Phenotypic characterization of some Pisifera Sh alleles in oil palm

Oil palm is mainly recognized as having three fruit types characterized by different shell thicknesses controlled by alleles at the SHELL locus (Sh). The alleles of Sh exhibit co-dominant monogenic inheritance, i.e., Dura (Sh/Sh) is thick-shelled, Pisifera (sh/sh) is shell-less, and Tenera (Sh/sh) has thin-shelled fruits. Pisifera palms are usually female-sterile. However, several Sh alleles are now known that confer a range of fruit and palm phenotypes. Verdant has germplasm with various Pisifera allelic constitutions: from homozygous combinations of AVROS (A/A), Nigeria (1/1), La Mé (3/3), Ghana (8/8), and heterozygous combinations (A/1, A/3, A/8, 1/3 and 1/8). Some heterozygous combinations of these Pisifera alleles are associated with fruit development and potential female fertility. Variation in morphology, fruit, and fertility is described for the allelic combinations of Sh giving the Pisifera phenotype. Fertile Pisiferas are potentially helpful for breeding and selection within the Pisifera germplasm. These phenotypes also provide information about different palm characters for the various Pisifera allelic combinations that may be useful when selecting parental lines for Pisifera breeding and commercial seed production.


Introduction
Narrowly based genetic resources of oil palm cause limitations in developing breeding programs.Commercial oil palms (Elaeis guineensis Jacq.) are generally classified into three types based on their fruit type, namely: Dura (kernel covered by a thick shell), Pisifera (no shell, kernel is "pea-like") and Tenera (thin shell)).The trait is controlled by a single gene (Sh) with Dura -Sh/Sh, Pisifera sh/sh, and Tenera Sh/sh.The alleles of the Sh are generally considered codominant [1].The Pisifera genotype (sh/sh) is usually associated with the female sterile phenotype.In commercial production of Teneras palms, it is used as the male (pollen) parent, with Dura being the female.
The Sh gene is probably the most critical in oil palm.It is a single gene that controls shell thickness but is likely to have pleiotropic effects on other traits, such as fertility and the underlying development of reproductive organs.Recently a range of alleles of Sh have been detected.These been classed as Pisifera alleles as they have no shells, and reduce fertility.These new alleles have potential in commercial Tenera seed production.Therefore, it is essential to study any pleiotropic effects that may impact commercial production.Characterization among different Pisifera Sh allele collections is essential.It is also interesting to study the interaction of the various allelic combinations.Phenotypic characterization to investigate the differences among Verdant's Pisifera collections has been carried out.Palm phenotypes were observed for vegetative traits (frond, petiole, and leaflet) and reproductive traits (bunch characters, female fertility, and fruit characteristics).In the case of female fertile Pisiferas, the fruits are shell-less with a "pea-like" kernel (hence the name).Fertile Pisiferas are of interest as they open the possibility of breeding and selection within the Pisifera gene pool.However, most Pisiferas are female sterile, and breeding for Pisifera improvement typically involves indirect approaches via Tenera sib-crosses.In addition, the shell-less kernel of fertile Pisiferas causes difficulties in seed propagation, meaning particular technologies are required for germination [3].

Materials and methods
Pisifera palms with nine different allelic constitutions were studied: AVROS (A/A), Nigeria (1/1), La Mé (3/3), Ghana (8/8), AVROS/Nigeria (A/1), AVROS/La Mé (A/3), AVROS/Ghana (A/8), Nigeria/La Mé (1/3) and Nigeria/Ghana (1/8).Phenotypic observations were carried out on mature palms at Timbang Deli Estate (Verdant Bioscience, Indonesia).At least four palms were observed for each Pisifera allelic constitution.Qualitative parameters were determined by adopting descriptive guidelines from East Asia Plant Variety Protection Forum [4] and included: palm canopy, palm height, trunk diameter, frond, rachis, petiole, and leaflet morphologies.Frond 17 was used for estimating leaf characters as described by Corley et al., [5].Female bunches were taken to record the number of fruits (especially kernels with an embryo).Corley and Tinker [6] classified fertile Pisifera palms as those producing many normal fruits and shell-less kernels.Table 1 shows there are differences in palm canopy for Pisifera allelic constitutions.Pisiferas which homozygous for the Ghana allele (8/8) shows a drooping canopy, as do the heterozygotes for the Ghana allele, i.e., 1/8 and A/8.All other Pisiferas showed a spreading canopy (figure 1).All of the Pisiferas studied also have leaflets at the frond's tip, as shown in Figure 2. In comparison, the absence of the leaflet at the frond's tip is shown by EAPVPF [4] (figure 3).Based on the data in Table 2, all Pisiferas showed medium width and depth of the petiole.The density of spines in the petiole varies: the homozygotes of La Mé (3/3) and Ghana (8/8) have a high density of spines, while in AVROS homozygotes (A/A) and heterozygotes (A/1 and A/3), they are sparse.There are differences in petiole color between the Pisifera genotypes.AVROS (A/A) and Ghana (8/8) homozygotes show a green petiole, Nigeria homozygote (1/1) shows a greenish-brown petiole, and homozygotes La Mé (3/3) has a brownish-green petiole and is long, as does the heterozygote 1/3 has brownish-green petiole also.However, the heterozygote 1/8 has a green petiole.Therefore, it seems appears that the "Green allele" in A/A and 8/8 are dominant to the other color alleles.The different petiole characters are shown in Figure 4. Regarding the leaflets, Pisiferas with La Mé 3/3 alleles have longer leaflets, but with medium width and leaflet number.However, Pisifera Nigeria (1/1) has narrow leaflet width.All of the Pisifera have green midrib color.Leaflet characters are shown in Figure 5, and the midrib is shown in Figure 6.Table 3 shows that only Pisifera 3/3 has a long bunch stalk (figure 7).Bunch shape varies among the Pisiferas from cordiform to ovate (figure 8) with no apparent pattern related to the Pisifera allele.Pisiferas female bunches with AVROS and La Mé alleles (A/A, 3/3, 1/3, A/1, A/3) have dense spines except for A/8, whereas all Pisiferas with the allele Ghana (8/8, 1/8, and A/8) show medium density.All Pisiferas studied have long spikes and oblong fruit shapes.The number of fertile fruit (with embryo present) per bunch varies.The presence of the embryo was checked by slicing the fruits.Table 3 shows the Pisifera that are homoallelic (A/A, 1/1, 3/3, 8/8) produced fruit but have no embryo.Kernels with embryos were only found in some Pisiferas with heterozygous alleles.Pisiferas with Nigeria/La Mé allele (1/3) had 8.2% fertile fruit with embryo, Pisiferas with AVROS/Nigeria allele (A/1) had 5.4%, and Pisiferas with AVROS/La Mé allele (A/3) had 18.2%.However, heteroallelic combinations with the Ghana allele i.e.Nigeria/Ghana (1/8) had sterile fruit and AVROS/Ghana (A/8) was mainly sterile, but showed 0.1% fruit with an embryo.Fertile Pisifera fruit are shown in Figure 9.

