Breeding using parents of restricted origins

Breeding using parental lines of restricted origins (BPROs) is a term used to give a description of populations that can be traced back to specific origins. Breeding material available at Verdant is obtained via Dami Oil Palm Research Station (Papua New Guinea). BPROs have been conserved and, or developed independently by selfing, sib crossing, and inter-progeny crossing within the BPRO. Deli Dura is a well-known female breeding population along with the famous AVROS male lines. Other breeding materials being conserved, targeted, and developed by Verdant include: Calabar, Sibiti, and Ekona materials, plus other breeding materials initially received from the former Binga Research Station, Democratic Republic of Congo. Verdant is also exploiting several lines of La Mé material. Breeding using marker-defined genotypes is deployed to target breeding using selected parents. Progeny and palms with distinct genetic variation and complementary traits can be identified by in-depth genotyping (e.g., Diversity Array Technology, DArT marker analysis) – PCoA plots generated from DArT show distinct diversity clusters within and among breeding materials. AVROS and Deli Dura breeding materials are separated by relatively large genetic distances, which may account, in part, for their success in producing commercial Teneras. The PCoA plots also show that Deli Duras are distinct from Dabou and Angola Duras. Variation in Pisifera germplasm is associated with allelic variation at the SHELL gene (Sh). Various Sh allelic combinations are found in the BPRO materials: A/A, 1/1, and 8/8.


Introduction
Oil palm originates from Africa, where cultivation was first established and later spread to Southeast Asia during the colonialization era [1].Indonesia and Malaysia are the extensive palm oil producing country in the world and together contribute more than 80% of oil palm production.
Production of palm is determined by good planting material and agronomic practices.With respect to breeding, there is a massive gap between the potential production of oil palm and current production levels.The estimated potential production of oil palm is around 18 tonnes of palm products per Ha per year [2]; however, the average world yield is below four tonnes of palm products per Ha per year [3].
Deli Dura is the best-known female parent used to produce high-yielding varieties.However, low genetic variation within Deli Dura may limit the maximum productivity of its descendants [4].On the other hand, there is also variation in commercial male parents as these are derived from various populations.An early study reported the breeding of parental lines from restricted origins (BPROs); several populations were known as restricted origin material, such as Deli, Ekona, La Mé, AVROS, and Calabar [5].
Oil palm breeding is entering into a new era termed, "breeding by genotype" which is based on the genotypic selection using DNA markers.Since the discovery of the Sh gene that controls the shell thickness, this along with SSR, and SNP markers is now used routinely in breeding [6], [7], [8], [9].This genetic variation and its association with yield and other traits, allows breeders to predict and select winning crosses and accelerate breeding progress.In the future, "breeding by genotype" is expected also to target secondary traits of interest, e.g., disease and pest resistance, oil quality, and traits for mechanical harvesting.
In this paper, we describe and discuss the genetic variation found in and between BPROs.

Genetic Materials
Representatives of Verdant's oil collection were genotyped using Diversity Array Technology (DArT); in total, 327 progenies were analysed with 5 -11 individuals per population.See Table 1.Genotyping by SNP analysis was carried out using DArTseqLD (low density, Diversity Arrays Technology Pty Ltd., Canberra, ACT, Australia).Two thousand markers were used to determine the diversity of the samples.The data were analyzed using Hamming distance matrix to calculate the genetic differences between the samples.DArT techniques applied in this work are described by more detail in Kilian et al., 2012 [10].

Genetic Distinction Analysis
Raw data from DArTSeq was used to analyze genetic diversity, genetic clustering, and distance between palms and populations.Between the Dura populations, the distance matrix ranged from 0.0188 -0.2396 (low values indicate more remarkable similarity, while high values indicate more distinct differences).Table 2. shows selected populations from six BPRO (not all data are shown as over 197 progenies were analyzed).The populations selected (table 1) had the average similarity score for that BPRO.The highest similarity within a selected population was the DM Deli population, whereas the selected Ekona population had the most notable diversity.Variation between the selected populations was most remarkable between the Sibiti and Ekona BPROs.
Table 3. shows variations in genetic similarities among selected Pisifera populations (AVROS, Ghana, Calabar, and La Mé).A total of 130 progenies were analyzed, but Table 3 provides data for only a representative progeny for each of the four BPROs (based on the Pisifera alleles present).The variation between Pisifera populations ranged from 0.0277 -0.2692.The slightest variation was found in the AVROS population (0.0277), and the most notable diversity was found between AVROS and La Me BPROs (0.2692).

