1. Introduction
Lantana (
Lantana camara L., Verbenaceae) is a popular ornamental plant, especially in the subtropical and tropical regions of the world. A survey of the nursery industry in Florida, United States of America (U.S.A.), revealed that hundreds of nurseries and greenhouse growers produced
L. camara, and the sales of
L. camara plants contributed more than
$40 million a year to the state’s economy [
1]. The popularity of
L. camara is due to its ability to bloom year-round, attract many species of butterflies, tolerate harsh environmental conditions, thrive with low maintenance requirements, and propagate easily [
2,
3]. The genus
Lantana is comprised of seven species, with six from South America and one from Ethiopia [
4]. Most of the lantana cultivars grown by the ornamental plant industry belong to the species
L. camara. Lantana camara originated in West Indies and was introduced to the tropical regions of the world by 1900 [
5,
6]. At present,
L. camara is found in more than 60 countries under hundreds of cultivar names [
7].
Lantana camara is also known to be one of the world’s most aggressive invader plant species [
2]. As an introduced species in the U.S.A.,
L. camara has escaped from cultivation and established itself in the wild through seed dispersal and hybridization with Florida native lantana species,
Lantana depressa [
8]. Establishment and spread of
L. camara in Florida has endangered
L. depressa. The Florida Exotic Pest Plant Council (FLEPPC) has placed
L. camara as a Category I invasive species in southern, central, and northern Florida (
https://www.fleppc.org/). Thus, it is urgent to take actions to control its invasiveness. Considerable efforts have been made to control the spread of invasive
L. camara using genetic sterilization and the development of sterile cultivars [
9,
10,
11]. A number of sterile cultivars have been released that possess greatly reduced male and female fertility and little potential to cross-pollinate with native lantana. Despite the success, the development of sterile cultivars has been severely complicated by the ability of many
L. camara cultivars to produce unreduced female gametes (UFGs). The production of UFGs (UFGP) has enabled
L.
camara to evolve multiple ploidy levels and many
L.
camara triploids and pentaploids to produce viable seeds, as diploids or tetraploids do. Although the formation of unreduced pollen is more frequent in some other plants [
12], it is relatively rare in
L. camara [
8]. Therefore, it is important to understand the genetic mechanism(s) underlying the development of UFGs in
L. camara.
The formation of unreduced gametes (2
n) is a common mechanism for the generation of polyploids in natural systems [
13]. Unreduced gametes have also been employed to engineer sexual polyploidization in several crop species [
14,
15] and have become a very valuable source of genetic variation for many plant breeding programs. There is on-going research on utilizing unreduced 2
n gametes to fix heterosis in hybrid seeds. During normal meiosis and gamete formation, the chromosome number in the mother cells is reduced by half. However, 2
n gametes can be formed due to the first division restitution (FDR) or the second division restitution (SDR) [
16]. The molecular mechanisms underlying unreduced gamete formation were studied mainly by analyzing mutants producing unreduced gametes. It was revealed that the formation of unreduced gametes was mainly caused by meiotic defects, including defects in meiotic division, spindle orientation, and cytokinesis [
13]. Several genes involved in the formation of unreduced gametes have been identified, mainly in
Arabidopsis, including
SWITCH1 (
SWI1)/
DYAD [
17],
CYCA1;2/TAM [
18],
OMISSION OF SECOND DIVISION 1 (
OSD1) [
19],
Arabidopsis Parallel Spindle1 (
AtPS1) [
20], and
Tetraspore/Stud (
TES/STUD) [
21]. More studies are needed to understand the mechanisms of unreduced gamete formation for utilization in plant breeding and improvement. The
L. camara plants with the ability to produce UFGs are valuable materials for deepening our understanding of the genetic mechanisms of UFG formation.
