Patent Application
20220167576
The ASCII file, entitled 90442SequenceListing.txt, created on Dec. 21, 2021, comprising 47,024,652 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.
The present invention, in some embodiments thereof, relates to methods of enhancing grain yield, plants and products generated thereby.
Altering the panicle architecture of rice was a major breeding target to improve grain yield during green revolution (Zhu et al., 2013). Compact dense panicle with more primary and secondary rachis branches was preferred, resulting in more number of grains and thus increased yield (Sweeney and McCouch, 2007). Rachis branching pattern in rice mainly depends on the inflorescence meristem activity abortion. Also, delay in rachis branch meristem conversion into terminal spikelet meristem, and next order rachis branch formation lead to an altered number of secondary rachis branches (Ikeda-Kawakatsu et al., 2009).
Molecular genetics mechanisms underpinning rice panicle architecture identified yield-enhancing genes that were functionally validated using mutants or the transgenic approach (Ying et al., 2014). Genes belonging to growth-promoting hormones and transcription factor families were characterized, which influenced rachis branching and the number of grains in a panicle (Xing and Zhang, 2010). Such genes affecting the rice panicle architecture include MOC1 (Li et al., 2003), FZP (Komatsu et al., 2003), Gn1a (Ashikari et al., 2005), APO1 (Ikeda-Kawakatsu et al., 2009), DEP1 (Huang et al., 2009), SPL14 (Miura et al., 2010), LAX (Tabuchi et al., 2011), SPIKE (Fujita et al., 2013), TAWAWA1 (Yoshida et al., 2013), PAY1 (Lu et al., 2013), FUWA (Chen et al., 2015); Ghd7 (Weng et al., 2014), OsNAM12.1 (Dixit et al., 2015), GRF4 (Sun et al., 2016), and DWARF11 (Wu et al., 2016) among others.
Increase in grain number targeted through more secondary rachis branches can create a tradeoff between yield and grain quality (Mohapatra, 2013). Inferior grains (in the lower part of the panicle) formed due to secondary rachis branches have inordinate protein content, poor palatability and reduced milling quality due to high chalk as compared to the grains located on the primary rachis branches (Matsue et al., 1994; Matsue et al., 1995). Paradoxically, the number of secondary rachis branches is more important for yield than the number of primary rachis branches (Adriani et al., 2016). Increase in the number of grains on upper secondary rachis branches can improve grain quality including lower reduction in grain weight (Wang et al., 2008). The trade-off between yield-enhancing factors and maintaining better grain quality must be considered to ensure wider adoption of high yielding rice varieties (Das et al., 2018). Genes that increase the number of secondary rachis branches bearing grains that are not compromised for quality traits are thus desirable for a meaningful increase in yield.
According to an aspect of some embodiments of the present invention there is provided a method of enhancing grain yield of a Gramineae plant, the method comprising:
(a) crossing a recipient plant with a donor plant comprising a sequence variation in a TPR allele, the sequence variation imparting enhanced grain yield; and
(b) selecting a progeny plant of the crossing comprising the sequence variation by identifying said variation, the progeny plant being characterized by an improved grain yield relative to the recipient plant.
According to an aspect of some embodiments of the present invention there is provided a method of enhancing grain yield of a Gramineae plant, the method comprising:
(a) crossing a recipient plant with a donor plant comprising a TPR allele of haplotype I; and
(b) selecting a progeny plant of the crossing comprising the TPR allele of haplotype I by identifying said variation, the progeny plant being characterized by an improved grain yield relative to the recipient plant.
According to some embodiments of the invention, the recipient plant is heterozygous for the sequence variation or haplotype I.
According to some embodiments of the invention, the recipient plant is null for the sequence variation or haplotype I.
According to some embodiments of the invention, the selecting is based on phenotypic screening for a grain yield- and/or grain quality-associated trait.
According to some embodiments of the invention, the selecting is by marker-assisted selection.
According to some embodiments of the invention, the marker-assisted selection is by a method selected from the group consisting of: allele-specific hybridization; Southern analysis; Northern analysis; in situ hybridization, deep-sequencing; and hybridization of primers followed by polymerase chain reaction amplification of a region of a marker.
According to some embodiments of the invention, the method further comprises:
(c) backcrossing the one or more of selected progeny plants to produce backcross progeny plants; and
(d) selecting a backcross progeny plant comprising the sequence variation or haplotype I, the progeny plant being characterized by an improved grain yield relative to the recipient plant.
According to some embodiments of the invention, (c) and (d) are repeated one or more times.
According to some embodiments of the invention, the donor and/or recipient plant is a rice plant.
According to some embodiments of the invention, the recipient plant is a rice plant selected from the group consisting of: the Indica rice group; the Japonica rice group; and the Glaberrima rice group.
According to some embodiments of the invention, the donor plant is a rice plant of the Indica rice group.
According to some embodiments of the invention, the recipient plant is a rice plant of the Indica rice group.
According to some embodiments of the invention, the recipient rice plant is of a variety comprising a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P.
According to some embodiments of the invention, the method comprises breeding the recipient plant, donor plant or progeny plant to comprise a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P.
According to some embodiments of the invention, the recipient plant or the progeny comprises a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P is show in Table A.
According to some embodiments of the invention, the grain quality-associated trait is selected from the group consisting of amylose content, chalkiness, gel temperature, viscosity, retrogradation, and cooking traits such as the cooked grain length or width.
According to some embodiments of the invention, the grain yield is manifested by at least one of TSP, USRB and head rice yield.
According to an aspect of some embodiments of the present invention there is provided a plant obtainable according to the method as described herein.
According to an aspect of some embodiments of the present invention there is provided an elite line of a Gramineae plant comprising a sequence variation in a TPR allele or a haplotype I, the plant being characterized by an improved grain yield as compared to a control plant.
According to some embodiments of the invention, the recipient rice plant comprises a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P.
According to some embodiments of the invention the plant is a rice plant.
According to an aspect of some embodiments of the present invention there is provided a part of the plant (e.g., seed, grain) as described herein.
According to an aspect of some embodiments of the present invention there is provided a processed product comprising DNA of the plant as described herein.
According to some embodiments of the invention, the sequence variation comprises at least one SNP selected from the group of SNPs shown in
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.
In the drawings:
The present invention, in some embodiments thereof, relates to methods of enhancing grain yield, plants and products generated thereby.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
Rice is the staple for nearly half the global population. Ensuring food security by increasing crop yield of rice by meeting the demographic consumer preferences is critical. The classical tradeoff between yield increases for reduced grain quality is unacceptable in rice as value proposition defined through milling, visual and palatable qualities remain as economic drivers. The present inventors report 188 genetic loci interlinking several panicle architecture traits that increase yield and maintain grain quality as desired by rice traders and consumers. Multi-omics, single- and multi-locus GWAS, gene regulatory network and gene set analyses identified novel genes influencing secondary branching at upper and bottom part of the panicle, leading to increased superior grains. Multiple non-synonymous SNPs in a nuclear-pore anchor protein-encoding gene (OsTPR) were specifically associated with superior haplotypes of increased upper secondary rachis branches (USRB) and grain number. These grains maintained the quality traits including low chalk. This confirmed a critical role for OsTPR in influencing yield while maintaining quality and suggest directed evolution under artificial selection towards the desirable haplotype composition of the elite indica lines.
The present inventors identified additional genes D11, MOC1 or MYB4P or alleles thereof that can function optionally together with OsTPR in increasing yield.
Thus, according to an aspect there is provided a method of enhancing grain yield of a Gramineae plant, the method comprising:
(a) crossing a recipient plant with a donor plant comprising a sequence variation in a TPR allele, the sequence variation imparting enhanced grain yield; and
(b) selecting a progeny plant of the crossing comprising the sequence variation by identifying said variation, the progeny plant being characterized by an improved grain yield relative to the recipient plant.