Principal Coordinates Analysis
The visualization of genetic diversity can be displayed using PCoA analysis [11]. Figure 1.displays seven clusters (A-F) for the BPROs germplasm analyzed.Each cluster represents a group of population.

Discussion
The DM Deli genotypes showed the highest similarities between the palms within all progenies tested.This may be a consequence of the wide use of DM Deli in breeding cycles, which may have promoted a genetic bottleneck.This presents a challenge for breeding, and DM Deli germplasm may be "improved" by introgression with the other Dura groups.The most remarkable genetic distinction was found between Ekona and Sibiti.
The AVROS Pisifera group also showed a narrow genetic base.However, variation was found between groups, with the most notable variation found in La Mé germplasm.Improvement of Pisiferas is not simple as these are often female sterile.Breeding for improvement usually involves extracting Pisiferas from Tenera sibs derived from crossing.
Genotyping of the Duras and Pisiferas derived from restricted origins has revealed similarities and distinctions.The material analyzed formed distinct clusters which generally corresponded to origins.Since these groups are associated with specific characteristics/traits, it is likely that further analysis will reveal markers associated with traits of interest.Of note, the DM Deli Duras and AVROS Pisiferas show the most distinct genetic compared to another restricted origin.This suggests that Teneras formed from Dura x AVROS will have a relatively high level of heterozygosity and may possess heterotic effects, such as high yield.From Table 4, DM Deli x AVROS produced the highest predicted diversity (0.2563).Secondary traits of interest are Ganoderma tolerance, long stalk, virescent, etc., and breeders aim to combine these with high yield.It may not be necessary to avoid crossing between genotypes with a narrow distance if attractive secondary traits can be combined.For example, crossing between Dabou Deli x La Mé has the potential to produce moderate Ganoderma tolerance also, the crossing between DM Deli Dura with Calabar Pisifera can produce virescent Teneras.However, these crosses are expected to have lower predicted yields than DM Deli x AVROS.
Realizing the potential of the Restricted Origins can be made by inter-crossing to introgress traits of interest.Table 1 shows that crossing between Ekona Dura x Sibiti Dura while Table 2 shows AVROS Pisifera and La Mé Pisifera may produce promising descendants based on genetic distance comparison.Genotypic data will allow breeders to predict winning crosses and to focus for crossing on germplasm with the highest potential.This will reduce the breeding cost as it will reduce the number of progenies that need to be field planted.As well as reducing the area needed for trials and, in the end, will reduce operational costs for running the breeding.However, the genotypic data must first to be correlated with traits, especially yield.

Genotyping Approaches in oil palm breeding
Markers for the Sh gene have had a massive impact on oil palm breeding in selecting Dura, Pisifera, and Tenera genotypes in segregating populations.Today several Pisifera alleles are known, and allelic combinations can be revealed (e.g., In the BPRO materials studied here, there are 4 Pisifera alleles: A=AVROS, 1=Nigeria, 3=La Mé, and 8=Ghana).A study on phenotypic associations of Pisifera alleles and allele combinations is reported in these proceedings [12].
Verdant aims to introgress traits of interest from BPRO materials but also preserves these as base populations as genetic resources for future breeding.
F1 Hybrid is a major breeding goal for Verdant.Many other crop species have been improved by the introduction of F1 Hybrids.The gap between current yield limits and potential yield in oil palm is large.Verdant's F1 Hybrids are discussed in the proceedings of this conference [13].

Conclusion
Oil palm breeding has entered the genomics era, and the determination of crossing programs is no longer limited to phenotypic data.It is undeniable that the use of genotypic data is a massive aid to breeders in determining winning crosses with additional benefits of accelerated breeding and cost and savings.However, the acid test is always performed in field trials and then in the plantation.

Acknowledgments
The staff of Verdant Bioscience carried out the practical work reported in this paper, while the genotypic data was obtained by DArT analysis (https://www.diversityarrays.com/).The paper is published with the permission of the Boards of Directors of Verdant Bioscience Private Limited, Singapore, and PT Timbang Deli Indonesia.

Figure 1 .
Figure 1.Display of genetic diversity using Principal Coordinate of Analysis.
TM, LGC Biosearch Technologies, UK).The DNA quality was assessed using gel electrophoresis.DNA samples were stored at -20 o C until required for DArTseq library construction.

Table 2 .
Genetic similarity/distinction among representative Dura populations (One selected progeny per BPRO).

Table 3 .
Genetic similarity/distinction between selected representative Pisifera populations (one progeny per population).

Table 4 .
Details of populations in clusters (A-G).

Table 5 .
Predicted genetic diversity from crosses between Dura and Pisifera groups.