Nucleic acid molecules and other molecules associated with soybean cyst nematode resistance Number:7,154,021 from the United States Patent and Trademark Office (PTO) owispatent The present invention is in the field of soybean genetics. More specifically, the invention relates to nucleic acid molecules from regions the soybean genome, which are associated with soybean cyst nematode resistance. The invention also relates to proteins encoded by such nucleic acid molecules as well as antibodies capable of recognizing these proteins. The invention also relates to nucleic acid markers from regions the soybean genome, which are associated with soybean cyst nematode resistance. Moreover, the invention relates to uses of such molecules, including, transforming soybean cyst nematode resistant soybean with constructs containing nucleic acid molecules from regions the soybean genome, which are associated with soybean cyst nematode resistance. Furthermore, the invention relates to the use of such molecules in a plant breeding program.<H resistance, associated, Nucleic, acid, mo, 7154021.html, Patent, pto, 7154021

Title: Nucleic acid molecules and other molecules associated with soybean cyst nematode resistance

Abstract: The present invention is in the field of soybean genetics. More specifically, the invention relates to nucleic acid molecules from regions the soybean genome, which are associated with soybean cyst nematode resistance. The invention also relates to proteins encoded by such nucleic acid molecules as well as antibodies capable of recognizing these proteins. The invention also relates to nucleic acid markers from regions the soybean genome, which are associated with soybean cyst nematode resistance. Moreover, the invention relates to uses of such molecules, including, transforming soybean cyst nematode resistant soybean with constructs containing nucleic acid molecules from regions the soybean genome, which are associated with soybean cyst nematode resistance. Furthermore, the invention relates to the use of such molecules in a plant breeding program.

Patent Number: 7,154,021 Issued on 12/26/2006 to Hauge, et al.

Inventors: Hauge; Brian M. (Beverly, MA), Wang; Ming Li (Lexington, MA), Parsons; Jeremy David (Cambridge, GB), Parnell; Laurence David (Cambridge, MA)
Assignee: Monsanto Technology LLC (St. Louis, MO)

Appl. No.: 09/754,853

Filed: January 5, 2001

Current U.S. Class: 800/267 ; 800/265; 800/266

Current International Class: A01H 1/00 (20060101)

Field of Search: 800/260,265,267,312

References Cited [Referenced By]

U.S. Patent Documents


5491081	February 1996	Webb
6096944	August 2000	Vierling et al.
6162967	December 2000	Webb
6228992	May 2001	Jessen et al.
6284948	September 2001	Jessen et al.
6300541	October 2001	Lightfoot et al.
6538175	March 2003	Webb
2002/0129402	September 2002	Lightfoot et al.
2002/0144310	October 2002	Lightfoot et al.
2003/0135881	July 2003	Webb