According to an alternative aspect there is provided a method of enhancing grain yield of a Gramineae plant, the method comprising:
(a) crossing a recipient plant with a donor plant comprising a TPR allele of haplotype I; and
(b) selecting a progeny plant of the crossing comprising the TPR allele of haplotype I by identifying said variation, the progeny plant being characterized by an improved grain yield relative to the recipient plant.
So that the invention may be more readily understood, certain terms are first defined. Other definitions are provided throughout the description.
As used herein, the term “plant” refers to an entire plant, its organs (i.e., leaves, stems, roots, flowers etc.), seeds, plant cells, and progeny of the same. The term “plant cell” includes without limitation cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores. According to a specific embodiment, the plant is a plant line.
According to a specific embodiment the plant line is an elite line. The phrase “plant part” refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calli, and the like. According to a specific embodiment, the plant part comprises the nucleic acid variation in a TPR allele as described below. According to a specific embodiment, the plant part is a seed.
As used herein, the phrases “progeny plant” refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof.
As used herein the term “Gramineae plant” refers to the cereal grass family, which cultivated species include but are not limited to maize (corn), wheat, rice, barley, rye, sorghum or millet.
According to a specific embodiment the Gramineae plant is a cultivated plant.
As used herein the term “rice” refers to a cultivated Oryza plant.
As used herein the term “cultivated Oryza plant” refers to a cultivated grass rice species having a diploid genome, 2n=24 (AA genome). Examples of domesticated Oryza species include but are not limited to, Oryza sativa (Asian rice) or Oryza glaberrima (African rice). The term may be interchanged with the term rice.
Domesticated Oryza varieties contemplated herein according to exemplary embodiments refer to long grain, short grain, white, brown, red and black.
There are three main varieties of Oryza sativa:
Indica: The indica variety is long-grained.
Japonica: Japonica rice is short-grained and high in amylopectin (thus becoming “sticky” when cooked), and is grown mainly in more temperate or colder regions such as Japan.
Javanica: Javanica rice is broad-grained and grown in tropical climates.
Other major varieties include Aromatic and Glutinos.
Allele mining using 3k rice genomes revealed that within the indica subgroup the superior haplotype 1 was enriched while the inferior haplotype 3 was least represented (
According to a specific embodiment, the donor plant is Indica.
According to a specific embodiment, the recipient plant is Indica or Japonica.
Within each variety, there are many recipient cultivars, each favored for particular purposes or regions. Any genetic background of domesticated Oryza e.g., Oryza sativa, can be used. Other varieties and germplasms which can be used according to the present teachings are selected from the group consisting of: IR64; Nipponbare; PM-36, PS 36, Lemont, γS 27, Arkansas Fortuna, Sri Kuning, IR36, IR72, Gaisen Ibaraki 2, Ashoka 228, IR74, NERICA 4, PS 12, Bala, Moroberekan, IR42, Akihikari, Nipponbare, IR20, IR56, IR66, NSIC Rc158, NSIC Rc222, and NSIC Rc238.
As used herein the term “maize” is also interchangeably referred to as “corn” “Zea maize L.” or “Zea maize subsp.”
As used herein “cultivated maize” refers to the conventionally grown Zea mays for human or animal food or beverages or as a source of raw materials, food supplements, chemicals or fuel.
The maize plant is diploid (2N=20) in nature.
Any genetic background of Zea maize can be used. A number of commercial varieties are available including, but not limited to:
Zea mays var. amylacea (typically used for producing corn flower)
Zea mays var. everta (typically used for producing pop-corn)
Zea mays var. indentata (Dent corn)
Zea mays var. indurata (Flint corn)
Zea mays var. saccharata and Zea mays var. rugosa (Sweet corn)
Zea mays var. ceratina (Waxy corn)
Zea mays (Amylomaize)
Zea mays var. tunicata Larrañaga ex A. St. Hil (Pod corn)
Zea mays var. japonica (Striped maize)
As used herein the term “wheat” is also interchangeably referred to as “Triticum L.” or “Triticum subsp.”.
As used herein the term “common wheat” is also interchangeably referred to as “Bread wheat” or “Triticum aestivum”.
As used herein the term “durum wheat” is also interchangeably referred to as “Macaroni wheat” or “Triticum durum Desf.” or “Triticum turgidum subsp. durum”.
Wheat is conventionally grown for human or animal food or beverages or as a source of raw materials, food supplements, chemicals or fuel. The common wheat plant is allohexaploid (6N=42) in nature, whereas the durum wheat is a tetraploid (4N=28).
Any genetic background of Triticum can be used. A number of commercial varieties are available including, but not limited to:
T. aestivum (95% of the wheat production, also known as common wheat, typically used for producing flour for baking)
T. aethiopicum (commonly known as Ethiopian wheat)
T. araraticum (commonly known as Armenian or Araratian wild emmer)
T. boeoticum (commonly known as Einkorn wheat)
T. carthlicum (commonly known as Persian wheat)
T. compactum (similar to common wheat)
T. dicoccoides (commonly known as Emmer wheat, Farro, Hulled wheat)
T. dicoccon (commonly known as Emmer wheat, Farro, Hulled wheat)
T. durum
T. ispahanicum (commonly known as Emmer wheat, Farro, Hulled wheat)
T. karamyschevii (commonly known as Emmer wheat, Farro, Hulled wheat)
T. macha
T. militinae
T. monococcum (commonly known as Einkorn wheat)
T. polonicum (commonly known as Polish wheat)
T. spelta (commonly known as Dinkel wheat)
T. timopheevii (commonly known as Zanduri wheat)
T. turanicum
T. urartu (commonly known as Einkorn wheat)
T. vavilovii
T. zhukovskyi
The term “crossed” or “cross” in the context of this invention means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, i.e., when the pollen and ovule are from the same plant or from genetically identical plants).
Although the present description refers in a more detailed manner to rice, other species of Gramineae are equally envisaged.
According to a specific embodiment, the donor plant and the recipient plant are from the same species (e.g., rice/rice).
According to a specific embodiment, the donor plant and the recipient plant are from different species (e.g., rice/corn).
According to a specific embodiment, the donor plant and the recipient plant are from the same cultivar (e.g., indica/indica).
According to a specific embodiment, the donor plant and the recipient plant are from different cultivars (e.g., indica/japonica)
As used herein “enhancing” or “increasing” refers to an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1.5 fold, 2 fold or more as compared to a control of the same genetic background without the sequence variation (i.e., control plant e.g., the recipient plant).
As used herein “reducing” or “decreasing” refers to a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1.5 fold, 2 fold or more as compared to a control of the same genetic background without the sequence variation (i.e., e.g., the recipient plant).
As used herein “grain yield” refers to the measure of the yield of grains per unit area of land cultivation.
Grain yield can be measured in pounds per acre, typically at 12.0% moisture. Grain yield of rice can be determined by the number of panicles per unit area, the number of fertile florets per panicle, and/or grain weight per floret. According to a specific embodiment, the grain yield is manifested by an increase in total spikelet number (TSP) per panicle.
According to a specific embodiment, the grain yield is manifested by an increase in total number of secondary rachis branches (TSRB).
According to a specific embodiment, the grain yield is manifested by an increase in upper secondary rachis branches (USRB).
According to a specific embodiment, the grain yield is manifested by an increase in middle secondary rachis branches (MSRB).
According to a specific embodiment, the grain yield is manifested by an increase in total number of primary rachis branches (TPRB).
According to a specific embodiment, the grain yield is manifested by an increase in number of upper primary rachis branches (UPRB).
