The chloroplast genome inheritance pattern of the Deli-Nigerian prospection material (NPM) × Yangambi population of Elaeis guineensis Jacq

Introduction

The oil palm (Elaeis guineensis Jacq.) is one of the most economically productive oil crops and produces the most versatile vegetable oil in the world. Global palm oil production is continuously increasing, with 2020 global annual production reaching 74.7 million metric tonnes (FAO, 2022). The crop thrives in the tropical environment, specifically, in a belt spanning the equator, with the major producers being Indonesia followed by Malaysia (Abubakar & Ishak, 2022). The palm oil demand will increase with the growing world population and in 2050 is expected to reach 240 million tonnes (Corley, 2009).

One of the ways to sustainably produce more palm oil is via plant breeding. For example, the discovery of shell gene inheritance led to significantly greater yields (Beirnaert & Vanderweyen, 1941) through the development of the tenera palm, a hybrid of the thick-shell dura and the shell-less pisifera, which produces a thin-shell fruit that contains 30% more oil than the dura variety (Seng et al., 2011). Since then, the tenera palm has been widely planted by plantations. Thus, continuous yield improvement through the genetic exploitation of various lineages is necessary for developing new varieties, especially in preparing the industry for climate change and improving agronomic traits. Currently, commercial seed production utilizes lineages from Algemeene Vereeniging van Rubberplanters ter Oostkust van Sumatra (AVROS), including dumpy AVROS, dumpy Yangambi, dumpy Yangambi x AVROS, Calabar, and Ekona (Rajanaidu et al., 2013), and Felda Global Ventures Holdings Berhad (FGV), with a focus on exploiting the Yangambi background.

Besides conventional breeding, various investigations have successfully enhanced plants’ agronomic traits by genetically modifying the nuclear genome (Řepková, 2010; An et al., 2022). Despite their frequent use, transgene expression and hereditary agronomic characteristics are challenging to regulate because of their nuclear biparental characteristics. There is also a risk that the transgene may spread among the plants’ wild relatives (Řepková, 2010) through pollen dispersion. The chloroplast, one of the organelles present in the plant cell, harbors an independent genome that is small in size, and has mostly been reported to be uniparentally inherited from maternal parents, and thus has a lower risk of being spread through pollen (Daniell, 2007; Birky, 2008; Park et al., 2021; An et al., 2022), making it a suitable genome for inheritance studies and genetic engineering. Examples of genetic engineering performed on the chloroplast genome to improve agronomic traits include herbicide detoxification (Bansal & Saha, 2012), resistance to stress, nutritional value enhancement, biopharmaceuticals, and vaccine development (Daniell et al., 2016).

The chloroplast genome in angiosperms is quite conserved and is made up of a typical quadripartite structure, with large single-copy (LSC) and small single-copy (SSC) regions, as well as two copies of inverted repeats (IRs) separating the LSC and SSC. The genome size is usually between 120 and 170 kb in length. Previously, the generation of E. guineensis chloroplast genome data has been performed in a small number of works focusing on genome characterizations and the deep phylogenomic analysis of palm species. Based on the first reference genome (NC_017602) (Uthaipaisanwong et al., 2012), E. guineensis chloroplast genome has 156,973 bp in size with 112 unique genes. Comparative chloroplast genomes analysis between E. guineensis and date palm species revealed chloroplast genomes size differences (1,489 bp). However, their chloroplast genome structural characteristics and gene contents are similar. In another study (Yao et al., 2023), E. guineensis chloroplast genome was utilized for deep phylogenomic study within Arecaceae species. In addition, the chloroplast genome sequence (ON248756) shows a few gaps (N) present in the genome alignment. Both studies (Uthaipaisanwong et al., 2012; Yao et al., 2023) showed the gene content and arrangement in chloroplast genome are highly conserved in the palm family. Comparative analysis of chloroplast genomes was done between three different palm species from Brazil (Francisconi et al., 2022). This study showed these three species’ nucleotide sequences are significantly varied from each other in term of insertion/deletion (InDels) and single nucleotide polymorphisms (SNPs). Although there are variations in the coding-regions among species involved, the region with high polymorphic diversity is the intergenic regions where most of the SNPs are found.