Foreign Patent Documents


WO 95 20669	Aug., 1995	WO
WO 01/51627	Jun., 2002	WO

Other References

Staub and Serquen 1996, HortScience 31(5):729-738. cited by examiner .
Forugoux-Nicol et al 1999, Plant Molecular Biology 40:857-872. cited by examiner .
Zhu et al 2003, Genetics 163:1123-1134. cited by examiner .
Rafalski 2002, Plant Science 162: 329-333. cited by examiner .
J.D. Thompson et al., Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, (1994) vol. 22, No. 22., pp. 4673-4680. cited by other .
Wen-Yuan Song, et al., A receptor Kinase-Like Protein Encoded by the Rice Disease Resistance Gene, Xa21., Science 270 (1995) pp. 1804-1806. cited by other .
Concibido et al., Cell Biology & Molecular Genetics RFLP mapping and Marker-Assisted Selected of Soybean Cyst Nematode Resistance in PI 209332., Crop. Sci. 36 (1996) pp. 1643-1650. cited by other .
Mansur et al., Generation Mean Analysis of Resistance to Race 3 of Soybean Cyst Nematode., Crop Sci. 33 (1993) pp. 1249-1253. cited by other .
Rao-Arelli et al., Genetic Relationships Among Soybean Plant Introductions for Resistance to Race 3 of Soybean Cyst Nematode., Crop Sc., 28 (1988) pp. 650-652. cited by other .
Concibido et al., Targeted Comparative Genome Analysis and Qualitative Mapping of a Major Partial-Resistance Gene to the Soybean cyst Nematode., Theor Appl. Genet. (1996) 93:234-241. cited by other .
Kobe et al., A Structural Basis of the Interactions Between Leucine-Rich Repeats and Protein Ligands., Nature vol. 374 (1995) pp. 183-186. cited by other .
Cregan et al., Two Simple Sequence Repeat Markets to Select for Soybean Cyst Nematode Resistance Conditioned by the Rhg1 Locus., Theor Appl. Genet. (1999) 99:811-818. cited by other .
Qui et al. RFLP Markers Associated with Soybean Cyst Nematode Resistance and Seed Composition in a `Peking` x `Essex` Population., Theor Appl. Genet 98:356-364 (1999). cited by other .
Concibido et al., Genome Mapping of Soybean Cyst Nematode Resistance Genes of `Peking`, PI 90763 and PI 88788 Using DNA markers., Crop Sci. 37:258-264 (1997). cited by other .
Jones et al., The Role of Leucine-Rich Repeat Proteins and Plant Defences, Adv. Bot. Res. Incorp. Ad. Plant Path 24:89-167 (1997). cited by other .
Matson and Williams, Evidence of a Fourth Gene For Resistance to the Soybean Cyst Nematode., Crop Sci. 5:447 (1965). cited by other .
Concibido, V.C., et al., "A Common Soybean Cyst Nematode Resistance Gene," Phytopathology, vol. 85(10):1140 (1995). cited by other .
Dong, K., et al., "Genetics of Soybean-Heterodera glycines Interactions," Journal of Nematology, vol. 29(4):509-522 (1997). cited by other .
Prabhu, R. R., et al., "Selecting Soybean Cultivars for Dual Resistance to Soybean Cyst Nematode and Sudden Death Syndrome Using Two DNA Markers," Crop Science, vol. 39(4):982-987 (Jul.-Aug. 1999). cited by other .
Concibido et al., "A Decade of QTL Mapping for Cyst Nematode Resistance in Soybean", Crop Science, 44(4):1121-1131 (2004). cited by other .
Lange et al., "A Plant DNA Isolation Protocol Suitable for Polymerase Chain Reaction Based Marker-Assisted Breeding", Crop Science, 38:217-220 (1998). cited by other .
Matthews et al., "Molecular Markers Residing Close to the Rhg4 Locus Conferring Resistance to Soybean Cyst Nematode Race 3 on Linkage Group A of Soybean", Theor. Appl. Genet., 97:1047-1052 (1998). cited by other .
Meksem et al.; "Clustering Among LOCI Underlying Soybean Resistance to Fusarium solani, SDS and SCN in Near-Isogenic Lines", Theor. Appl. Genet., 99:1131-1142 (1999). cited by other .
Meksem et al., "Conversion of AFLP Bands into High-Throughput DNA Markers", Mol. Genet. Genomics, 265:207-214 (2001). cited by other .
Meksem et al., `"Forrest` Resistance to the Soybean Cyst Nematode is Bigenic: Saturation Mapping of the Rhg1 and Rhg4 LOCI", Theor. Appl. Gene., 103:710-717 (2001). cited by other .
Meksem et al., "Two Large-Insert Soybean Genomic Libraries constructed in a Binary Vector: Applications in Chromosome Walking and Genome Wide Physical Mapping", Theor. Appl. Gene., 101:747-755 (2000). cited by other .
Meksem et al., "High-Throughput Genotyping for a Polymorphism Linked to Soybean Cyst Nematode Resistance Gene Rhg4 by Using Taqman.TM. Probes", Molecular Breeding, 7:63-71 (2001). cited by other .
Bell-Johnson et al., "Biotechnology Approaches to Improving Resistance to SCN and SDS: Methods for High Throughput Marker Assisted Selection", Soybean Genetics Newsletter, 25:115-117 (1998). cited by other .
Boutin et al., "RFLP Analysis of Cyst Nematode Resistance in Soybeans", Soybean Genetics Newsletter, 19:123-127 (1992). cited by other .
Chang et al., "Association of Loci Underlying Field Resistance to Soybean Sudden Death Syndrome (SDS) and Cyst Nematode (SCN) Race 3", Crop Sci., 37(3):965-971 (1997). cited by other .
Concibido et al., "DNA Marker Analysis of Loci Underlying Resistance to Soybean Cyst Nematode (Heterodera Glycines Ichinohe)", Crop Sci., 34(1):240-246 (1994). cited by other .
Concibido et al., "RFLP Mapping of Cyst Nematode Resistance Genes in Soybeans", Soybean Genetics Newsletter, 20:136-139 (1993). cited by other .
Mahalingam et al., "Cytological Expression of Early Response to Infection by Heterodera gylcines Ichinohe in Resistant PI 437654 Soybean", Genome, 39:986-998 (1996). cited by other .
Mahalingam et al., "DNA Markers for Resistance to Heterodera glycines I. Race 3 in Soybean Cultivar Peking", Breeding Science, 45:435-443 (1995). cited by other .
Meksem et al., "A High-Resolution Map of the Vicinity of the R1 Locus on Chromosome V of Potato Based on RFLP and AFLP Markers", Mol Gen Genet, 249:74-81 (1995). cited by other .
Webb et al., "Genetic Mapping of Soybean Cyst Nematode Race-3 Resistance LOCI in the Soybean P1 437.654", Theor Appl Genet, 91(1):574-581 (1995). cited by other.