According to a specific embodiment a cross between the claimed haplotype (in Samba Mahsuri) to an OsMOC1 genotype the following values were obtained (see breeding scheme on
The determination of the plant architecture as mentioned by the above parameters can be done as follows: the length of each panicle starting from the first node until the last node is measured and divided by three—resulting to bottom, middle, and upper levels within the panicle. The sum of rachis branches based on category of primary or secondary is calculated for each level. This provides measurements for primary rachis branches (PRB) at the bottom (BPRB), middle (MPRB), and upper (UPRB) levels of the panicle. Similarly, this approach is used for secondary rachis branches (SRB) at the bottom (BSRB), middle, (MSRB), and upper (USRB) levels of the panicle.
According to a specific embodiment, the grain yield is manifested by an increase in percentage of head rice yield. The weight of head rice, expressed as a percentage of the original rough rice weight, is defined as the head rice yield (HRY) and may vary from 0, where all kernels are broken to a theoretical maximum of approximately 70% (no kernels are broken, 30% waste consisting of husk and bran). According to a specific embodiment, the grain yield is manifested by an increase in weight of filled spikelets per plant.
According to a specific embodiment, the grain yield is manifested by an increase in bulk grain weight per a predetermined number of plants.
According to a specific embodiment, the grain yield is manifested by an increase in number of panicles per plant.
According to a specific embodiment, there is an overall increase in spikelets (filled and unfilled) e.g., when superior alleles of the four central hub genes are pyramided (see
According to a specific embodiment, the increase in grain yield does not compromise grain quality.
Hence grain quality in a plant comprising the sequence variation in the TPR allele is about the same as or better than that of a control plant of the same genetic background without the variation.
As shown in Example 3, there is an association of TPR (OsTPR) with increased USRB and TSP without compromising the grain quality. Additionally, analysis of grain quality traits indicated that the amylose content, gel temperature, viscosity, retrogradation, etc., and cooking traits such as the cooked grain length and width (
Rice grain quality is typically composed of several factors, including grain appearance and milling, storing, eating, cooking, and nutritional qualities; appearance quality is mostly determined by grain shape and endosperm opacity (or chalkiness) [Zhang Q F. Strategies for developing green super rice. Proc Natl Acad Sci USA. 2007; 104:16402-16409. doi: 10.1073/pnas.0708013104; Wan X Y, Weng J F, Zhai H Q, Wang J K, Lei C L, Liu X L, Guo T, Jiang L, Su N, Wan J M. Quantitative trait loci (QTL) analysis for rice grain width and fine mapping of an identified QTL allele gw-5 in a recombination hotspot region on Chromosome 5. Genetics. 2008; 179:2239-2252. doi: 10.1534/genetics.108.089862.].
Embodiments of the invention therefore relate to any manifestation of a grain quality. Such include but are not limited to amylose content, chalkiness, gel temperature, trough, viscosity, breakdown, raw grain shape, raw grain length, raw grain width, retrogradation, and cooking traits such as the cooked grain shape, cooked grain length or width, final viscosity, peak viscosity, pasting temperature.
According to a specific embodiment, the quality trait refers to the raw grain and is manifested by chalkiness, grain size and/or grain shape.
Chalk. An opaque region of the rice kernel resulting from loose packing of the starch granules. Chalk may occur throughout or in a part of the kernel.
Chalkiness is a major constraint in rice production because it is one of the key factors determining grain quality and price. Its reduction is a major goal, and the primary purpose of this study was to dissect the genetic basis of grain chalkiness.
Chalkiness parameters include chalkiness rate, white core rate, white belly rate, chalkiness area, white core area, and white belly area
According to a specific embodiment, chalk is less than 5%.
According to a specific embodiment, the chalkiness of the progeny plant is similar to that of the recipient or better (i.e., reduced chalkiness by at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, as compared to the same genetic background not carrying the variation i.e., control).
As used herein “amylose content” which refers to percent apparent amylose content is typically measured by a continuous flow analyzer.
Apparent Amylose Percent. The most important grain characteristic that describes cooking behavior in each grain class, or type, i.e., long-, medium- and short-grain. The percentage of the endosperm starch of milled rice that is amylose. Standard long grains contain 20% to 23% amylose. Rexmont type long grains contain 24% to 25% amylose. Short and medium grains contain 16% to 19% amylose. Waxy rice contains 0% amylose.
According to a specific embodiment the amylose content is intermediate, i.e., 20-25%.
As used herein “gel temperature” or gel (gelatinization) temperature refers to the temperature at which rice starch granules imbibe water and lose crytallinity in an irreversible manner. It can be measured using the alkali spreading method. According to a specific embodiment, moderate gel temperature is preferred, as well known to the skilled artisan.
As used herein “viscosity” refers to the flow resistance of a rice flour suspension. It can be measured using a Rapid Visco Analyzer (RVA). According to a specific embodiment an intermediate viscosity is preferred as well known to the skilled artisan.
As used herein “retrogradation” refers to the appearance of starch clumps. Retrogradation refers to the crystallinization of amylose chains at room temperature which results to the hardening of rice after cooking. This parameter can be measured using RVA.
Grain Length (L). Length of a rice grain is measured in millimeters and can be measured using a SeedCount SC5000 Image Analyzer.
Grain Width (W). Width of a rice grain is measured in millimeters and can be measured using a SeedCount SC5000 Image Analyzer.
According to a specific embodiment the grain length and/or grain width are about the same as that of the control.
According to a specific embodiment, the grain characteristic is selected from high head rice recovery (HRR) and low chalkiness (less than 5%).
As mentioned the method involves transferring a TPR nucleic acid sequence comprising the superior allele (as an example for sequence variation) to a donor plant.
As used herein “translocated promoter region” gene or “TPR” belongs to the nuclear-pore complex that mediate the transport of RNA and other cargo between the nucleus and the cytoplasm. In Arabidopsis the protein was shown to play a role in RNA homeostasis, flowering time and auxin signaling.
The Arabidopsis thaliana homolog AtTPR (AT1G79280) is typically represented by the sequence SEQ ID NO: 9, NM_001198507, SEQ ID NO: 10 NP_178048.2, for the nucleotide and amino acid sequences, respectively. The amino acid sequence is shown in
The present inventors have identified the rice homolog OsTPR, represented by the sequence SEQ ID NO: 7 NC_029257.1, SEQ ID NO: 8 XP_015625577.1, for the nucleotide and amino acid sequences, respectively. The gene is located on chromosome 2: 31009161 to 31033605 bp; minus strand. The amino acid sequence is shown in
As used herein a sequence variation in a TPR allele, refers to at least one mutation that can be any of a deletion, substitution, deletion, insertion, inversion of one or more nucleotides in the OsTPR reference sequence.
The variation can be at least 90% identical, but not full identity with the OsTPR allele, in the DNA or protein level.
As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g. charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have “sequence similarity” or “similarity”. Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff J G. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A. 1992, 89(22): 10915-9].
Identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.
Hence according to a specific embodiment, the variation can arise from different cultivars of the species (e.g., indica to japonica) or different species.
According to some embodiments of the invention, the identity is a global identity, i.e., an identity over the entire amino acid or nucleic acid sequences of the invention and not over portions thereof.
As mentioned, this variation that may be of a single base to several hundreds or thousands of nucleotides e.g., 1-200 nucleotides confers the enhanced grain yield.
Methods of validating such variations and their effect in the phenotypes are well known in the art and described in length in the Examples section which follows.
The at least one mutation may be in the coding sequence.
Alternatively or additionally, the at least one mutation may be in the non-coding sequence.