Lantana camara also possesses a number of other characteristics for plant science research, including tolerance to drought [
22,
23], salt [
24], and allelopathy [
25]. These traits imply that
L. camara may be less prone to diseases or pathogens. The nucleotide-binding site leucine-rich repeat (NBS-LRR) genes play an important role in conferring disease resistance in plants [
26]. The identification of genes controlling these characteristics would provide a genomic resource for plant breeding. Nevertheless, only very limited genomic information is available for
L. camara. Up to date, there is only one transcriptome dataset available at the National Center for Biotechnology Information (
https://www.ncbi.nlm.nih.gov/). Very few molecular markers are available for fingerprinting
L. camara cultivars, differentiating its hybrids with native lantana species, or investigating its population structure and genetic diversity [
7]. Therefore, more genomic resources are much needed to expedite the development of sterile
L. camara cultivars, identify cryptic interspecific hybrids, and understand its invasiveness.
In this study, we applied RNA sequencing to compare the transcriptomes of young ovaries of two
L. camara genotypes differing in gamete formation. One genotype, Landmark White Lantana (LWL) produces normal reduced (
n) female gametes (non-UFG-producing or non-UFGP) [
27], while the other genotype, GDGHOP-36 (GGO), forms 100% unreduced (
2n) female gametes (UFG-producing or UFGP) [
8]. We revealed candidate genes associated with the formation of UFGs. We further identified gene families related to stress tolerance and disease resistance genes in
L. camara. Our findings not only significantly enriched the genomic resources of
L. camara, but also provided insight into the formation of UFGs in
L. camara.
3. Discussion
As an important ornamental plant and an invasive species,
L. camara is understudied and has very limited genomic resources. The development of sterile triploid lantana cultivars is an effective approach to controlling their invasiveness [
9]. However, this is hindered by the formation of UFGs frequently observed in
L. camara [
8]. Currently, little is known about the underlying genetic mechanisms of UFG formation in lantana. Our study investigated the transcriptome profiles of young ovaries of a lantana line producing UFGs and another cultivar producing normal gametes. The transcriptome analysis significantly enriched the genomic resources of lantana by contributing transcript sequences and molecular markers in a transcriptome-wide manner. The comparative analysis between the two genotypes revealed genes associated with female gamete production, as well as those orthologous to genes known to control unreduced gamete production. The development of molecular markers located within those genes and the markers that potentially change gene functions provided good candidates for discovering the mutations controlling the formation of UFGs. In addition, the identification of disease resistance genes and orthologous gene families associated with stress (drought/salt) tolerance and allelopathy would facilitate understanding the special characteristics of lantana and provide a genomic resource for plant breeding. Results from this study help us gain very valuable insight into the genetic basis of UFG formation in
L. camara and may enable the development of novel genetic tools for manipulating gamete formation in plant breeding, genetic improvement, and invasive species management.
The ovary is an organ where female gametes are produced. There have been a few transcriptome studies performed on ovary tissues to investigate gene regulations of fruit development in tomato (
Solanum lycopersicum) [
28,
29], wild tomato (
Solanum pimpinellifolium) [
30], and rice (
Oryza sativa) [
31], to elucidate carpel fusion mechanisms in maize (
Zea mays), to find genes regulating embryo and pod development in peanut (
Arachis hypogaea) [
32], to find genes responsive to freezing stress in almond (
Prunus dulcis Mill.) [
33], and to identify genes controlling ovary coloration in Asiatic hybrid lilies (
Lilium spp.) [
34]. By focusing on the 29,383 transcripts containing full-length CDS, the N50 increased to 2206 bp. The unique transcript sequences and those containing full-length CDS provide a valuable resource for further studies of genes expressed in ovaries of lantana. Functional annotation revealed that most of those sequences had significant hits in the five major databases (NR, NT, Kyoto Encyclopedia of Genes and Genomes or KEGG, GO, and Swiss-Prot) (
Table S1), which facilitated the identification of transcripts associated with gamete formation.
Gamete formation involves a series of cell divisions, and meiosis is particularly critical for the production of gametes with reduced chromosome numbers. Defects in these cell cycles, early meiotic events, spindle orientation, or cytokinesis can all lead to the formation of unreduced gametes [
13]. Through gene family analysis, there were 10 lantana gene families that were orthologous to genes known to control unreduced gamete production in
Arabidopsis. Furthermore, a total of 214 lantana transcript sequences were associated with gamete production based on annotated functions. These genes can be further studied in the future to understand the UFG formation in lantana. They can also potentially serve as candidates for gene editing. Among the 925 identified TF-encoding sequences, the most abundant TF family bHLH may play an important role during the development of lantana ovaries. As supported by studies in
Arabidopsis, bHLH TFs, such as
CRABS CLAW (
CRC),
SPATULA (
SPT), and
HECATE (
HEC), were reported to regulate the development of gynoecium, the female reproductive organ [
35,
36].
