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When two organisms have significant difference in the genetic materials in their respective genomes, these two organisms can be said to be genetically different. For example, a naturally-occurring nucleic acid, polypeptide, or cell present in a living animal is not isolated, but the same polynucleotide, polypeptide, or cell separated from some or all of the coexisting materials in the natural system, is isolated, even if subsequently reintroduced into the natural system.

Either the given nucleic acid or the complementary nucleic acid can be determined from any specified polynucleotide sequence. The homologous molecules can be termed homologs. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins or sequences thereof. Higher levels of sequence identity, e. Methods for determining sequence identity percentages e.

INTRODUCTION

Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Sci U. Alignment is also often performed by inspection and manual alignment. Similarly, nucleic acids can also be described with reference to a starting nucleic acid, e.

When one molecule is said to have certain percentage of sequence identity with a larger molecule, it means that when the two molecules are optimally aligned, said percentage of residues in the smaller molecule finds a match residue in the larger molecule in accordance with the order by which the two molecules are optimally aligned. In another embodiment, DNA sequences that are substantially identical to the candidate genes may be used to generate a transgenic plant that is more resistant to SCN than the host.

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USA Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions i. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by to yield the percentage of sequence identity.

Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about residues, and most preferably the sequences are substantially identical over at least about residues.

In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding regions. Analogous minor variation can also include amino acid deletion or insertion, or both.

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Quantitative Trait Loci (Qtl) : Methods and Protocols - elintrandoltio.cf

Guidance in determining which amino acid residues can be substituted, inserted, or deleted without eliminating biological or immunological activity can be found using computer programs well known in the art, for example, DNASTAR software. A variety of additional terms are defined or otherwise characterized herein. In practicing the instrumentalities described herein, many conventional techniques in molecular biology, microbiology, and recombinant DNA are optionally used.

These techniques are well known to those of ordinary skill in the art. For example, one skilled in the art would be familiar with techniques for in vitro amplification methods, including the polymerase chain reaction PCR , for the production of the homologous nucleic acids described herein. The markers that are genetically linked to the QTLs disclosed herein, or other similarly placed markers, may be used in soybean breeding for marker-assisted selection of resistance to different races of SCN, including but not limited to races 1, 2, 3, 5, 14 and LY1.

Selection for resistant strains based on two markers that flank a QTL is generally more reliable than selections based on one marker linked to the QTL. Flanking-marker selection relies on two markers thereby reducing the number of false positive due to recombination between one marker and the QTL, and consequently, reducing the probability selecting a line that is susceptible.

It is to be recognized, however, that when markers are closely linked to a QTL, results from single-marker selections may be acceptable. According to another aspect of the present disclosure, a new phenotypically superior SCN-resistant soybean plant may be produced which possesses desirable traits but is substantially free from undesirable characteristics currently associated with SCN-resistant soybean lines.

The first step for producing such an improved soybean line may involve providing one or more plants from a parental soybean plant line which comprises in its genome one or more molecular markers disclosed above that are genetically linked to SCN resistance. Preferably, the parental plant line is purebreeding for one or more of such molecular markers. The second step in this method comprises introgressing SCN resistance into a recipient soybean plant line which is not resistant or less resistant to SCNs by performing marker-assisted selection based upon the one or more molecular markers in the parent mentioned in the first step.

The progeny thus obtained may be backcrossed to obtain a new improved plant having the one or more molecular markers in their genome which are associated with SCN resistance. Plants thus selected may then be used to develop new SCN-resistant recombinant soybean plant lines, for example, by single seed descent.

Alternatively, the plant may optionally be further treated to selective breeding by performing additional backcrosses and selections, based upon the presence of one or more of the markers, for multiple generations. The presence of one or more of the markers may be readily determined by single nucleotide polymorphism SNP analysis of the plant's genomic DNA. Marker-assisted selection may also be used to confirm previous selection for SCN resistance or susceptibility made by challenging plants with SCNs in the field or greenhouse and scoring the resulting phenotypes.