According to a specific embodiment, the sequence variation comprises at least one single nucleotide polymorphism (SNP) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least two single nucleotide polymorphisms (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least three single nucleotide polymorphisms (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least three single nucleotide polymorphisms (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least four single nucleotide polymorphisms (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least five single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least six single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least seven single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least eight single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least nine single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least ten single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least ten to 20 single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least ten to 30 single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation comprises at least ten to 50 single nucleotide polymorphism (SNPs) selected from the group of SNPs of TPR and optionally other genes shown in Table A below.
According to a specific embodiment, the sequence variation is as set forth in haplotype I (AGGATCA, SEQ ID NO: 1).
According to a specific embodiment haplotype I and genetic variations in other genes are provided in Table A below:
Thus, the SNP can be located in an intron, a regulatory (e.g., promoter), and/or a coding region.
The at least one mutation can be in a functional domain. An example of a functional domain may be a promoter, a TPR domain, a binding region for SPL3/SPL14.
Exemplary SNPs include, but are not limited to:
snp_02_31010249, snp_02_31013745, snp_02_31019010 (SNP within the TPR domain), snp_02_31023080, snp_02_31024549, snp_02_31026954, snp_02_31035009 (SNP in promoter region).
As used herein “haplotype” relates to a set of SNP alleles that tend to always occur together (i.e., that are associated statistically).
The at least one mutation can be in a structural domain of the DNA, RNA or protein.
Haplotype and gene structure in
Now the superior haplotype, AGGATCA, is a mixture of both reference and alternative alleles. Here, the alleles in bold (A and C) are from the reference while the ones not in bold are alternative alleles. The underlined allele (T) is an alternative allele which refers to the SNP which has both a missense and splice variant annotation (snp_02_31024549). The r-square values of snp_02_31024549 with the neighboring reference alleles, snp_02_31023080 and snp_02_31026954, are 0.623 and 0.564, respectively. Most of the seven SNPs have high D′ (0.986 to 1.0) but the r-square values are variable (0.56 to 0.986).
Motif connection with haplotype SPL3/SPL14 in Arabidopsis bind specifically to the consensus nucleotide sequence 5′-TNCGTACAA-3′. For motif searching, the input sequence consists of a stretch of 31 bp, having the upstream SNP, snp_02_31035009 (G/A), in the middle (15th SNP) and is reverse transcribed for the software used. Snp_02_31035009 overlaps with the eighth position of the motif (5′-TNCGTACAA-3′) having either T(A) for lines with high USRB (HUSRB) or C(G) for lines with low USRB (LUSRB). The alleles in parentheses are the reverse complement of the nucleotides in the genomic sequence. These alleles are colored in orange in the haplotype sequence, with A for HUSRB lines and G for LUSRB lines.
As used herein a “donor plant” which may also be referred to as a “non-recurrent parent” comprises the variation in the TPR gene.
According to a specific embodiment, Samba Mahsuri is a donor plant.
As used herein “a recipient plant” which may also be referred to as a “recurrent parent” refers to a cultivar e.g., an elite line, that could be made better (e. in terms of yield) by adding sequence variation.
The recipient plant may not comprise the variation at all, accordingly the recipient plant is null for said sequence variation, e.g., haplotype I.
According to other embodiments, the recipient plant may comprise a single allele or more but not all alleles which comprise the variation, in such a case the plant is heterozygous for the variation. In certain case, such a plant may be simply subjected to selfing to render it homozygous for the variation.
Accordingly, the recipient plant is heterozygous for the sequence variation e.g., haplotype I.
As mentioned, once progeny plants are selected for the trait, the progeny may be crossed to the recurrent parent (it is ‘crossed back’ to the recurrent parent, hence the term backcross). The progeny of this cross is selected for the trait of interest and then crossed back to the recurrent parent. This process is repeated for as many backcrosses as are needed to create a line that is the recurrent parent with the sequence variation from the donor parent. The goal of backcrossing is to obtain a line as identical as possible to the recurrent parent with the addition of the sequence variation that has been added through breeding.
According to an embodiment of the invention, the recipient rice plant may comprise a genetic background that comprises a sequence variation in a gene selected from the group consisting of D11, MOC1 and MYB4P (D11, MOC1, MYB4P, D11+MOC1, D11+MYB4P, MOC1+MYB4P, D11+MYB4P+MOC1). The present inventor identified a superior phenotype in terms of yield when variations in any of these genes were added to the TPR allele variation (see
MOC1 encodes a GRAS family transcription regulator which controls tillering, number of rachis branches, and spikelets in rice (Li, X., Qian, Q., Fu, Z., Wang, Y., Xiong, G., Zeng, D., Wang, X., Liu, X., Teng, S., Hiroshi, F., Yuan, M., Luo, D., Han, B., Li, J. 2003. Control of tillering in rice. Nature. 422: 618-621. DOI:10.1038/nature01518). An exemplary sequence is provided in SEQ ID NO: 13 or 14.
MYB4P encodes an R2R3 MYB family transcription factor which plays a role in cell differentiation, abiotic response, and phosphate acquisition (www(dot)uniprot(dot)org/uniprot/Q2R2L6; www(dot)ncbi(dot)nlm(dot)nih(dot)gov/pubmed/24813725) Yang, W. T., Baek, D., Yun, D. J., Hwang, W. H., Park, D. S., Nam, M. H., Chung, E. S., Chung, Y. S., Yi, Y. B., Kim, D. H. 2014. Overexpression of OsMYB4P, an R2R3-type MYB transcriptional activator, increases phosphate acquisition in rice. Plant Physiol Biochem. 80:259-67. doi: 10.1016/j.plaphy.2014.02.024. An exemplary sequence is provided in SEQ ID NO: 15 or 16.
According to a specific embodiment the recipient rice plant is of a variety comprising a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P.
According to a specific embodiment the method further comprises breeding the recipient plant, donor plant or progeny plant to comprise a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P.
According to a specific embodiment the recipient plant or said progeny comprises a sequence variation in a gene selected from the group consisting of D11, MOC1, and MYB4P is shown in
Such genetic backgrounds are available in the IRIS series shown in
Exemplary haplotypes are shown in Table A above.
Other sequence variations are described in
According to an additional or an alternative embodiment, the recipient plant is characterized by high amylose content (e.g., comprising a sequence variation which reduces expression of a starch branching enzyme IIb (sbeIIb, e.g., LOC_Os02g32660)), and optionally a sequence variation in MOC1. Such an exemplary line is IR36ae. According to a specific embodiment, the cross is between Samba Masuri and IR36ae (see
Numerous ways are known in the art for crossing Gramineae plants or species thereof. The following refers to rice but in a non-limiting manner such that it can encompass other Gramineae or crosses therebetween (interspecific crossing).
Thus, according to one embodiment, for producing a rice plant with improved yield, protoplast fusion can be used for the transfer of nucleic acids from a donor plant to a recipient plant. Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells of which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi-nucleate cell. The fused cell, that may even be obtained with plant species that cannot be interbred in nature, is tissue cultured into a hybrid plant exhibiting the desirable combination of traits. More specifically, a first protoplast can be obtained from a rice plant or other plant line that exhibits improved grain yield. For example, a protoplast from a superior Indica line e.g., IR64. A second protoplast can be obtained from rice or other plant variety, preferably a popular Japonica rice cultivar e.g., Nipponbare. Additionally, the second protoplast may be from a rice variety that comprises commercially desirable characteristics, such as, but not limited to disease resistance, insect resistance, weed resistance, etc. The protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art.
Alternatively, embryo rescue may be employed in the transfer of a nucleic acid comprising the TPR variation as described above from a donor plant to a recipient plant. Embryo rescue can be used as a procedure to isolate embryos from crosses wherein plants fail to produce viable seed. In this process, the fertilized ovary or immature seed of a plant is tissue cultured to create new plants.