Primary Examiner: Kruse; David H
Attorney, Agent or Firm: Kelley; Thomas E. Lavin, Jr.; Lawrence M. Arnold & Porter LLP

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority under 35 U.S.C. .sctn.119(e) of U.S. Application No. 60/174,880, filed Jan. 7, 2000, the disclosure of which is herein incorporated by reference in its entirety.

Claims

What is claimed is:

1. A method of introgressing an allele into a soybean plant comprising (A) crossing at least one SCN resistant soybean plant having an rhg1 SCN resistant allele with at least one SCN sensitive soybean plant having an rhg1 SCN sensitive allele in order to form a segregating population, (B) screening said segregating population with one or more nucleic acid markers to determine if one or more soybean plants from said segregating population contains a deletion of 19 nucleotides corresponding to SEQ ID NO: 2 and encompassing position 48881, and (C) selecting, if present, one or more soybean plants of said segregating population containing said deletion.

2. The method according to claim 1, wherein said one or more soybean plants of said segregating population have a yellow soybean seed.

3. A method of introgressing an allele from a first soybean plant comprising a polymorphism relative to a second soybean plant into a selected soybean plant comprising screening with one or more nucleic acid markers a population of soybean plants formed by a cross of said first and said second soybean plant and selecting a soybean plant, wherein said allele is an allele having one or more polymorphisms in a protein coding region corresponding to nucleotides 45163 to 45314, 45450 to 45509, 46941 to 48763 or 48975 to 49573 of SEQ ID NO: 2 thereby introgressing said allele from said first soybean plant comprising a polymorphism into said selected soybean plant.

4. The method according to claim 3, wherein said polymorphisms selected from the group consisting of 45173, 45309, 47057, 47140, 47208, 47571, 47617, 47796, 47856, 47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681, 49012, and 49316 of SEQ ID NO: 2.

5. The method according to claim 3, wherein said introgressing said allele into said selected soybean plant results in a yellow soybean seed.

6. A method of introgressing an allele comprising a polymorphism into a soybean plant lacking said polymorphism comprising screening a population of soybean plants with one or more nucleic acid markers and selecting a soybean plant, wherein said one or more nucleic acid markers are capable of detecting one or more polymorphisms located at a position in SEQ ID NO: 2 selected from the group consisting of 45173, 45309, 47057, 47140, 47208, 47571, 47617, 47796, 47856, 47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681, 49012, and 49316, thereby selecting a soybean plant comprising said polymorphism.

7. The method according to claim 6, wherein said introgressing said allele comprising a polymorphism into said soybean plant results in one or more soybean plant having a yellow soybean seed and said polymorphism.

