olukolu B, Kole P, Abbott AG (2010b) Association mapping of fruit traits and phytomedicine contents in a structured population of bitter melon (Momordica charantia L.). In: Thies JA, Kousik S, Levi A (eds) Cucurbitaceae 2010 Proceedings. American Society of Horticultural Science, Alexandria, VA, pp 42-45
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: An F2 population of bitter melon (Momordica charantia; Family Cucurbitaceae) was developed by controlled selfing of a single F1 hybrid plant that had been developed previously from crossing of a horticultural variety, Taiwan White, which belongs to the botanical variety Momordica charantia var. charantia by a medicinal genotype, CBM-12, belonging to the botanical variety Momordica charantia var. muricata. Taiwan White bears very large, elongate, white, glossy and non-muricated (non-spiny) fruits but with low content of an anticancer phytomedicine, cucurbitacin-B and two antidiabetes phytomedicines, charantin and plant insulin. In contrast, CBM-12 produces small, round, dull and muricated (spiny) fruits but with high content of cucurbitacin-B, charantin and plant insulin. These two parental genotypes have also exhibited contrasting qualitative variation with regard to seed color (black vs. brown) and stigma color (yellow vs. green). The F2 population consisting of 140 individuals was grown under controlled conditions in greenhouses. This population exhibited Mendelian segregation for color (green, light green, white), shape (round, oblong, elongate), luster (dull, glossy) and surface (spiny, non-spiny) of fruits; seed color (black, brown); sigma color (yellow, green); and quantitative variation for number, length, maximum diameter and fresh weight of fruits. Fresh young leaves from the F2 individuals have been collected and stored for DNA extraction and molecular marker analysis. Fruit samples from each F2 plant have been collected, chopped, freeze-dried, powdered and stored for extraction and quantification of cucurbitacin-B, charantin and plant insulin. F3 seeds from each of the F2 plants have been collected to develop recombinant inbred lines following single-seed descent strategy. Through this project, four high school students were also mentored under the Summer Program for Research Interns-2009 sponsored by the SC LIFE Project of the Clemson University that is funded by an award from the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Program to the Clemson University. The results obtained from the experiments of this project were presented in the International Symposium on Molecular Markers in Horticulture held from July 29 to August 1, 2009 at Corvallis (Oregon); the Eighth Annual Research Poster Presentation of the Summer Program for Research Interns on 17 July 2009 at Clemson University; several meetings of the South Carolina Center for Botanical Medicine held at Charleston and Clemson (South Carolina) participated by many scientists from Clemson University, Medical University of South Carolina, University of South Carolina and USDA Vegetable Laboratory (Charleston, South Carolina) and many fruits and vegetable growers; and also submitted for the International Cucurbitaceae Symposium held during 20-24 September 2009 at Hunan, China. The goals, achievements and potential impact of the project were highlighted in the South Carolina Farmers - Summer 2009 magazine and the Independentmail.com, published on 15 July 2009 from Anderson (South Carolina). PARTICIPANTS: Four colleagues from the Clemson University including Bode A. Olukolu, Phullara Kole, Prof. Albert G. Abbott and Prof. Kenneth R. Marcus assisted me on this project. Four high school students were trained on areas related to this project under the 2009 Summer Program for Research Interns funded by an award from the Howard Hughes Medical Institute Undergraduate Biological Sciences Education Program to the Clemson University. They include McKenzie Alexander, Sravya Durbha, Angela Luo and Hiral Shah. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Molecular marker analysis using the DNA of the F2 individuals will facilitate construction of the first genetic linkage map for bitter melon. The phenotypic data on the six qualitative traits including color, luster, surface and shape of fruits; seed color and stigma color will be useful for locating the position of the genes controlling these characters on the genetic map. The metric data on the fruit traits including number, length, maximum diameter and fresh weight will enable detection of quantitative trait loci (QTL) controlling these polygenic characters on the genetic linkage map. Similarly, metric data on the contents of cucurbitacin-B, charantin and plant insulin will facilitate detection of the QTLs controlling their contents. Therefore, molecular markers closely linked to the genes governing the six qualitative traits and QTLs controlling the six quantitative traits can be used in future breeding programs to incorporate all the desirable genes and QTLs into the genetic background of Taiwan White leading to the development of duel-purpose varieties (DPVs) possessing superior fruit yield and quality traits coupled with high content of cucurbitacin-B, charantin and plant insulin. Development of DPVs will lead to large-scale cultivation of bitter melon in many US states as the presently cultivated varieties, grown in only eight states now, have low content of these phytomedicines. Large-scale cultivation will augment the income of limited-resource farmers. Besides consumption as functional food vegetable, the fruits can also be processed to prepare extracts, powders and pills. This post-harvest processing will facilitate development of agro-industries leading to generation of income and employment. Large-scale cultivation of DPVs will also benefit people involved in the retail markets, seed production, transport, pharmaceutical industries and national and international exporters. The incidence of cancer and diabetes is increasing at an alarming rate in the US. According to the American Cancer Society Surveillance Research 2008, about 565,650 Americans were expected to die of cancer and the estimated new cases of cancer were 1,437,180. According to the National Diabetes Fact Sheet of 2007, 23.6 million Americans have diabetes. Another 57 million people were estimated to have pre-diabetes, a condition that increases the risk of developing type-2 diabetes, the most common form of the disease, as well as heart disease and stroke. Therefore, development of DPVs will be useful to mitigate the problems of cancer, diabetes and its related diseases, such as obesity, hypertension and stroke. The recombinant inbred lines being developed now will be used to construct a genetic linkage map and locating QTLs controlling contents of other phytomedicines including the anticancer bioactives, alpha- and beta-momorcharin, momordin, lycopene and beta-carotene. The concepts and strategies employed for development of DPVs in bitter melon will serve as a model for genetic improvement in other medicinal plants and nutraceutical crops. Financial support for the investigations was obtained from the Clemson University and USDA-AFRI through a NRI-CGP grant.
Publications
- Kole, C., Olukolu, B., Kole, P., and Abbott, A.G. (2009a). Characterization of a Momordica charantia germplasm with AFLP and SSR markers. In: International Society of Horticultural Science International Symposium on Molecular Markers in Horticulture, Oregon State University, Corvallis, OR, July 29-Aug 1, 2009, p 67.
- Kole, C., Olukolu, B., Kole, P., and Abbott, A.G. (2009b). Towards molecular breeding of dual-purpose varieties in bitter melon (momordica charantia L.). In: 4th International Cucurbit Symposium, September 21-26, 2009, Changsha, Hunan, China.
- Kole, C., Olukolu, B., Kole, P., and Abbott, A.G. (2010). Towards phytomedomics with bitter melon (Momordica charantia L.) as a model. International Conference on the Status of Plant & Animal genome research, san Diego, CA, January 9-13, P164.
- Luo, A., Shah, H., Olukolu, B., Kole, P., Abbott, A.G., and Kole, C. (2009). Genetic variability in bitter melon (Momordica charantia) with regard to the content of an antidiabetic phytomedicine charantin. 8th Annual Research Poster Presentation. 2009 Summer Program for Research Interns, Clemson University, July 17, 2009, p 15.
- Shah, H., Luo, A., Olukolu, B., Kole, P., Abbott, A.G., and Kole, C. (2009). Estimation of plant insulin content in a bitter melon (Momordica charantia) germplasm. 8th Annual Research Poster Presentation. 2009 Summer Program for Research Interns, Clemson University, July 17, 2009, p 20.
- Alexander, M., Durbha, S., Olukolu, B., Kole, P., Abbott, A.G., and Kole, C. (2009). Genetic divergence in bitter melon with regard to DNA markers, fruit quality traits and therapeutic bioactives. 8th Annual Research Poster Presentation. 2009 Summer Program for Research Interns, Clemson University, July 17, 2009, p 3.
