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 Tropical Plant Genetic Resources and Disease Research: Hilo, HI
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ARS Home Pacific West Area Hilo, Hawaii Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center Tropical Plant Genetic Resources and Disease Research Research





- Matsumoto Brower, Tracie
- Keith, Lisa
- Wall, Marisa
- Yu, Qingyi
- 2023
- Publications
- Plant Genetic Resources, Genomics and Genetic Improvement (301)

Research Project: Conservation, Management, and Genetic Improvement of Tropical and Subtropical Fruit, Nut, and Beverage Crop Germplasm for the Pacific Region **Location:
                            Tropical Plant Genetic Resources and Disease Research** 2023 Annual Report **Objectives**
                                

This project aims to address the following objectives. Objective 1:  Conduct research to develop genetic resource maintenance, evaluation, or characterization methods and, in alignment with the overall NPGS Plan, apply them to priority tropical and subtropical fruit (e.g., papaya, pineapple), nut (e.g., macadamia), and beverage (e.g., coffee) crop genetic resources to avoid backlogs in genetic resource and information management. Sub-objective 1.A: Develop novel tools and strategies to isolate, identify, characterize, and manage common and emerging plant pathogens of macadamia and other tropical and subtropical crops to maintain optimum health of the current collection and distribute clean germplasm. Sub-objective 1.B: Evaluate genetic diversity and population structure of tropical and subtropical fruit, nut, and beverage germplasm collections and apply genomic tools in germplasm management. Sub-objective 1.C: Conduct research to improve propagation methods, enable safety duplication, increase the number of cryopreserved accessions, and digitize relevant information.

Objective 2:  Acquire, distribute, and maintain the safety, genetic integrity, health, and viability of priority tropical and subtropical fruit (e.g., papaya, pineapple), nut (e.g., macadamia), and beverage (e.g., coffee) crop genetic resources and associated descriptive information.

Objective 3:  Breed genetically enhanced germplasm that broadens the diversity available for improving selected crops, such as coffee and papaya, by incorporating superior traits from cultivars, landraces, and wild relatives into adapted genetic backgrounds and gene pools. Sub-objective 3.A:  Identify and validate genomic regions in the coffee genome harboring coffee leaf rust (CLR) resistance genes and develop and breed CLR-resistant coffee cultivars that maintain high cupping quality. Sub-objective 3.B: Develop molecular tools to identify and breed new P. palmivora tolerant Carica papaya. **Approach**
                                

This project is responsible for collecting, maintaining, evaluating, and distributing germplasm of tropical and subtropical fruit, nut, and beverage crops. Crops include pineapple (Ananas), breadfruit (Artocarpus), starfruit (Averrhoa), peach palm (Bactris), pili nut (Canarium), papaya (Carica and Vasconcellea), coffee (Coffea), longan (Dimocarpus), lychee (Litchi), macadamia (Macadamia), acerola (Malpighia), rambutan and pulasan (Nephelium), and guava (Psidium). 

Objective 1 will determine the fungal pathogens and species of Phytophthora that exist on macadamia and other tropical crops through the identification and characterization of host-pathogen interaction. A collection of Phytophthora isolates will be established to understand the diversity of pathogens that may affect the macadamia accessions.  In vitro, fungicide tests will be conducted to identify the efficacy of fungicides and will be confirmed in vivo using seedling and field trials.  In addition, we will develop high-resolution SNP panels for the designated crops at the Hilo genebank to assess genetic diversity and population structure, identify gaps for future collections, and confirm genotype identity. Also, improve propagation methods for clonal crops to enable accession safety in the Hilo genebank while increasing the storage length of seeds to enable greater regeneration and potential expansion of the Carica and wild relative collections.
 
Objective 2, will manage and operate a clonal germplasm genebank of 15 genetically diverse fruit and nut crops, and provide scientists, domestically and internationally, with high-quality, disease-tested scion, seed, and leaf samples for characterization and research. New germplasm will be acquired through public and private institutions. Clonal crops have a minimum of two plants per accession and, where possible, maintain additional propagules for distribution. We aim to initiate and maintain all clonal accessions in tissue culture and investigate methods for cryopreservation. Plants grown in tissue culture will be acclimatized into the greenhouse every few years to ensure the plant can be successfully regenerated into the field and ensure the phenotype of the accession is unchanged. Germplasm stored as seeds will be maintained in our cold storage facility and also cryopreserved at Fort Collins. We will also serve as backup for the avocado and cacao collections for Miami and Mayagüez repositories, respectively. 

Objective 3, will identify the genomic regions in coffee linked to or conferring coffee leaf rust (CLR) resistance to improve the effectiveness of the molecular breeding platform for developing new coffee varieties through analysis of an F2 segregating population with SNPs markers to identify quantitative trait loci (QTLs). The Carica collection will be screened for P. palmivora using an inoculated seedling assay. We will also determine if a cross between Phytophthora susceptible ‘SunUp’ and tolerant ‘Waimanalo’ will identify tolerant genes in C. papaya to facilitate the identification of new cultivars for the papaya industry. This will be accomplished through Bulked segregant analysis Ribonucleic Acid-sequencing (BSR-Seq). **Progress Report**
                                

This report documents the progress for this new project, “Conservation, Management, and Genetic Improvement of Tropical and Subtropical Fruit, Nut, and Beverage Crop Germplasm for the Pacific Region” which began in March 2023 and continues the research of 2040-21000-016-000D, “Management, Characterization, and Evaluation of Pacific Tropical and Subtropical Fruit and Nut Genetic Resources and Associated Information”. For additional information, please see the report for the previous project. 