8. A method of introgressing an rhg1 SCN resistant allele into a non-resistant soybean plant comprising (A) crossing at least one SCN resistant soybean plant having said rhg1 SCN resistant allele corresponding to an rhg1 SCN resistant allele present in Peking with at least one SCN sensitive soybean plant having an rhg1 SCN sensitive allele in order to form a segregating population, (B) screening said segregating population with one or more nucleic acid markers to identify said rhg1 SCN resistant allele, wherein said one or more nucleic acid markers are capable of detecting a polymorphism located at a position in SEQ ID NO: 2 corresponding to nucleotides between 45163 and 49573, and (C) selecting one or more members of said segregating population having said rhg1 SCN resistant allele.

9. The method according to claim 8, wherein said one or more members of said segregating population have a yellow soybean seed.

10. The method according to claim 8, wherein said one or more nucleic acid markers are capable of detecting a single nucleotide polymorphism or INDEL mutation.

11. The method according to claim 8, wherein said one or more nucleic acid markers are capable of detecting one or more polymorphisms located at a position in SEQ ID NO: 2 selected from the group consisting of 45173, 45309, 47057, 47140, 47208, 47571, 47617, 47796, 47856, 47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681, 49012, 49316, and 46703.

12. The method according to claim 8, wherein said one or more nucleic acid markers are capable of detecting single nucleotide polymorphisms.

13. The method according to claim 12, wherein said single nucleotide polymorphisms are located at a position in SEQ ID NO: 2 selected from the group consisting of 45173, 45309, 47057, 47140, 47208, 47571, 47617, 47796, 47856, 47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681, 49012, and 49316.

14. The method according to claim 8, wherein said one or more nucleic acid markers are capable of detecting INDEL mutations.

Description

FIELD OF THE INVENTION

The present invention is in the field of soybean genetics. More specifically, the invention relates to nucleic acid molecules from regions of the soybean genome, which are associated with soybean cyst nematode (SCN) resistance. The invention also relates to proteins encoded by such nucleic acid molecules as well as antibodies capable of recognizing these proteins. The invention also relates to nucleic acid markers from regions of the soybean genome, which are associated with SCN resistance. Moreover, the invention relates to uses of such molecules, including, transforming SCN sensitive soybean with constructs containing nucleic acid molecules from regions in the soybean genome, which are associated with SCN resistance. Furthermore, the invention relates to the use of such molecules in a plant breeding program.

BACKGROUND OF THE INVENTION

The soybean, Glycine max (L.) Merril (Glycine max or soybean), is one of the major economic crops grown worldwide as a primary source of vegetable oil and protein (Sinclair and Backman, Compendium of Soybean Diseases, 3.sup.rd Ed. APS Press, St. Paul, Minn., p. 106. (1989)). The growing demand for low cholesterol and high fiber diets has also increased soybean's importance as a health food.

Prior to 1940, soybean cultivars were either direct releases of introductions brought from Asia or pure line selections from genetically diverse plant introductions. The soybean plant was primarily used as a hay crop in the early part of the 19th century. Only a few introductions were large-seeded types useful for feed grain and oil production. From the mid 1930's through the 1960's, gains in soybean seed yields were achieved by changing the breeding method from evaluation and selection of introduced germplasm to crossing elite by elite lines. The continuous cycle of cross hybridizing the elite strains selected from the progenies of previous crosses resulted in the modem day cultivars.

Over 10,000 soybean strains have now been introduced into the United States since the early 1900's (Bernard et al., United States National Gennplasm Collections. In: L. D. Hil (ed.), World Soybean Research, pp. 286 289. Interstate Printers and Publ., Danville, Ill. (1976)). A limited number of those introductions form the genetic base of cultivars developed from the hybridization and selection programs (Johnson and Bernard, The Soybean, Norman Ed., Academic Press, N.Y., pp. 1 73 (1963)). For example, in a survey conducted by Specht and Williams, Genetic Contributions, Fehr eds. American Soil Association, Wisconsin, pp. 49 73 (1984), for the 136 cultivars released from 1939 to 1989, only 16 different introductions were the source of cytoplasm for 121 of that 136. Certain soybean strains are sensitive to one or more pathogens. One economically important pathogen is SCN.