Alternatively, plants can be analyzed by SNP analysis to determine the presence of the molecular markers disclosed herein in order to confirm the presence of a genomic locus associated with SCN resistance. This method can be applied to a soybean plant, soybean seeds or other tissues. With the completion of the soybean genome sequencing project, genes within each QTL may be identified using the genomic information that are publicly available, for example, at the website maintained by NCBI.


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Alternatively, markers linked to each of the mapped QTL may also be used in positional cloning of genes that reside within those QTL. Positional cloning first involves creating a physical map of a contig contiguous overlapping of cloned DNA inserts , in the genomic region encompassing one or more marker loci and the target gene. The target gene is then identified and isolated within one or more clones residing in the contig. Having a clone of a gene allows it to be used in genetic studies, transformation, and the development of transgenic plants and novel phenotypes.

It is to be recognized to that resistance to one race of SCN, or to multiple races, is likely to be controlled by more than one gene or more than one genomic locus. Genes found to be associated with SCN resistance can also be introduced into a parental plant by transformation in order to create a transgenic plant carrying the SCN resistance gene s.


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  • Transformation of a plant cell can be accomplished by a variety of different methodology. Any method for the introduction of foreign DNA or RNA into a plant cell and for expression therein may be used within the context of the present disclosure. The following nonlimiting examples report general procedures, reagents and characterization methods that teach by way of example, and should not be construed in a narrowing manner that limits the disclosure to what is specifically disclosed.

    Materials and Methods

    Those skilled in the art will understand that numerous modifications may be made and still the result will fall within the spirit and scope of the present invention. By way of example, to obtain F RILs, one would start from a genetically segregating F2 population of a biparent cross.


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    • At maturity, one single seed of each F2 plant was harvested and individually recorded, i. These F2 seeds were grown to produce F3 plants. Again, one single seed of each F3 plant was harvested. The process was repeated until F6 generation was reached. The F6 plants were grown and harvested in bulk. At this F7 stage, most of the genes in the genome are homozygous as a result of several generations of genetic recombination.

      DNA extraction and molecular marker analysis were performed as described below. Each reaction contained ng of, genomic DNA, 0. The allele of each marker was analyzed with GeneMapper 3. The resulting double-stranded PCR products were immobilized onto paramagnetic particles followed by steps of washing and denaturing. The hybridized SAM was rinsed and dried for 20 min in dark. The clusters of homozygote and heterozygous genotypes for each SNP were manually checked and identified for polymorphisms between the two parental lines.

      Soybean cyst nematode SCN bioassays were performed in the greenhouse at the University of Missouri-Columbia according to established methods Arelli et al.

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      More particularly, soybean seeds were germinated for three days in germination pouches and then seedlings were transplanted into micropots one plant in each micropot filled with steam-pasteurized sandy soil. Five plants of each indicator lines and five single-plant replications per RIL two independent experiments for all races were planted together in random way.

      Thirty days after transplanting, roots were harvested and washed to collect female nematode. Nematode cysts were then counted under a stereo-microscope. Data processing and analysis were performed as described in the following text. Linkage analysis was performed with the computer program JoinMap 3.

      A likelihood of odds LOD threshold score of 3. Chromosome numbers Chr. Molecular genetic linkage analysis was performed using the methodology described below. Nearly fluorescent-labeled SSR markers were surveyed for DNA polymorphisms between two parental lines of each genetic population. Of these, from to markers, which produced mainly co-dominant loci, were polymorphic. These markers were utilized for genotyping of the F or RIL mapping populations. For each genetic population, a molecular genetic linkage map encompassing 20 soybean chromosomes and fragments was constructed.

      These chromosomes and fragments covered approximately 1, to cM. Overall, marker order and genetic distance between markers in the constructed genetic map had a linear relationship with the composite linkage map Song et al. These QTLs may be defined by flanking markers on the genome sequence. The genomic regions encompassed by these QTLs contain many genes encoding proteins or metabolites that may play a role in SCN resistance. These genes may also include networking regulators or transcription factors.

      The gene identifiers of these genes are listed in FIG. In addition, about genetic markers have been identified within this region which may be used for fine mapping the QTLs with NILs.