Developing a Genomic Framework for Studying Agronomic and Nutritional Traits in Pineapple
Ashley Yow, PhD Final Seminar
Wednesday, October 19, 2022, 1:00 pm
(Under the direction of Dr. Massimo Iorizzo and Dr. Penny Perkins-Veazie, Co-chairs)

Zoom Link:  https://ncsu.zoom.us/j/96976460563?pwd=TGdZYy9FN3RkSnZucGJuWHFnWm81QT09
Meeting ID:  969 7646 0563
Passcode:  549106

 Yow_PhD.Abstr.pdf

Pineapple (Ananas comosus (L.) Merr.) is the second most important tropical fruit crop globally, and ‘MD2’ is the most important cultivated variety. High-quality genomic resources, including genome assemblies and annotations are necessary for studying important traits in fruit crops like pineapple. Two of these important traits include bromelain enzyme content and cold stress tolerance. Bromelain enzymes are papain-family proteases with high medicinal value due to their proteolytic activity and anti-inflammatory effects and cold stress-induced flowering is one of the major bottlenecks for pineapple production. For these reasons, we aimed to develop an improved pineapple genome assembly and annotation, and to identify bromelain enzyme genes and elucidate mechanisms for cold stress tolerance in pineapple.

In this work, we utilized PacBio and Hi-C data to develop a new, high-quality genome assembly and gene prediction for MD2. Compared to the previous MD2 assembly, major improvements included a 26.6-fold increase in contig N50 length, phased chromosomes, and >6000 new genes. The new MD2 assembly also included 161.6 Mb additional sequences and >3000 extra genes compared to the F153 genome. The new assembly also facilitated the comparison of the two individual haplotypes of the MD2 genome and provided a strong foundation for studying FAR1-RELATED SEQUENCE (FRS) genes, bromelain enzyme genes, and the relationship between cold stress and precocious flowering in pineapple.

The haplotype diversity of the MD2 genome indicated that a high level of heterozygosity in this pineapple contributes to maintaining functional alleles. A total of 82 FRS genes were identified in the new MD2 genome and it appeared that this gene family was expanded in pineapple and rice, and that their expansion contributed to evolving specialized functions in reproductive tissues.

A total of 71 papain-family protease genes were identified in the new MD2 genome, and were used to specifically identify the subgroup of genes that code for bromelains. Orthologous and phylogenetic analyses allowed us to determine that bromelain genes are clustered into one orthogroup (189) and one phylogenetic clade (VI). Duplication and synteny analysis allowed us to determine the most likely origin of the bromelain subfamily genes within the larger papain family of proteases in pineapple. By utilizing a combination of approaches, including genomic, transcriptomic, and proteomic analyses, 4 genes were identified as bromelain genes in this work, which can be used for future research on bromelain.

Two different pineapple genotypes, one susceptible (MD2) and one tolerant (Dole-17) to precocious flowering, were used to identify genes and gene networks that are likely associated with cold stress response and stress-mediated regulation of flowering time in pineapple. Through differential expression and weighted gene co-expression network analyses, a number of genes were identified that were likely responsible for differences in cold response between genotypes and were primarily involved in ethylene signaling, carbohydrate accumulation, and physiological adaptation to cold. Given the results of this study, we hypothesize that MD2 likely has a higher susceptibility to cold stress that induces ethylene production, which would in turn, cause earlier induction of flowering.

The new MD2 assembly will serve as a new reference for genetic and genomic studies in pineapple and the genes and gene families identified here can serve as a foundation for performing applied research studies on bromelain and for developing new varieties of pineapple with higher bromelain content, increased cold tolerance, and delayed or more synchronized flowering time.