WO2012123766A1 - Procédé d'obtention de souches généalogiques - Google Patents

Procédé d'obtention de souches généalogiques Download PDF

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Publication number
WO2012123766A1
WO2012123766A1 PCT/GB2012/050598 GB2012050598W WO2012123766A1 WO 2012123766 A1 WO2012123766 A1 WO 2012123766A1 GB 2012050598 W GB2012050598 W GB 2012050598W WO 2012123766 A1 WO2012123766 A1 WO 2012123766A1
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WO
WIPO (PCT)
Prior art keywords
tenera
palms
oil
dura
homozygous
Prior art date
Application number
PCT/GB2012/050598
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English (en)
Inventor
Brian Peter FORSTER
Peter Douglas Savaria Caligari
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Bioproperties Pte. Ltd
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Publication date
Application filed by Bioproperties Pte. Ltd filed Critical Bioproperties Pte. Ltd
Publication of WO2012123766A1 publication Critical patent/WO2012123766A1/fr

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Classifications

    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10Seeds
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Definitions

  • This invention relates to a process for obtaining breeding lines of oil palm, and to lines and hybrids obtainable thereby
  • Oil palm is a major world crop. It provided one-quarter of the world's production of vegetable oils in 2007. As well as providing edible oil, it has many other uses;
  • V ⁇ hybrids typically exhibit heterosis (hybrid vigour) and are genetically uniform, allowing optimization of agronomic practices (precision farming).
  • Fi hybrid varieties are created by large scale crossing between two complementary and homozygous parents to generate Fi hybrid offspring that are heterozygous, genetically uniform and elite. For example, in 1933, only about 1% of maize grown in the USA was hybrid, a value that has now increased to 95% due to the greatly increased yields achieved; during the period from 1920 to 2000, the yield of hybrid corn rose from about 25 to 140 bushels per acre.
  • telomeres are converted by conventional means (for example, colchicine treatment) to homozygous doubled haploids, suitable for use as homozygous parents in producing genetically uniform hybrids. Occasionally haploid palms will double spontaneously.
  • zygosity analysis indicates that a small proportion of the seeds from self or open pollinations are highly homozygous (HH), even completely homozygous. These (if necessary after further zygosity testing) are taken to be doubled haploids (DHs) or classed as highly homozygous depending on the extent of homozygosity (see definitions below). Both HH and DH lines are valuable for breeding purposes.
  • the thickness of the lignified endocarp (seed shell) of oil palm fruits is a property of great commercial importance. It is under control of a single gene. This gene has two allelic forms, referred to as Sh and sh.
  • the original cultivated oil palms all possessed thick endocarps and are termed 'dura ' . They are homozygous for the endocarp thickness gene, with genotype Sh/Sh.
  • the original cultivated dura palm varieties exhibited a low oil extraction ratio (OER) in the range of 12-16%.
  • sh a mutant allele of the endocarp thickness gene was later discovered, termed sh. This yielded virtually no lignified endocarp in its homozygous form, sh/sh, giving rise to a type of palm known as pisifera, but in the heterozygous state (Sh/sh) gave rise to an
  • Such palms are termed 'tenera'. Modern commercial oil palm varieties are all of the heterozygous 'tenera' type.
  • Pisifera palms are often female-sterile and therefore commercial crossing is done predominantly with dura as the female parent and pisifera as the male parent.
  • oil palms useful as breeding stock which comprises selecting non-tenera progeny from a population obtained by crossing dura with pisifera oil palm, and testing the selected progeny for homozygosity, discarding heterozygous oil palms.
  • the oil palms are also tested for ploidy, for example by flow cytometry, and only diploid palms are retained.
  • the retained palms are completely homozygous (doubled haploids).
  • Highly homozygous oil palm plants obtained by the process of the invention may be preserved and propagated as breeding lines by self-fertilization or by clonal propagation. They may be used to produce hybrids of uniform properties by crossing with other highly homozygous lines, which conveniently have complementary properties. Hybrids typically exhibit improved properties, e.g., hybrid vigour. Choosing the most suitable parent lines to cross in order to obtain superior properties is generally initially a matter of trial and error but can take into account complementary characteristics if data is available. Loci contributing to heterosis (hybrid vigour) may be found subsequently and aid parental selection.
  • Non-tenera progeny may be selected in one of two main ways: by phenotypic or genetic marker tests. At immature stages of palm development, it is generally not possible to distinguish between the phenotypes of tenera, dura and pisifera: it is necessary to grow the population to maturity (fruit production), at which stage the phenotypic differences in the shell thickness of fruit can readily be observed and non-tenera individuals selected. This may take 2-4 years after field planting: but since most of the population is tenera, and is useful directly as such, this does not impose much extra burden or inconvenience on the oil palm producer.
  • Figure 1 is a graph showing a homozygosity analysis of 140 palms comparing results obtained with 31 markers and with 111 markers;
  • Figure 2 is a block diagram of the process of analysing the samples
  • Figure 3 shows dura, tenera and pisifera fruit, in section.
  • non-tenera palms were found among the 50,899 screened. 153 of these non-tenera palms were then analysed according to the scheme shown in Figure 2. First they were tested for ploidy by flow cytometry, as described below. Flow Cytometry
  • Leaf samples of about 1 cm were taken from confirmed haploid and diploid plants with a chromosome count of 16 (n) and 32 (2n), respectively. These were used as standards.
  • Analyte preparation Cell suspensions were prepared according to the method of Arumuganathan and Earle (Arumuganathan K, Earle ED, Estimation of DNA contents of plants by Flow Cytometry, Plant Molecular Biology Reporter 1991; 9(3): 229-233, 1991) with the following modifications.
  • Leaf samples (about 1 cm ) were sliced by chopping with a sharp clean razor-blade (20-30 chops), in a plastic 9 cm diameter Petri dish containing 1.5 ml of cold (5 °C) CyStain® UV Ploidy solution modified by addition of 6.48 mM dithiothreitol (DTT) and 1% (v/v) polyvinylpyrrolidone (PVP-40) (Sigma- Aldrich, USA). The addition of DTT and PVP-40 were found to reduce background counts ('noise') in output histograms of particle fluorescence in the analyte. Slicing was done in low light conditions as DAPI is light sensitive.
  • DTT dithiothreitol
  • PVP-40 polyvinylpyrrolidone
  • the cell suspensions were then incubated at 5 °C for 1 hour in the dark. Each suspension was filtered through a 40 ⁇ Cell Tries® (Partec, Germany), sieved into a 3.5 ml x 12 mm Rohren tubes (Sarstedt, Germany) and a further 0.5 ml of cold (5 °C) CyStain® UV Ploidy solution was added. The final suspension was then mixed thoroughly by pumping analyte in and out of a pipette.
  • Ploidy results so obtained are shown in Table 1 below. 8 plants were found to be triploid, the remaining 147 being diploid. Plants identified as diploid were tested for zygosity.
  • the first screen used a cascade of 10 highly polymorphic microsatellite or simple sequence repeat (SSR) markers, which are shown in Table 2 below. Plants were tested with the first SSR marker: those found to be homozygous using this marker were taken forward to be tested with the second SSR marker: and so on until all 10 SSR markers had been used.
  • This set of 10 markers represents genetic loci on different chromosomes. Using this set of 10 markers, 29 out of the 153 non-teneras sampled proved to be homozygous and deemed candidate HH or DH (see Table 1 below).
  • HH highly homozygous
  • An individual palm is classified as highly homozygous (HH)when it is scored as having 20 unlinked homozygous loci where the parent with the same shell thickness phenotype is heterozygous at each locus analyzed. Since dura DHs 10 are expected to arise from a dura haploid set of chromosomes, only markers heterozygous in the dura parent are informative. Likewise, pisifera DHs require 20 heterozygous unlinked markers in the pisifera parent to be homozygous.
  • An individual palm is classified as doubled haploid (DH) when it has 28 unlinked homozygous loci regardless of the parental genotypes.
  • 'Unlinked' refers to loci that are on separate linkage groups (chromosomes) or where the minimum distance between any two loci in the same linkage group is
  • the class of palms that are highly homozygous includes the class of palms that are completely homozygous (DH, doubled haploid), doubled haploids being a special case of HH.
  • Table 3 gives probabilities of homozygous lines occurring by normal sexual processes (meiosis and fertilization). An individual is classed as HH when the probability of 20 or more unlinked markers, heterozygous in parental lines, all being homozygous is less than 1 in 1 million.
  • Figure 1 shows that using a relatively small number of markers (31) provides a good estimate of homozygosity: results from this set are highly correlated with data from a set of over 100 markers.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Botany (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Physiology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