Traditional breeding techniques can also be used to introgress a nucleic acid sequence of the variation in the TPR allele into a recipient rice plant in which a higher grain yield is desirable. In one method, related to pedigree breeding, a donor rice plant comprising a nucleic acid sequence variation as discussed is crossed with a rice plant in which a higher grain yield is desirable. The resulting plant population (representing the F1 hybrids) is then self-pollinated and set seeds (F2 seeds). The F2 plants grown from the F2 seeds are then screened for improved grain yield and optionally grain quality. The population can be screened or selected for improved grain yield in a number of different ways. For example, the population can be screened by field evaluation over several seasons.
An embodiment relates to a rice plant having improved grain yield, or part thereof, comprising within its genome the TPR variation, wherein the variation is not in its natural genetic background. The rice plants having improved grain yield described herein can be of any genetic type such as inbred, hybrid, haploid, dihaploid. Further, the plants of the present invention may be heterozygous or homozygous for the variation. As mentioned, although the TPR variation may be transferred to any rice plant in order to provide for a plant having improved grain yield, the methods and plants described herein are related to Gramineae family.
Inbred rice lines having improved grain yield can be developed using the techniques of recurrent selection and backcrossing, selfing and/or dihaploids or any other technique used to make parental lines. In a method of selection and backcrossing, improved grain yield can be introgressed into the recipient plant (which as mentioned is also termed “a recurrent parent”) by crossing the recurrent parent with a first donor plant (which is different from the recurrent parent and referred to herein as the “non-recurrent parent”). The recurrent parent is a plant in which an increase in grain yield is desirable. Optionally, the recurrent parent possesses commercially desirable characteristics, such as, but not limited to disease resistance, insect resistance, weed resistance, etc. The non-recurrent parent comprises a nucleic acid sequence with the TPR sequence variation. The non-recurrent parent can be any plant variety or inbred line that is cross-fertile with the recurrent parent (or fertilization may be aided by means which are well known in the art). The progeny resulting from a cross between the recurrent parent and non-recurrent parent are backcrossed to the recurrent parent. The resulting plant population is then screened (also termed here as “selected”). The population can be screened in a number of different ways. F1 hybrid plants that exhibit improved grain yield and comprise the requisite nucleic acid sequence for the variation are then selected and selfed and selected for over a number of generations in order to allow for the rice plant to become increasingly inbred. This process of continued selfing and selection can be performed for zero to five or more generations. The result of such breeding and selection is the production of lines that are genetically homogenous for the variation associated with improved grain yield as well as other genes associated with traits of commercial interest.
Instead of using phenotypic screens, marker assisted selection (MAS) can be performed using one or more of the herein described molecular markers (e.g., for haplotype I), hybridization probes, or polynucleotides to identify those progeny that comprise the nucleic acid variation. The MAS is used for by identifying said variation.
Molecular markers can include restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), single nucleotide polymorphisms (SNP) or simple sequence repeats (SSR). A primary motivation for development of molecular markers in crop species is the potential for increased efficiency in plant breeding through marker assisted selection (MAS) and marker assisted backcrossing (MABC). Genetic marker alleles are used to identify plants that contain a desired genotype at one or more loci and that are expected to transfer the desired genotype, along with a desired phenotype to their progeny. Genetic marker alleles can be used to identify plants that contain a desired genotype at one locus or at several unlinked or linked loci (e.g., a haplotype) and that would be expected to transfer the desired genotype, along with a desired phenotype to their progeny.
After a desired phenotype, e.g., improved grain yield and a polymorphic chromosomal locus are determined to segregate together, it is possible to use those polymorphic loci to select for alleles corresponding to the desired phenotype: a process called marker-assisted selection (MAS). In brief, a nucleic acid corresponding to the marker nucleic acid is detected in a biological sample from a plant to be selected. This detection can take the form of hybridization of a probe nucleic acid to a marker, e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, or the like. A variety of procedures for detecting markers are described herein. After the presence (or absence) of a particular marker and/or marker allele in the biological sample is verified, the plant is selected, i.e., used to make progeny plants by selective breeding.
Screening a large number of plants for improved grain yield can be expensive, time consuming and unreliable. Use of the genetically-linked nucleic acids described herein as genetic markers for improved grain yield is an effective method for selecting plants capable of fertility restoration in breeding programs. For example, one advantage of marker-assisted selection over field evaluations for improved grain yield is that MAS can be done at any time of year regardless of the growing season. Moreover, environmental effects are irrelevant to MAS.
Another use of MAS in plant breeding is to assist the recovery of the recurrent parent genotype by backcross breeding. Backcross breeding is the process of crossing a progeny back to one of its parents. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent into an otherwise desirable genetic background from the recurrent parent. The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting variety. This is often necessary, because donor parent plants may be otherwise undesirable. In contrast, varieties which are the result of intensive breeding programs may merely being deficient in one desired trait such as improved grain yield. Backcrossing can be done to select for or against a trait.
Markers corresponding to genetic polymorphisms between members of a population can be detected by numerous methods, well-established in the art (e.g., restriction fragment length polymorphisms, isozyme markers, allele specific hybridization (ASH), amplified variable sequences of the plant genome, self-sustained sequence replication, simple sequence repeat (SSR), single nucleotide polymorphism (SNP) or amplified fragment length polymorphisms (AFLP)).
The majority of genetic markers rely on one or more properties of nucleic acids for their detection. For example, some techniques for detecting genetic markers utilize hybridization of a probe nucleic acid to nucleic acids corresponding to the genetic marker. Hybridization formats include but are not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays. Markers which are restriction fragment length polymorphisms (RFLP), are detected by hybridizing a probe (which is typically a sub-fragment or a synthetic oligonucleotide corresponding to a sub-fragment of the nucleic acid to be detected) to restriction digested genomic DNA. The restriction enzyme is selected to provide restriction fragments of at least two alternative (or polymorphic) lengths in different individuals and will often vary from line to line. Determining a (one or more) restriction enzyme that produces informative fragments for each cross is a simple procedure, well known in the art. After separation by length in an appropriate matrix (e.g., agarose) and transfer to a membrane (e.g., nitrocellulose, nylon), the labeled probe is hybridized under conditions which result in equilibrium binding of the probe to the target followed by removal of excess probe by washing. Nucleic acid probes to the marker loci can be cloned and/or synthesized. Detectable labels suitable for use with nucleic acid probes include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes and colorimetric labels. Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents and enzymes. Labeling markers is readily achieved such as by the use of labeled PCR primers to marker loci.
The hybridized probe is then detected using, most typically, autoradiography or other similar detection technique (e.g., fluorography, liquid scintillation counter, etc.). Examples of specific hybridization protocols are widely available in the art.
Amplified variable sequences refer to amplified sequences of the plant genome which exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. Once identified, the presence of specific variable sequence can be used to predict phenotypic traits. Preferably, DNA from the plant serves as a template for amplification with primers that flank a variable sequence of DNA. The variable sequence is amplified and then sequenced.
In vitro amplification techniques are well known in the art. Examples of techniques include in vitro methods, including the polymerase chain reaction (PCR), the ligase chain reaction (LCR), O,.beta.-replicase amplification and other RNA polymerase mediated techniques (e.g., NASBA). Essentially any RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase.
Oligonucleotides for use as primers, e.g., in amplification reactions and for use as nucleic acid sequence probes, are typically synthesized chemically according to the solid phase phosphoramidite triester method, or can simply be ordered commercially.
Alternatively, self-sustained sequence replication can be used to identify genetic markers. Self-sustained sequence replication refers to a method of nucleic acid amplification using target nucleic acid sequences which are replicated exponentially in vitro under substantially isothermal conditions by using three enzymatic activities involved in retroviral replication: (1) reverse transcriptase, (2) Rnase H and (3) a DNA-dependent RNA polymerase. By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target.
