An integrative study of Cyperaceae: focused on expressions of floral MADS-box genes and the structural evolution of chloroplast genomes

Abstract

Cyperaceae is the third largest family in monocots and the seventh largest family in angiosperm containing 88 genera and ca. 5,500 species worldwide. They are well known as a material for the paper (papyrus; Cyperus papyrus L.) in ancient Egypt and are economically and environmentally useful plants. The family is placed in Poales and is distinguished by highly reduced flowers and complex inflorescences. However, many studies in Poales are mainly focused on Poaceae, and the comprehensive studies of Cyperaceae are insufficient. Four subjects were included in this study: 1) the MADS-box genes of Carex dickinsii as a representative of Cyperaceae were identified using transcriptome analysis to understand the evolution and diversification of flowers in Cyperaceae, and their expression patterns were confirmed in each organ. 2) the genome sizes were estimated from 43 taxa of Korean Carex, representing 15 sections. 3) the entire chloroplast (cp) genome of C. lupulina as a representative of the Carex was determined. 4) the cp genome structure of C. lupulina was compared with those of related taxa to understand the evolution and diversification of the cp genomes in Cyperaceae. Carex L., with more than 2,000 species, is the largest genus in Cyperaceae and distinguished by unisexual flowers without perianth. The pistillate flowers of Carex are characterized by the presence of a perigynium which is a sac-like organ surrounding the perianthless female flower and the staminate flowers consist of three or fewer stamens subtended by a scale. In this study, a total of 48 MADS-box genes [40 type II (35 MIKCC and five MIKC*-type) and eight type I MADS-box genes] were identified in C. dickinsii. The MADS-box genes of C. dickinsii were expressed in leaves, pistillate scales, perigynia, carpels, staminate scales, and stamens. Especially, E function genes were expressed in pistillate scales, perigynia, and staminate scales, which are specialized organs in Carex. Therefore, these organs are considered as a part of flower in Carex. Considering different expression patterns of floral MADS-box genes, the evolutionary origins of pistillate scale and staminate scale are different although their morphology is identical. The genome sizes of 43 Korean Carex were estimated using flow cytometry. The number of taxa represent 24.4% of Korean Carex. The average genome size of Carex species based on current study and data from the Plant DNA C-value Database (release 7.1) is 0.47 pg (1C). The genome size of C. cuspidate is the largest (1C=1.64 pg), and that of C. brownii, C. kobomugi, C. nubigena, and C. paxii were identical and the smallest (1C=0.20pg). The large genomes are frequently found in the subg. Carex, especially in sect. Aulocystis, sect. Digitatae, sect. Glaucae, sect. Paniceae, and sect. Siderostictae. The subg. Vignea had relatively small genome in which the sect. Phleoideae had the smallest genome. The complete cp genome of C. lupulina was sequenced based on the Nanopore platform. The cp genome of C. lupulina is 246,165 bp in size which is the second largest in the reported cp genomes in angiosperm (NCBI organelle DB; accessed on July 2019). The cp genome consists of LSC (99,356 bp), SSC (10,293 bp), and two inverted repeats (each 68,258 bp), and it has a total of 123 genes including 63 unique protein-coding genes, 29 unique tRNA genes, and 4 rRNA genes. To address the structural evolution of cp genomes in Cyperaceae, cp genome of C. lupulina was compared with previously reported cp genomes from relative taxa. Compare to the sister groups of Cyperaceae, there are tree inversions (rps4~part of the accD: 12~13 kb, rps12~trnL-CAA: 6~7 kb, and ndhF: ca. 2.2 kb), two translocations (part of the accD: 0.2~1.4 kb and rps14~trnS-GGA: 7.5~9 kb), one inverted translocation (ndhG~ycf: 5~11 kb), three gene losses (accD, infA, and rps18), and one IR expansion (ndhG~ycf1: 10~11 kb). In Carex, there are one inversion (part of the clpP~rps11: ca. 10 kb), two translocations (rps4~part of the clpP: ca. 15 kb and rps16: ca. 1.2 kb), three inverted translocations (psbK~atpI: ca. 8 kb, rps14~psaA: ca. 5 kb, and trnQ-UUG: ca. 140 bp), seven gene losses (clpP, rpl23, rpoA, rps3, ycf1, ycf2, and trnT-GGU), and one gene duplication (trnfM-CAU). Autapomorphic characters of C. lupulina were two large inverted translocations (rps8~petD: ca. 30 kb and psbA~matK: ca. 5.2 kb), eight gene losses (ndhD, petA, rpoC2, rps2, rps7, rps16, ycf4, and ycf68), and five gene duplication (rps14, trnfM-CAU, trnG-GCC, trnR-UCU, and trnT-AGU). The number of repeat sequences in Cyperaceae are higher than that of Typhaceae and Eriocaulaceae. The repeats are more distributed in the LSC region than in other regions except for oligonucleotide repeats of C. lupulina that are frequently distributed in the IR region. Also, the repeats are frequently located in the non-coding region such as IGS (intergenic spacer region). Accumulation of repeats led to increase of size of the cp genomes in Cyperaceae. Especially in C. lupulina, many repeats in the IR region causes an increase in the size of the IR region, and are the main reason for the increase in the size of the cp genome. In addition, IR expansion into SSC region by the duplication of ndhG~ ycf1 region increases the size of the cp genome in Cyperaceae. And, these repeats can be found at the borders of rearrangements in Carex. It seems that these repeats correlated with structural changes in Carex. This study is the first report for 1) 48 floral MADS-box genes in Carex and their expression patterns in floral organs, 2) for genome size from 43 taxa of Carex in Korea, 3) for the complete cp genome of C. lupulina, and 4) for the structural changes in Cyperaceae. The results of this study will provide the basis for understanding the evolution and diversification of flowers in Carex and the structural evolution of cp genome in Cyperaceae.