La présente invention porte sur un procédé pour obtenir des palmiers à huile utiles en tant que stock de géniteurs, lequel procédé consiste à sélectionner une descendance non ténérale à partir d'une population obtenue par croisement de la dure-mère avec un palmier à huile pisifera, et à tester la descendance sélectionnée pour l'homozygosité, supprimant les palmiers à huile hétérozygotes. Les palmiers à huile peuvent également être testés pour la ploïdie, par exemple au moyen d'un cytomètre de flux, et seulement les palmiers diploïdes sont retenus. L'invention porte également sur un procédé pour produire des palmiers à huile hybrides génétiquement uniformes dans lequel une substance parente hautement homozygote est croisée avec un second parent hautement homozygote pour produire un hybride.
PCT/GB2012/050598 2011-03-17 2012-03-19 Procédé d'obtention de souches généalogiques WO2012123766A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB1104564.8 2011-03-17
GBGB1104564.8A GB201104564D0 (en) 2011-03-17 2011-03-17 Process for obtaining breeding lines

Publications (1)

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WO2012123766A1 true WO2012123766A1 (fr) 2012-09-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015010008A1 (fr) * 2013-07-18 2015-01-22 Malaysian Palm Oil Board Procédés de détection d'allèles shell de palmier à huile

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1972692A1 (fr) * 2007-03-19 2008-09-24 Sumatra Investment Corporation Pte. Ltd. Procedes de production de palmiers à huile haploides et haploides doublees
WO2008114000A1 (fr) 2007-03-19 2008-09-25 Sumatra Investment Corporation Pte. Ltd. Procédés de production de palmiers à huile haploïdes et dihaploïdes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1972692A1 (fr) * 2007-03-19 2008-09-24 Sumatra Investment Corporation Pte. Ltd. Procedes de production de palmiers à huile haploides et haploides doublees
WO2008114000A1 (fr) 2007-03-19 2008-09-25 Sumatra Investment Corporation Pte. Ltd. Procédés de production de palmiers à huile haploïdes et dihaploïdes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ARUMUGANATHAN K; EARLE ED: "Estimation of DNA contents of plants by Flow Cytometry", PLANT MOLECULAR BIOLOGY REPORTER, vol. 9, no. 3, 1991, pages 229 - 233

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015010008A1 (fr) * 2013-07-18 2015-01-22 Malaysian Palm Oil Board Procédés de détection d'allèles shell de palmier à huile

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