In most angiosperm, chloroplast genome are maternally inherited. Nonetheless, biparental inheritance of the chloroplast genome in angiosperms has been reported several times (Mason, Holsinger & Jansen, 1994; Hansen et al., 2007; Barnard-Kubow, McCoy & Galloway, 2017). Previous investigations on E. guineensis chloroplast genome did not highlight in detail the chloroplast genome inheritance pattern among E. guineensis individuals and their parental individuals. It is therefore pertinent to study the inheritance pattern of the chloroplast genome among E. guineensis individuals, especially in those of parental and F₁ progenies in a breeding programme.

In this study, the parents and progenies of E. guineensis in an ongoing breeding research programme by FGV were used. Based on simple sequence repeat (SSR) analysis performed by FGV, a few individuals in the progeny showed ambiguous relationships with their putative parents. As such, this article aims to clarify inheritance patterns among Deli-Nigerian prospection material (NPM) × Yangambi, specifically the GB33 population (J4-25 × ML-161), by comparing their chloroplast genome structure to that of their putative parents. By observing and comparing nucleotide variations between maternal and paternal parent of these F₁ progenies, any regions that signals possible mutation could be highlighted for future reference in breeding program.

Materials & Methods

Results

SNP and InDel characteristics

The variations detected from ML-161 were only 15 SNPs and nine InDels when compared to J4-25 (Table S3). Coverage depth for each nucleotide variation is listed in Table S3. For most of the F₁ progenies, the chloroplast genome was the exact copy of J4-25 except for six F₁ progenies (GB33-47, GB33-46, GB33-31, GB33-11, GB33-41 and GB33-17). The variations showed by the F₁ progenies with J4-25 contained only one nucleotide variation for each individual. Figure 1 shows variations among chloroplast genomes compared to the J4-25 individual which were further described in detail through Table S4. Figure 2 depicts the circular visualisation of the J4-25 chloroplast genome and nucleotide variations compared to other study samples. Although the number of variations between ML-161 and J4-25 are significant, Table 1 shows that all variations in the protein coding region are synonymous.

Discussion

This study revealed chloroplast genome inheritance patterns among 23 E. guineensis tenera individuals in a single population. The generation of E. guineensis chloroplast genome data has been performed in a small number of works focusing on genome characterizations (Uthaipaisanwong et al., 2012) and the deep phylogenomic analysis of palm species (Yao et al., 2023). However, these investigations did not highlight in detail the chloroplast genome inheritance pattern among E. guineensis individuals from parent individuals. Various analyses have shown that maternal inheritance of the chloroplast genome is particularly prevalent among angiosperms, for example, in cucumber (Park et al., 2021), spinach (She et al., 2022) and banana (Fauré et al., 1994). There is also evidence that the chloroplast genome from angiosperm plants can be inherited paternally and biparentally (Shrestha et al., 2021). In the present study, the paternal chloroplast genome (ML-161) displayed a more significant number of variations than the maternal one, J4-25. Based on coverage depth analysis, the reliability of these nucleotide sequences had a high confidence level (>100×) (Table S4). All variations in ML-161 were single-nucleotide except for one position in ndh D–psa C, which demonstrated two variations. Nucleotide variations, either single-nucleotide polymorphisms (SNPs) or insertion–deletion mutations (indels) between sequences, mostly occurred in the non-coding region (intron and intergenic spacer).

Despite being assembled independently in de novo mode, 17 F₁ progenies inherited the exact sequences of their maternal chloroplast genome (J4-25). Conversely, six of the F₁ progenies demonstrated a single variation in nucleotide position, all within intergenic regions. High sequence variations in the chloroplast intergenic regions have been consistently reported in many works (Hamilton, 1999; Abdullah Shahzadi et al., 2019; Gichira et al., 2019). The data generated from the present study confirm this earlier work, showing that intergenic regions have a higher evolutionary rate than the coding region. Although variations also occurred within the coding region, they were synonymous, which means that there were no amino acid changes in the transcription process.