There are many different types of molecular markers, including amplified fragment length polymorphisms (AFLP), allele-specific hybridization (ASH), single nucleotide polymorphisms (SNP), simple sequence repeats (SSR) and isozyme markers. SSR data is generated by hybridizing primers to conserved regions of the plant genome which flank the SSR sequence. PCR is then used to amplify the repeats between the primers. The amplified sequences are then electrophoresed to determine the size and therefore the di-, tri and tetra nucleotide repeats.
The presence of the variation in the genome of a plant exhibiting a preferred phenotypic trait is determined by any method listed above, e.g., RFLP, AFLP, SSR, etc. If the nucleic acids from the plant are positive for a desired genetic marker, the plant can be selfed to create a true breeding line with the same genotype or it can be crossed with a plant with the same marker or with other desired characteristics to create a sexually crossed hybrid generation.
As mentioned, selection can be also or alternatively based on phenotypic screening for a grain yield- and/or grain quality-associated trait, such as described above.
Once the appropriate selections are made, the process is repeated. The process of backcrossing to the recurrent parent and selecting for improved grain yield (or the variation or both) is repeated for approximately 2-5 or more generations. The progeny resulting from this process are heterozygous for the variation. The last backcross generation can then be selfed in order to provide for homozygous pure breeding progeny for improved grain yield.
The rice lines having improved grain yield described herein can be used in additional crossings to create hybrid plants having improved grain yield. For example, a first inbred rice plant having improved grain yield produced by methods described herein can be crossed with a second inbred rice plant possessing commercially desirable traits such as, but not limited to, disease resistance, insect resistance, weed resistance, etc. This second inbred rice line may or may not have relatively improved grain yield.
Regardless of the selection method, following trait selection and backcrossing the genome of the cultivated Gramineae plant e.g., rice plant of the recurrent parent is recovered to at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98%. That is, the plant of the invention has a genome being at least 85%, e.g., 85-99.9% that of the recurrent parent e.g., Oryza sativa.
Also provided are such methods, wherein the recovery of the recurrent rice plant's genome (e.g., Oryza sativa) is between 92% and 97%.
A rice plant having improved grain yield, or a part thereof, or an elite line obtainable by a method described herein is also an embodiment of the present invention.
According to an embodiment, the plant is not Samba Mahsuri.
According to a specific embodiment, the plant is not a breeding line.
According to a specific embodiment, the plant is not any of the below lines:
Also contemplated is an elite line of a Gramineae plant comprising a sequence variation in a TPR allele or a haplotype I, the plant being characterized by an improved grain yield as compared to a control plant (excluded are the lines described above).
Also provided are processed rice products which are produced from the plants described herein and preferably contain the nucleic acid sequence conferring the improved out-crossing rate described herein. Also provided are methods of processing the rice (e.g., to produce meal) or other processed products.
Food Characteristics:
Rice starch is a major source of carbohydrate in the human diet, particularly in Asia, and the grain of the invention and products derived from it can be used to prepare food. The food may be consumed by man or animals, for example in livestock production or in pet-food. The grain derived from the rice plant can readily be used in food processing procedures, and therefore the invention includes milled, ground, kibbled, cracked, rolled, boiled or parboiled grain, or products obtained from the processed or whole grain of the rice plant, including flour, brokers, rice bran and oil. The products may be precooked or quick-cooking rice, instant rice, granulated rice, gelatinized rice, canned rice or rice pudding. The grain or starch may be used in the production of processed rice products including noodles, rice cakes, rice paper or egg roll wrapper, or in fermented products such as fermented noodle or beverages such as sake. The grain or starch derived therefrom may also be used in, for example, breads, cakes, crackers, biscuits and the like, including where the rice flour is mixed with wheat or other flours, or food additives such as thickeners or binding agents, or to make drinks, noodles, pasta or quick soups. The rice products may be suitable for use in wheatfree diets. The grain or products derived from the grain of the invention may be used in breakfast cereals such as puffed rice, rice flakes or as extruded products.
Dietary Fiber:
Dietary fiber, in this specification, is the carbohydrate and carbohydrate digestion products that are not absorbed in the small intestine of healthy humans but enter the large bowel. This includes resistant starch and other soluble and insoluble carbohydrate polymers. It is intended to comprise that portion of carbohydrates that are fermentable, at least partially, in the large bowel by the resident microflora.
Non-Food Applications:
Rice is widely used in non-food industries, including the film, paper, textile, corrugating and adhesive industries, for example as a sizing agent. Rice starch may be used as a substrate for the production of glucose syrups or for ethanol production.
DNA detection in the processed products can be performed using methods which are well known in the art and are described in some detail hereinabove.
It will be appreciated that the present teachings contemplate the protection of cultivated Gramineae plants such as cultivated rice plants and will not in any way encompass wild Gramineae per se.
Applicant notes that all varieties designated IRIS are not restricted for use and can be provided by Applicant to 3rd party for research purposes under a Standard Material Transfer Agreement (“SMTA”).
It is expected that during the life of a patent maturing from this application many relevant markers will be developed and the scope of the term marker is intended to include all such new technologies a priori.
As used herein the term “about” refers to ±10%.
The terms “comprises”, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
The term “consisting of” means “including and limited to”.
The term “consisting essentially of” means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
As used herein, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof.
Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
As used herein the term “method” refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.
Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, “Molecular Cloning: A laboratory Manual” Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique” by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.
Phenotype Data Measurement and Transformation
A total of 310 diverse resequenced indica rice accessions grown in the experimental station of the International Rice Research (IRRI), Laguna, Philippines (14° N, 121° E) during the 2015 dry season was harvested before full grain ripening, and then phenotyped using Panicle Traits Phenotyping (P-TraP) Tool (Al-Tam et al. 2013). The phenotype values were transformed using WarpedLMM (Fusi et al. 2014), a transformation tool which ensures Gaussian distribution of residuals by incorporating all available covariates and genotype profile (i.e. individual genetic loci and polygenic genetic background) of each accession in order to increase GWAS power in identifying important genomic regions and increase the accuracy in estimating heritability and predicting phenotypic values.
Genotype Data Filtering
Genotype data consisting of 15,099,561 bi-allelic SNPs from The 3,000 Genomes Rice Project was filtered for the 310 resequenced indica accessions using PLINK 1.9 (Chang et al. 2015). Markers with missing call rate more than 0.20% were excluded, followed by samples with missing call rate more than 0.20%. Moreover, variants with minor allele frequency less than 10% were filtered out to include only common variants and to control false-positive results (Tabangin et al., 2009). A total of 747,087 high quality SNPs were retained from 309 samples after filtering.
Single Locus GWAS Analysis
The marker-based Balding-Nichols kinship matrix was calculated through the Efficient Mixed-Model Association eXpedited (EMMAX) ver. Beta (Kang et al. 2010) using the emmax-kin function. The kinship matrix was considered as covariate while the SNPs were treated as fixed effects for the variance component linear mixed model implemented in EMMAX. The EMMAX associations between the filtered genotype data and transformed phenotype data for each trait were then computed. Manhattan and QQ plots were created using R (ver. 1.0.153) (R Core Team, 2018) in order to visualize the significant genomic regions and assess the reliability of the EMMAX associations, respectively. The red line represents the Bonferroni threshold (7.17) which assumes independence among all the tests (Tabangin et al., 2009). However, this correction method is extremely stringent and renders high rate of false negative results; hence, it reduces true discovery rate. In this study, SNPs with q-value less than 0.05 were considered to be significant in order to increase the discovery rate and control the rate of false positive results (Lin and Lee, 2015). The blue line in the Manhattan plot represents the SNP with the highest significant p-value whose q-value is still less than 0.05. All SNPs within the red and blue lines in the Manhattan plot were considered to be significant.