Direct comparison of the transcriptome profiles of young ovaries between two lantana genotypes differing in gamete production enabled a glance at the expressions of genes related with gamete production. To identify the genes that are actively expressed (likely to be functional in ovaries) in one genotype and not expressed (unlikely to be functional in ovaries) in the other, we specifically focused on those with an FPKM value ≥1 in one genotype and FPKM = 0 in the other genotype. Overall, there were 5224 transcripts actively expressed in GGO and not expressed in LWL. As LWL forms reduced gametes, the absence of these gene expressions in LWL may indicate that they are not required for normal gamete production. However, we cannot exclude the possibility that some genes related with UFG formation are among these 5224 genes. GO enrichment analysis showed that no GO terms were enriched for these genes, whereas there were 43 GO terms enriched in the 4314 transcripts actively expressed in LWL and not expressed in GGO. These genes were likely playing normal functions in the ovaries of LWL, but not in the ovaries of GGO due to the absence of expressions. They potentially included the genes whose absence of expressions were caused by the formation of UFGs or led to the formation of UFGs in GGO. Among these 4314 transcripts, there were 11 transcripts associated with gamete formation-related biological processes. Moreover, we found three enriched GO terms related with the telomere. During meiosis, chromosome pairing at prophase is required for subsequent chromosome segregation that reduces the chromosome number before gamete formation [
37]. The rapid prophase chromosome movement is led by telomeres, which is important to chromosome pairing and synapsis. The telomeres cluster prior to the initiation of synapsis [
38]. Therefore, the disturbed telomere migration could lead to failures of synapsis and chiasma formation [
39]. Out of the total five transcripts associated with these three telomere-related GO terms, four were not expressed in GGO, but all five were actively expressed in LWL, indicating likely that telomere activities during meiosis is disrupted in GGO. This may imply a very important role of telomeres in unreduced gamete formation. These 15 transcripts seem to be good candidates for the future study of gamete production in lantana (and other plants).
Since the lantana genome reference is not available, we annotated the effects of sequence variants using the transcriptome as a reference. Particularly, we focused on the polymorphisms in genes associated with gamete production and the genes that were orthologs to those known to control unreduced gamete production in other plants. Toward this end, we identified 83 SNPs and seven indels in 19 genes associated with gamete formation and nine orthologs likely controlling unreduced gamete formation. Variant annotations indicated that 25 SNPs and two indels out of these variants likely have an impact on gene functions. More studies are needed to identify the causal mutations leading to UFG formation in lantana.
Toward further understanding and better utilization of the special characteristics of lantana, including its disease resistance, drought/salt tolerance, and allelopathy, we discovered the putative disease resistance (NBS) genes, and gene families related with drought/salt tolerance and allelopathy for the first time in lantana. In total, we identified 91 NBS genes and three gene clusters based on phylogenetic analysis. Similarly, based on a previous study that clustered NBS genes from multiple species, three major groups were obtained, including two groups of the non-TIR NBS genes and one TIR group [
40]. However, no TIR domain was identified within the NBS genes in this study, which was likely due to the incomplete coverage of NBS genes or a specific feature of lantana NBS genes. In addition, we identified 291 gene families related to drought/salt tolerance and four gene families related to allelopathy, which enriched the gene pool of stress tolerance in plants and can be further explored in the future.
Molecular markers are an important genomic resource that have many applications, such as cultivar fingerprinting, identification of cryptic interspecific hybrids, genetic mapping, genetic diversity analysis, and phylogenetic analysis [
41]. A major issue caused by the invasiveness of
L. camara is its hybridization with
L. depressa, making
L. depressa an endangered species [
42]. Molecular markers based on SSRs and indels can be easily developed into PCR-based genotyping tools, which will be invaluable to many applications, including identifying cryptic hybrids resulting from unintended natural pollination or crossing between
L.
camara and
L.
depressa and protecting the native lantana species in ecological conservation and restoration. In this study, we catalogued a total of 9513 SSRs in lantana, with primers designed for each. Moreover, we identified 165,229 SNPs and 9984 indels that were polymorphic between LWL and GGO, and further annotated their effects. Some of the variations could lead to codon changes, including start or stop codons, and they may significantly impact gene functions and lead to changes in lantana phenotype.