In support of Sub-objective 1A, research continued to assess and re-evaluate germplasm screening using leaf and seedling bioassays to evaluate macadamia accessions for tolerance or resistance to Phytophthora spp. Seven accessions were evaluated using seedling bioassays. In addition to the multiple diagnoses from the increase in trees exhibiting macadamia quick decline (MQD) symptoms caused by P. tropicalis and P. heveae, many trees show slow decline symptoms. To determine if the cause was biological, whole trees showing slow decline symptoms were felled, sectioned, and dissected. Several fungal isolates were recovered from upper canopy samples and identified by morphological and molecular methods. Proof of pathogenicity studies are ongoing. In addition, research is being conducted to develop novel strategies to identify and develop disease resistance in papaya. Sixteen Phytophthora palmivora isolates were collected from papaya farms at different locations on Hawaii Island. 

In support of Sub-objective 1B, research is being conducted on the genotypic evaluation of 91 pineapple germplasm accessions using resequencing data. The analysis resulted in identifying a total of 7,944,346 high-quality single nucleotide polymorphisms (SNPs) distributed throughout the pineapple genome. Subsequently, population structure analysis was performed to gain insights into the genetic composition of the accessions. The assessment revealed distinct genetic components in the wild A. comosus var. microstachys, highlighting its unique genetic makeup. In contrast, the domesticated varieties, namely A. comosus var. comosus, A. comosus var. bracteatus, and A. comosus var. erectifolius, exhibited evidence of gene flow. This suggests the influence of genetic exchange and breeding efforts in shaping the genetic profiles of the domesticated pineapple varieties. To facilitate the identification and differentiation of major pineapple cultivars, a panel of 123 high-quality SNPs was specially designed. This SNP panel can serve as a reliable tool for distinguishing between different cultivars and enhancing genetic identification and breeding programs. Additionally, a set of 540 SNPs was carefully selected to estimate the level of heterozygosity in the pineapple germplasm collection. For avocado, 75 germplasm accessions were genotyped using 94 SNPs. Also, leaf tissue samples were collected from 151 litchi and 78 longan germplasm accessions and sent to an ARS scientist in Beltsville, Maryland, for genotypic evaluation and international germplasm collections. 

In support of Sub-objective 1C, research is being conducted to improve propagation methods, enable safety duplication, increase the number of cryopreserved accessions, and digitize relevant information. Fourteen accessions were sent to ARS in Fort Collins, Colorado, for long-term cold storage at 4 degrees C and cryopreservation. 

In support of Objective 2, to acquire, distribute, and maintain the safety, genetic integrity, health, and viability of priority tropical and subtropical fruit (papaya, pineapple), nut (macadamia), and beverage (coffee) crop genetic resources and associated descriptive information, distribution of thirteen requests for forty-three items was sent to requestors, including four accessions of papaya to India. Five accessions of coffee were received from cooperators and placed in tissue culture or a greenhouse. Seventy-six pineapples were harvested and planted. Fifty-seven tissue culture pineapple plants were screened for Pineapple Mealybug Wilt Virus 1, 2, 3, and 6. 

In support of Sub-objective 3A, research is being conducted to identify the genome regions that regulate coffee leaf rust (CLR) resistance utilizing next-generation sequencing (NGS)-based bulk segregant analysis (BSA) and comparative bulk transcriptome analysis. Also, 24 of 76 coffee accessions have undergone one round of CLR resistance screening via leaf disk assay. Two of the 24 accessions were completely resistant to CLR. The remaining accessions were susceptible to varying degrees and produced urediniospores. Methods testing for the genotyping aspect of this sub-objective is ongoing. To assess gene expression changes in response to CLR infection, we conducted real-time PCR using time-series samples, focusing on immune-responsive genes. Based on these findings, we selected important time points for further comparative bulk transcriptome analysis. ARS researchers performed DNA extraction on 152 F2 mapping population samples, which will be utilized for bulk segregant analysis. Additionally, genome sequencing of two CLR-resistant Catimor coffee plants and two CLR-sensitive arabica coffee plants has been done by our ARS collaborator in Beltsville, Maryland. These data sets serve as crucial resources for mapping the genomic regions that harbor CLR-resistant genes. **Accomplishments**
                                

1. Development of molecular markers for pineapple cultivar identification. ARS scientists in Hilo, Hawaii, developed molecular markers for differentiating pineapple cultivars. The genetic evaluation of 91 pineapple germplasm accessions provided valuable insights into the genetic diversity and structure of the national pineapple collection. The development of molecular markers further contributes to the efficient management and utilization of pineapple genetic resources. These findings lay the foundation for targeted breeding programs and conservation efforts to enhance pineapple production and preserve genetic diversity.


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