SCN accounts for roughly 40% of the total disease in soybean and can result in significant yield losses (up to 90%). SCN is the most destructive pest of soybean to date and accounts for an estimated yield loss of up to $809 million dollars annually. Currently, the most cost effective control measures are crop rotation and the use of host plant resistance. While breeders have successfully developed SCN resistant soybean lines, breeding is both difficult and time consuming due to the complex and polygenic nature of resistance. The resistance is often race specific and does not provide stability over time due to changing SCN populations in the field. In addition, many of the resistant soybean varieties carry a significant yield penalty when grown in the absence of SCN.

SCN, Heterodera glycines Ichinohe, was identified on soybeans in the United States in 1954 at Castle Hayne, N.C. Winstead, et al., Plant Dis. Rep. 39:9 11 (1955). Since its discovery the SCN has been recognized as one of the most destructive pests in soybean. It has been reported in nearly all states in which soybeans are grown, and it causes major production problems in several states, being particularly destructive in the Midwestern states. See generally: Caldwell, et al., Agron. J. 52:635 636 (1960); Rao-Arelli and Anand, Crop. Sci. 28:650 652, (1988); Baltazar and Mansur, Soybean Genet. Newsl. 19:120 122 (1992); Concibido, et al., Crop. Sci., (1993). For example, sensitive soybean cultivars had 5.7 35.8% lower seed yields than did resistant cultivars on SCN race-3 infested sites in Iowa. (Niblack and Norton, Plant Dis. 76:943 948 (1992)).

Shortly after the discovery of SCN in the United States, sources of SCN resistance were identified (Ross and Brim, Plant Dis. Rep. 41:923 924 (1957)). Some lines such as Peking and Plant Introduction (PI) PI88788, were quickly incorporated into breeding programs. Peking became widely used as a source of resistance due to its lack of agronomically undesirable traits, with Pickett as the first SCN resistant cultivar released (Brim and Ross, Crop Sci. 6:305 (1966)). The recognition that certain SCN resistant populations could overcome resistant cultivars lead to an extensive screen for additional sources of SCN resistance. PI88788 emerged as a popular source of race 3 and 4 resistance even though it had a cyst index greater than 10% (but less than 20%) against race 4, and Peking and its derivatives emerged as a popular source for races 1 and 3. PI437654 was subsequently identified as having resistance to all known races and its SCN resistance was backcrossed into Forrest. Currently there are more than 130 PIs known to have SCN resistance.

SCN race 3 is considered to be the prominent race in the Midwestern soybean producing states. Considerable effort has been devoted to the genetics and breeding for resistance to race 3. While both Peking and PI88788 are resistant to SCN race 3, classical genetics studies suggest that they harbor different genes for race 3 resistance (Rao-Arelli and Anand, Crop Sci. 28:650 652 (1988)). Crosses between PI88788(R) and Essex(S) segregate 9(R): 55(S) in the F.sub.2 population and 1(R): 26(Seg): 37(S) families in the F.sub.3 generation, suggesting that resistance to race 3 in PI88788 is conditioned by one recessive and two dominant genes, where as Peking and PI90763 resistance is conditioned by one dominant and two recessive genes. Based on reciprocal crosses, Peking, Forrest, and PI90763 have genes in common for resistance to SCN race 3 (Rao-Arelli and Anand, Crop Sci., 28:650 652 (1988)). A cross between Peking and PI88788 segregates 13(R):3(S) in the F.sub.2 generation, indicating a major difference between the parents for race 3 resistance. Generation mean analysis based on four crosses between resistant and sensitive genotypes; A20 (R), Jack (R), Cordell (R) and A2234 (S), suggests that an additive genetic model is sufficient to explain most of the genetic variation of race 3 SCN resistance in each cross, while the analysis of the pooled data indicates the presence of dominant effects as well (Mansur, Carriquiry and Roa-Arelli, Crop Sci. 33:1249 1253 (1993)). This analysis further indicates that race 3 resistance is probably under the genetic control of three, but not more than four genes.