- Durbha, S., Alexander, M., Olukolu, B., Kole, P., Abbott, A.G., and Kole, C. (2009). Screening of bitter melon (Momordica charantia) hybrids employing AFLP and SSR markers. 8th Annual Research Poster Presentation. 2009 Summer Program for Research Interns, Clemson University, July 17, 2009, p 9.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: A Bitter Melon (Momordica charantia; Family Cucurbitaceae) germplasm consisting of 22 varieties of diverse pedigrees and geographical origin (India, China, Japan, Thailand, Taiwan and Hong Kong) was established in July 2008 in a greenhouse at the Clemson University. Observations recorded on five fruit characters including color (green vs. white), shape (long vs. round), luster (glossy vs. non-glossy), surface (spiny vs. non-spiny) and size (large vs. small) revealed contrasting variation among the accessions. Amplified Fragment Length (AFLP) analysis of DNA extracted from young leaf tissues of these accessions led to the detection of 250 molecular markers. Cluster analysis based on the fruit traits and the AFLP markers evidenced for wide genetic divergence in the germplasm. Quantification of the two antidiabetic phytomedicines, charantin and plant insulin extracted from lyophilized fruits of the 22 genotypes revealed wide genetic variation. Two parental genotypes were selected that were found to have high genetic distance and contrasting fruit characters. Crossing between plants of these two parents led to the establishment of 40 putative F1 hybrid plants from 15 F1 fruits. Hybridity of these plants were verified by comparing their AFLP banding patterns with those of their parents. One F1 plant is being maintained in a greenhouse for production of an F2 segregating population to use in construction of a molecular linkage map and mapping of polygenes (Quantitative trait loci, QTLs) controlling the content of the two bioactives. The results of these investigations were presented in a meeting of the South Carolina Center for Botanical Medicine at Charleston on August 26, 2008 that was attended by many scientists from the Clemson University, the Medical University of South Carolina and the US vegetable Laboratory of USDA and also many growers. Works of this project was also highlighted in the "Clemson University Public Service Impact Magazine" and in the Newsletter of the South Carolina Center for Botanical Medicine. Many scientists of the Clemson University and the Medical University of South Carolina visited the greenhouse to discuss about the project and its progress. Some of them have collected leaf tissues from many bitter melon accessions to conduct researches on extracts and the phytomedicines. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: In the original project we mentioned just as examples three model plants, bitter melon, periwinkle and Echinacea. As we recived funds through an "Incentive Project" from the Institute of Nutraceutical Research of the Clemson University and a Seed Grant sponsored by USDA-NRI-CGP in 2008 to work on bitter melon, we are working on bitter melon as the model medicinal plant for the project.
Impacts
This project aims at developing dual-purpose bitter melon varieties with higher content of two antidiabetic phytomedicines, charantin (CHR) and plant insulin (PLIN), along with better fruit quality traits employing molecular breeding. The development of an F1 hybrid of a variety with desirable fruit traits (while color, glossy, smooth surface, long shape and large size) but with low content of the two bioactives and a variety with higher bioactive content will facilitate development of an F2 segregating population for mapping the polygenes (QTLs) controlling bioactive contents and identification of molecular markers for using in marker-assisted backcross breeding of selected F2 plants with the variety with superior fruit traits. Bitter melon is sold in many retail stores in the US as a popular exotic vegetable and its extracts and powders are sold by many private companies. As a member of Cucurbitaceae, it is close to melon, watermelon and cucumber in agroclimatic requirements, and can be cultivated in the many US states. Cultivation of dual-purpose varieties will lead to fortified avenues of income for limited-resource farmers. Use of bitter melon extracts and powders requires post-harvest processing such as extraction, quantification, drying, quality assessment and packaging. Hence, its cultivation will foster the growth of small-scale industries leading to generation of employment and strengthened agricultural economy. Fruits of bitter melon are consumed as vegetable and medicine in many countries. Cultivation of dual-purpose bitter melon crop will meet domestic and international demands. Therefore, it will benefit people involved in agriculture, seed production, small-scale industries, retail market, transport and national and international export. Growing of bitter melon varieties with high bioactive content will pave the way for its use by diabetic and pre-diabetic people. The prevalence data released on 26th October 2005 by the Centers for Disease Control and Prevention (CDC) indicated that nearly 21 million Americans (7% of the US population) were affected by diabetes. Another 41 million people were estimated to have pre-diabetes, a condition that increases the risk of developing type-2 diabetes as well as heart disease and stroke. Once the ultimate goals of developing varieties with high charantin and plant insulin are attained, similar strategies can be followed to map genes and QTLs controlling content of other bioactives such as momordin, α- and β-momorcharin, cucurbitacin-B, lycopene and MP-30 those have anticancer, antiviral (including anti-HIV) and antimicrobial properties. These strategies can also be employed to improve bioactives in other nutraceutical crops. The outputs including germplasm and F1 hybrid besides scientific information could be achieved by using project grants funded by the Institute of Nutraceutical Research of Clemson University and USDA-NRI. The goals, outcome achieved so far and their potential impacts have been appreciated by many scientists from and outside the Clemson University, growers and general public who visited the greenhouse and had comprehensive interaction.
Publications
- No publications reported this period
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