PLINK 1.9 (Chang et al. 2015) software and HaploView (Barrett et al., 2005) software were used to define haplotype blocks with regions inherited without significant recombination in the diversity population. Haplotype blocks were determined using the blocks function in PLINK 1.9. HaploView was used to determine tag SNPs based on LD coefficient (D′≥0.80). The tag SNPs per block and the haplotype groups formed therewith were further visualized in boxplots for USRB.
Gene-Level and Gene-Set Analyses
Gene and gene-set analysis were performed using Multi-marker Analysis of GenoMic Annotation (MAGMA) ver. 1.06 in order to simultaneously determine the joint effect of multiple markers (de Leeuw et al. 2015). All significant SNPs previously identified from GWAS results were mapped to genes from the Rice Genome Annotation Project (MSU v7) based on genomic location (Kawahara et al. 2013). The SNP-wise mean model was used in MAGMA for the gene-level analysis, incorporating the p-values of SNPs computed from EMMAX and the filtered genotype data to estimate the LD between SNPs. After computing for the correlations between neighbouring genes and other gene-level metrics, competitive gene-set analysis was implemented as a linear regression model in order to test whether or not the mean association of the genes in the gene-set would be greater than that of the genes not in the gene-set. This approach used to create different sets of genes by removing one gene from the set of the top ten genes based on p-value for every gene-set.
Multi-Locus GWAS Analysis
Multi-locus GWAS was performed using Multi-Locus Random-SNP-Effect Mixed Linear Model (mrMLM) function in R, as described in Misra et al. (2018). Inputs were transformed phenotype data, filtered genotype data, and kinship matrix for each panicle trait: TSP, DSP, BRSB, DSBRB, MRSB, DMSRB, USRB, DUSRB, TSRB, and TPRB. The SNP data used in slGWAS was re-coded into ped and map files using recode function in PLINK 1.9, and then converted into diploid hapmap file using TASSELS (Bradbury et al., 2007). The methods implemented were mrMLM and FAST multi-locus random-SNP-effect EMMA (FASTrEMMA) considering a critical LOD score equals 3 for significant quantitative trait nucleotide (QTN), a search radius of 20 to identify potentially associated QTN, and restricted maximum likelihood (REML) for the likelihood parameter in FASTrEMMA. The QTNs identified with mlGWAS methods were validated by slGWAS results.
Targeted Association Analysis
Biallelic SNPs within 2 kb upstream and 1 kb downstream of known panicle-related genes were extracted by aligning resequencing data of 309 accessions against reference genome Nipponbare and tested its association with TSP, TPRB, TSRB and sub-component traits (USRB, MSRB, BSRB). SNPs with p-values <0.05 were considered significant and used to form haplotype groups. Pairwise comparison of haplotype groups were performed with t-test and p-values were adjusted using Holm's method. Results were visualized using boxplots created using the ggplot2 package (Wickham, 2016) in R.
Association Networks of Genetic Variants Identified Through mlGWAS and Targeted Association
The Cytoscape (Shannon et al., 2003) has been used to visualize the inter-relationship of SNPs associated to USRB, MSRB, BSRB, TSRB, TPRB and TSP traits. The SNPs with LOD score of 3.0 or above identified using mlGWAS with significant beta values (p<0.05) obtained from slGWAS and SNPs identified for known panicle architecture related genes through targeted association with significant beta values (p<0.05) has been merged together and transform as network input file (Source node:Traits,Target nodes-SNPs,Interaction-b values). Each trait has been grouped separately and shown as big circle nodes with distinct colors. The SNPs associated with each and across traits has been shown as out-degree edges highlighted in blue (multi locus) and red color (targeted association) with solid circles at each node.
Coexpression Network Analysis
The publicly available microarray data (GSE19024 and GSE41556) were extracted as CEL files from the six panicle developmental stages [Panicle1 (less than 1 mm), Panicle2 (3-5 mm), Panicle3 (10-15 mm) including three biological replicates, Panicle4 (40-50 mm), Panicle5 (Heading stage) with two biological replicates from two rice varieties Zhenshan 97 & Minghui 63] and additional heading stage of Nipponbare with 3 biological replicates. GCRMA normalization of all microarray samples was performed using the justGCRMA function (Wu et al. 2018) in R software. A total of 47 samples and 5041 panicles related genes were selected from Knetminer (Hassani-Pak, 2017) database based on published reports, and differentially expressed genes across the developing panicles in rice (Kudo et al. 2013).
Weighted gene correlation network analysis WGCNA package (Langfelder and Horvath, 2008) in R was used to identify the clusters (modules) of densely connected correlated genes. First the correlation matrix (coefficient=<0.75) was transformed into a matrix of connection strengths (an adjacency matrix) by raising the correlation matrix to the soft power β(beta) of 6. The adjacency function αij=Power(sij, β0)=|sij| β where sij is the co-expression similarity, and aij represents the resulting adjacency that measures the connection strengths. The power β is chosen using the scale free topology criterion proposed in (Zhang and Horvath, 2005). The adjacency matrix is converted to TO (topological overlap) matrix. Further the hierarchical clustering was done on TO similarity using cutreeDynamic function to identify modules. The eigenvectors for each module were calculated and clustered Modules with similar eigenvalue at threshold 0.25 has been merged using merge Close Modules function.
The edge weight indicates the strength of the co-expression from all nodes to all nodes. However, selection of a stringent threshold after module detection is required to remove noise. The genes were shown as nodes and the connection strength as weighted edges. Degree of connectivity was calculated to identify intramodular hubs. Nodes with an adjacency value of zero are removed from the modules and the edges with weight >=0.30089 were only selected. The genetic variants strongly associated with different panicle architecture traits were traced in the gene networks and highlighted its interactions. The visualization of co-expression network was done using Cystoscape (Shannon et al. 2003).
Cis Motif Searching
Potential binding sites of transcription factors that may alter the expression of candidate genes were determined using TomTom (Gupta et al., 2007). Probe sequences of specific length (30 bp) overlapping each of the significant SNPs detected with targeted association results were reverse transcribed and then used as input, considering an E-value<10. The motifs were compared with entries in JASPAR Core (2018) Plants Non-Redundant DNA Database (Khan et al. 2018).
Protein-Protein Interactions
Predicted protein-protein interactions of OsTPR were determined using STRING-DB ver. 10.5 (Szklarczyk et al., 2017) at high confidence (0.700) for the minimum required interaction score including less than 10 interactors for the first shell, and zero interactors for the second shell. The active interaction sources which served as potential evidence suggesting functional link include protein-protein experimental/biochemical data, associations in curated public databases, gene-fusion, co-expression data and text mining data (PubMed abstracts) were highlighted.
Gene Prediction
According to MSU 7.0 annotation, LOC_Os02g50790 and LOC_Os02g50799 were identified as two consecutive genes matching similar biological function showing homology to nucleoprotein TPR proteins. To confirm the annotation and gene models, the genomic sequence was extracted where these two genes are present in the genome. This whole sequence was used as input to the FGENESH for predicting gene model using standard parameters (Solovyev et al., 2006). Monocot sequences of Maize, Rice, Wheat and Barley were used as training set. The gene model prediction was validated with another abinitio method named as AUGUSTUS (Stanke et al., 2003).