RFLP analysis of segregating populations between resistant and sensitive lines; PI209332 (R), PI90763 (R), PI88788 (R), Peking (R) and Evan (S), identified a major SCN resistance QTL (rhg1) which maps to linkage group G (Concibido et al., Theor Appl. Genet. 93:234 241 (1996)). In this study, rhg1 explains 51.4% of the phenotypic variation in PI209322, 52.7% of the variation in PI90763, 40.0% of the variation in PI88788 and 28.1% of the variation in Peking. This major resistance QTL was assumed be one and the same in all of the mapping populations employed. However, as pointed out by the authors, it is possible that the genomic interval contains distinct but tightly linked QTLs. In a related study using PI209332 as the source of resistance, Concibido et al., Crop Sci. 36:1643 1650 (1996), show that a QTh on linkage group G (rhg1) is effective against the three SCN races tested, explaining 35% of the phenotypic variation to race 1, 50% of the variation to race 3, and 54% of the variation to race 6. In addition to the major QTL on linkage group G, 4 other QTLs mapping to linkage groups D, J, L and K were identified, with some of the resistance loci behaving in a race specific manner.

Concibido et al. (Crop Sci. 37:258 264 (1997)) found significant association of marker C006V to a major QTL on linkage group G (rhg1) and resistance to race 1, race 3 and race 6, in Peking and PI90763 (Evan X Peking, Evan X PI90763) and races 3 and 6 in PI88788 (Evan X PI88788), in agreement with the previous study based on the P209332 source of resistance (Concibido et al., Crop Sci. 36:1643 1650 (1996)). The resistance locus near C006V was effective against all races tested in all of the resistance sources. While statistically significant against all races, this locus accounts for different proportions of the total phenotypic variation with the races tested. For example, in PI90763 the resistance locus near C006V explains more than three times the phenotypic variation against race 1 than against race 3. The variability can be attributed to differences in the genetic backgrounds, variability among the SCN populations or may be a reflection of the limited size of the plant populations which were employed. This study further identified three additional independent SCN resistance QTLs; one near the RFLP marker A378H mapping to the opposite end of linkage group G from C006V (rhg1), one near the marker B032V-1 on linkage group J and a third linked to A280Hae-1 on linkage group N. Comparisons between the different SCN races indicated that some of the putative SCN QTLs behave in a race specific manner.

PI437654 was identified as having resistance to all known races. Based on analysis of 328 recombinant inbreed lines (RIL) derived from a cross between PI437654 and BSR101, Webb reported six QTLs associated with SCN resistance on linkage groups A2, C1, G, M, L25 and L26 (U.S. Pat. No. 5,491,081). An allele on linkage group G, presumed to be rhg1, is involved with certain SCN races tested (races 1, 2, 3, 5 and 14), and has the largest reported phenotypic effect on resistance to every race. In contrast, the QTLs on linkage groups A2, C1, M, L25 and L26 act in a race specific manner. The QTL on linkage group L25 was reportedly involved with four of the five races, while the QTLs on linkage groups, A2, C1 and L26 were each involved in resistance to two of the five races (U.S. Pat. No. 5,491,081). Webb further reports data that the resistance to any of the five races is likely to result from the combined effects of the QTL involved in each race (U.S. Pat. No. 5,491,081).

Qui et al. (Theor Appl Genet 98:356 364 (1999)) screened 200 F.sub.2:3 families derived from a cross between Peking and Essex and identified RFLP markers which are associated with SCN resistance QTLs on linkage groups B, E, I and H. The three QTLs on linkage groups B, E and H jointly account for 57.7% of the phenotypic variation to race 1, the QTLs on linkage groups H and B account for 21.4% of the variation to race 3, while the QTLs on linkage groups I and E are associated with resistance to race 5 accounting for 14.0% of the phenotypic variation. In contrast to previous mapping studies which use Peking as the source of resistance, no significant association was detected to the rhg1 locus on linkage group G. The authors point out that the marker Bng122, which has been shown to have significant linkage to rhg1, is not polymorphic in the population employed (Concibido et al., Crop Sci. 36:1643 1650 (1996)).