Discussion
In our study, different Pisifera allelic constitutions are associated with different phenotypic characters.Pisiferas are generally defined as being female sterile.In the case of female fertile Pisifera, the fruit is shell-less.The shell is an endocarp.It is a maternal trait that may be linked to a defect in the formation of the ovary and leading to female sterility.It is interesting in this respect that fertile fruits (with an embryo) are associated with a fiber ring (rudimentary endocarp) which indicates some ovule development (figure 9c).Morphological traits studied here were palm canopy, petiole, leaflet, and bunch traits.Pisiferas with the Ghana allele (8) was associated with a canopy character that tends to droop.Nigeria (1) and La Mé (3) alleles were associated with a brownish frond color.One possibility is that the alleles in AVROS (A) and Ghana (8) are dominant and give a green phenotype.La Mé is associated with long leaflets and bunch stalk.However, phenotypic performance is a combination of genotypes at the specific locus with genes in the background, and environmental influences to produce the observed phenotype.A study of phenotypic characters of different Sh alleles with the same or similar genotypic backgrounds is suggested.Also, oil palm is an ancient tetraploid [7] and a second (homoeologous) Sh locus cannot be ruled out.Fertile fruit found in some heteroallelic combinations is of interest as it potentially provides a new opportunity in breeding within the Pisifera germplasm.

Acknowledgments
The author would like to thank the Breeding and Biotechnology Departments of Verdant Bioscience for supporting the project, and also Orion Biosains for their services of Sh allele determination.This paper is published with the permission of the Boards of Directors of Verdant Bioscience Private Limited, Singapore, and PT Timbang Deli Indonesia, Indonesia.The authors also extend their appreciation to members and workers of the Breeding team for all assistance with trial observations.

Figure 3 .
Figure 3.Comparison of: the present and absent leaflets at the tip by EAPVPF [4].

Figure 6 .
Figure 6.Midrib color of: a) Ghana, b) AVROS, c) La Mé, and d) Nigeria.a b c d a b c d

Figure 7 .Figure 8 .
Figure 7. a) long stalk and b) medium stalk length Pisifera bunches.a b

Figure 9 .
Figure 9. Pisifera fruit: a. Parthenocarpy fruit (no shell/endocarp and no kernel), b.Fruit with the empty kernel, c.Fruit with kernel, d.Fruit with embryo.
[1] Babu B K, Mathur R K, Kumar P N, Ramajayam D, Ravichandran G, Venu M V B and Babu S S 2017 Development, identification and validation of CAPS marker for SHELL trait which governs dura, pisifera, and tenera fruit forms in oil palm (Elaeis guineensis Jacq.)PLoS One 12 1-16 [2] Singh R, Low E-T L, Ooi L C-L, Ong-Abdullah M, Ting N-C, Nookiah R, Ithnin M, Marjuni M, Mustaffa S, Yaakub Z, Amiruddin M D, Manaf M A A, Chan K-L, Halim M A A H, Sanusi M S N M, Lakey N, Sachdeva M, Bacher blaire, Garner P A, MacDonald J D, Smith S W, Wischmeyer C, Budiman M A, Beil M, Stroff C, Reed J, Brunt A van, Berg H, Ordway J M a b c d

Table 1 .
Palm traits of the various Pisifera allelic combinations.
Note: Medium refers to a comparison with other palms in the same block.

Table 2 .
Petiole and leaflet characters among the various allelic constitutions.

Table 3 .
Bunch and fruit traits of the various Pisiferas (percentages of fruit with/without kernel or embryo are given, all other fruits are parthenocarpic).