Multiple Sequence Alignment and Phylogenetic Analysis
Multiple sequence alignment was performed for the protein sequence of the candidate gene to decipher the phylogeny among the close relatives. Overall 13 species were under consideration including two sub-species of Oryza sativa-japonica and indica. Protein sequence of newly predicted Oryza sativa spp. japonica TPR gene from MSU7 was used to identify orthologous protein in other species including Oryza sativa spp. indica (Zhang et al., 2016). Whole proteome sequences from Ensemble database (www(dot)plants(dot)ensembl(dot)org/index(dot)html) for other species were used to identify the orthologous gene using locally installed NCBI-BLAST (Altschul et al., 1990). Multiple sequence alignment was performed using CLUSTALW (Thompson et al., 1994) after identification of candidate protein. MEGA X (Kumar et al., 2018) was used to create the NEIGHBOR-joining phylogenetic tree with 1000 bootstrap. Evolutionary distances were calculated using poisson correction method. Percentage of total replicates (1000 bootstrap) used to form the branches in the bootstrap test presented near to the branches. Another multiple sequence alignment of candidate protein was performed with 8 contrasting lines using CLUSTALW where 4 samples with higher number of upper secondary rachis branches and 4 samples with low number of upper secondary rachis braches were considered. Alignment was presented with highlighted domain and important non-synonymous SNPs.
Nucleotide Diversity Calculation
7 SNP positons for genic region and 15 SNP position for whole LD block region were downloaded from the SNP-seek (Alexandrov et al., 2015) data for whole 3k set. Whole population was separated into different sub-population based on classification present at SNP-seek database. Minor allele frequency was calculated for each SNP at each sub-population level to verify how many candidate SNPs are present in other sub-populations. Nucleotide diversity computed using VCF tools (Danecek et al., 2011) and presented as line plot.
Using P-TRAP software 41 traits for panicle architecture were phenotyped in 310 indica accessions. The accessions represented diverse countries and exhibited a high heritability of H2=0.69 for total number of secondary rachis branches (TSRB) and a wide range of phenotypic variation in this trait (
Single-locus GWAS (slGWAS) underpinned genomic regions with significant association to various panicle architecture traits through 747,087 high quality bi-allelic SNP markers from 309 resequenced indica samples. The genotyping rate was 99.98% and minor allele frequency was greater than 10%. Genomic association was most pronounced for variation in USRB and a clear candidate region was demarcated by the slGWAS peak (
The 197 SNPs on chromosome 2 were mapped within a 126.4 kb interval with five distinct linkage disequilibrium (LD) blocks (
To identify multi-marker cumulative effect, gene-set analysis was performed. Based on the gene level analysis, top 10 genes significantly associated to USRB with highest p-values were identified (not shown). The strongest association (p=0.013498) with USRB was detected in the gene set consisting of LOC_Os02g50700, LOC_Os02g50790 and LOC_Os02g50799. The two latter candidate genes (LOC_Os02g50790 and LOC_Os02g50799) were annotated in public databases as two separate genes with homology to nuclear pore anchor proteins. Both were related to flower development through gene ontology. However, gene model prediction using ‘AUGUSTUS’ with indica training set confirmed these to be a single gene of 24,444 bp encoding a protein of 2050 amino acids (
Haplotype analysis for OsTPR formed from the significant SNPs revealed that lines with high USRB contained the ‘superior’ haplotype1 (AGGATCA, SEQ ID NO: 1). In comparison, lines with low USRB were associated with ‘inferior’ haplotype2 (TATTCGG, SEQ ID NO: 2), haplotype3 (TATTCCG, SEQ ID NO: 3) and haplotype4 (TATACCG, SEQ ID NO: 4;
Importantly, lines of haplotype 1 had low chalk content and those with haplotype 3 had high chalk content. This result suggested an association of OsTPR with increased USRB and TSP without compromising the grain quality. Additionally, analysis of grain quality traits indicated that the amylose content, gel temperature, viscosity, retrogradation, and cooking traits such as the cooked grain length and width (
Allele mining using 3k rice genomes revealed that within the indica subgroup the superior haplotype 1 was enriched while the inferior haplotype 3 was least represented (
Since slGWAS does not account for small to medium effect loci, multi-locus GWAS (mlGWAS) analysis was used to identify the integrated effect of multiple markers on the phenotype and determine genomic regions of significant association with panicle branching traits and number of total spikelets. Additional 208 loci were identified across the 12 chromosomes for significant associations with different panicle architecture traits (not shown).
Among them were several important transcription factors and hormone responsive genes (
Many SNPs found in the 37 genes (transcription factor and hormone responsive genes previously cloned useful in yield/flower/panicle traits) exhibited small to intermediate effect associations with primary and secondary rachis branching traits (not shown). To identify the association between sub-traits of branching with TSP, association networks for genetic variants identified from slGWAS, mlGWAS and targeted association approaches were delineated.
Interestingly, DWARF11 and MOC1 contribute to increase in total spikelets due to increased number of rachis branches of MSRB, TPRB and BSRB (
To overcome any spurious association sometimes predicted by GWAS, genetic variants identified through slGWAS, mlGWAS, and targeted association approaches were linked to the transcriptome co-expression networks data at panicle development stage. This elucidated increased connectivity of nodal genes linked to key candidate genes as the central hub. The co-expression analysis of 5,041 genes, expressed during panicle development, clustered them into 17 modules (
Further evidence for the importance of OsTPR protein came from protein-protein interactions (
Methodology (Shown in
The F5-derived F6 Recombinant Inbred lines (RILs) has been developed using single seed descent (SSD) method by employing the hybridization between high amylose mutant IR36ae (possess inferior haplotype of OsTPR TATTCGG haplotype SEQ ID NO: 2) and superior haplotype of OsMOC1 CG haplotype) and Samba Mahsuri (possess superior haplotype of OsTPR AGGATCA haplotype SEQ ID NO: 1) and inferior haplotype of OsMOC1 GA haplotpe). The Sambha Mahsuri parent is one of the most popular varieties consumed mainly across India due to its excellent cooking and eating qualities.
A total of 369 F3 progenies were selected and eventually forwarded to advanced generation until F5, utilizing single seed descent (SSD) method through rapid generation advancement. During the 2019 wet season a field experimental trial was conducted by growing a set of selected 298 F5 progenies in randomized complete block design with 2 replications, which were eventually termed as F5:6 RILs population. Out of them, a total of 280 samples from SM×IR36ae F5 population was successfully phenotyped using GIMP and P-TRAP software. Genotype data for the candidate genes OsTPR and OsMOC1 identified previously using in silico approaches were obtained using the KASP genotyping assay technology. In the diversity panel GWAS analysis, AGGATCA (SEQ ID NO: 1) was the superior haplotype identified for TPR based on the reference genome Nipponbare. In the current genotype data generated using KASP genotyping assay technology, the complementary alleles of TPR haplotype TCCTAGT were generated since the TPR gene is in the negative strand (3′ to 5′). Samples with missing genotyping data for the significant SNPs in each candidate gene were removed. In addition, lines containing heterozygotes were also removed when forming the haplotypes. Lines possessing haplotypes with homozygous alleles for OsTPR and OsMOC1 are 210 and 251 samples, respectively. A total of 195 samples have complete phenotype and genotype data for OsTPR and OsMOC1 haplotypes. Comparison of haplotype effects of OsTPR, OsMOC1, and combination of OsTPR and OsMOC1 with measured 21 panicle architecture phenotype data was done using t-test or Wilcoxon test. Boxplots were constructed using ggplot package in R after removing heterozygotes.
Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
It is the intent of the applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.
This application is a Continuation of PCT Patent Application No. PCT/IB2020/055840 having International filing date of Jun. 21, 2020, which claims the benefit of priority under 35 USC § 119(e) of U.S. Provisional Patent Application Nos. 62/926,585 filed on Oct. 28, 2019 and 62/864,548 filed on Jun. 21, 2019. The contents of the above applications are all incorporated by reference as if fully set forth herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 62926585 | Oct 2019 | US | |
| 62864548 | Jun 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/IB2020/055840 | Jun 2020 | US |
| Child | 17557130 | US |