Accessibility to genomics data in recent decades has demonstrated the existence of plastid sequence polymorphisms among different plant populations and genera (Kim et al., 2015; Henriquez et al., 2020; Wei et al., 2021). Plastid genome characteristics have been particularly useful in assisting evolutionary and diversity studies among interspecific individuals. Nevertheless, when variations exist among intraspecific individuals, different approaches or interpretations are required. Previous works have reported the occurrence of intraspecific variations in the chloroplast genomes of several crop plants: the ‘Red Fuji’ apple (Miao et al., 2022), the castor bean (Muraguri et al., 2020), red clover (Li et al., 2019) and Korean ginseng (Jo et al., 2016). As a perennial plant, E. guineensis has the potential to develop intra-individual genomic variation because of its ability to independently grow the same organ at different times (Sun et al., 2019). Nonetheless, this assumption is currently theoretical and requires further analysis.

Further assessment in haplotype analysis involves the reference genome available in the GenBank database. Although the variant is different, ML-161 and J4-25 clearly showed a much closer relationship compared to the reference genome. It is particularly important to keep track of genetic inheritance patterns of haplotypes in crop breeding pedigrees to support new genomic combinations with desired traits (Varshney et al., 2021). In jujube breeding, chloroplast genome haplotypes were previously used as a guide for the next breeding plan in order to produce fruits with highly desirable traits (Hu et al., 2022). For a well-studied crop plant such as E. guineensis, only certain pedigrees are chosen because they carry the desired traits for commercial seed production. Evidently, in E. guineensis, the maternal inheritance pattern is quite robust despite minimal variation compared to ML-161. The highly conserved sequence displayed by the F₁ progenies demonstrates that there is considerably high potential for transferring the genetic characteristics from the maternal unit to the progenies without mutation.

SSR sequences have been known to be very useful in conducting population studies. The polymorphisms in the chloroplast SSR (cpSSR) usually vary across species and loci (Amiteye, 2021). SSRs from the chloroplast genome are desirable because they are easy to isolate with polymorphic characteristics, particularly in the intergenic spacer region. A comparative analysis of plastid SSRs among palm trees recently showed that most of the detected SSRs are mononucleotides within the non-coding region (Silveira et al., 2022). Similarly, the SSRs observed in the present investigation were mostly from single copy intergenic spacer regions, with 36 being mononucleotide repeats. These SSRs have the potential to be developed as molecular markers especially for genotype discrimination purposes. Despite report on low discriminatory ability of E. guineensis restriction fragment length polymorphism (RFLP) markers from chloroplast region (Jack, Dimitrijevic & Mayes, 1995), the discriminatory ability of these SSR markers for this species can still being assessed in the future. Low diversity was expected among samples in this study because the plant samples employed were intraspecific, in addition to the clonal inheritance and non-recombinant nature of the chloroplast genome. All four repeat types (palindrome, forward, reverse and complementary) are present in the chloroplast genome. The results are consistent with repeat types of three other palm family of genus Euterpe (Francisconi et al., 2022) in which palindrome repeat has the highest number, followed by forward repeat, reverse repeat and finally complementary repeat. This revealed high conservation of gene structure in palm species specifically among genus Euterpe and Elaeis species.

Conclusions

This study has presented the chloroplast genome assembly of 25 E. guineensis individuals from short-read paired-end raw data. Maternal inheritance among the F₁ progenies is robust with only a few individuals showing a variation in the sequence. Although there are intraspecific variations between male and female plants, they are minimal compared to the reference genome. Genotype evidence shows that in E. guineensis, chloroplast genome sequences are uniparentally inherited through the maternal parent, thus demonstrating their high potential for use to ensure the passing of the desired transgene to the progenies.

Supplemental Information