It has been reported that the rhg1 locus on linkage group G is necessary for the development of resistance to any of the SCN races. There have been efforts to develop molecular markers to identify breeding lines harboring the rhg1 SCN resistant allele. One of the most commonly used markers for marker assisted selection (MAS) of rhg1 is an SSR locus that co-segregates and maps roughly 0.4 cM from rhg1. This SRR marker, BARC-Satt.sub.--309 is able to distinguish most, if not all, of the SCN sensitive genotypes from those harboring rhg1 from important sources of resistance such as Peking and PI437654. Two simple sequence repeat markers have been reported that can be used to select for SCN resistance at the rhg1 locus (Concibido et al., Theor Appl Genet 99: 811 818 (1999)). Satt.sub.--309 was also effective in distinguishing SCN resistant sources PI88788 and PI209332 in many, but not all, sensitive genotypes. In particular, Satt.sub.--309 can not be used for MAS in populations developed from "typical" southern US cultivars (e.g., Lee, Bragg and Essex) crossed with resistance sources PI88788 or PI209332.

Matson and Williams have reported a dominant SCN resistance locus, Rhg4, which is tightly linked to the `i` locus on linkage group A2 (Matson and Williams, Crop Sci. 5:447 (1965)). The QTL reported by Webb on linkage group A2 maps near the `i` locus and is considered to be Rhg4 (U.S. Pat. No. 5,491,081). Webb concludes that only two loci on linkage groups A2 (Rhg4) and G (rhg1) explain the genetic variation to race 3.

SUMMARY OF THE INVENTION

The present invention includes and provides a method for the production of a soybean plant having an rhg1 SCN resistant allele comprising: (A) crossing a first soybean plant having an rhg1 SCN resistant allele with a second soybean plant having an rhg1 SCN sensitive allele to produce a segregating population; (B) screening the segregating population for a member having an rhg1 SCN resistant allele with a first nucleic acid molecule capable of specifically hybridizing to linkage group G, wherein the first nucleic acid molecule specifically hybridizes to a second nucleic acid molecule that is linked to the rhg1 SCN resistant allele; and, (C) selecting the member for further crossing and selection.

The present invention includes and provides a method of investigating an rhg1 haplotype of a soybean plant comprising: (A) isolating nucleic acid molecules from the soybean plant; (B) determining the nucleic acid sequence of an rhg1 allele or part thereof; and, (C) comparing the nucleic acid sequence of the rhg1 allele or part thereof to a reference nucleic acid sequence. The present invention includes and provides a method of introgressing SCN resistance or partial SCN resistance into a soybean plant comprising: performing marker assisted selection of the soybean plant with a nucleic acid marker, wherein the nucleic acid marker specifically hybridizes with a nucleic acid molecule having a first nucleic acid sequence that is physically linked to a second nucleic acid sequence that is located on linkage group G of soybean A3244, wherein the second nucleic acid sequence is within 500 kb of a third nucleic acid sequence which is capable of specifically hybridizing with the nucleic acid sequence of SEQ ID NO: 5, 6, complements thereof, or fragments thereof having at least 15 nucleotides; and, selecting the soybean plant based on the marker assisted selection.

The present invention includes and provides a method for the production of a soybean plant having an Rhg4 SCN resistant allele comprising: (A) crossing a first soybean plant having an Rhg4 SCN resistant allele with a second soybean plant having an Rhg4 SCN sensitive allele to produce a segregating population; (B) screening the segregating population for a member having an Rhg4 SCN resistant allele with a first nucleic acid molecule capable of specifically hybridizing to linkage group A2, wherein the first nucleic acid molecule specifically hybridizes to a second nucleic acid molecule linked to the Rhg4 SCN resistant allele; and, (C) selecting the member for further crossing and selection.

The present invention includes and provides a method of investigating an Rhg4 haplotype of a soybean plant comprising: (A) isolating nucleic acid molecules from the soybean plant; (B) determining the nucleic acid sequence of an Rhg4 allele or part thereof; and (C) comparing the nucleic acid sequence of the Rhg4 allele or part thereof to a reference nucleic acid sequence.

The present invention includes and provides a method of introgressing SCN resistance or partial SCN resistance into a soybean plant comprising: performing marker assisted selection of the soybean plant with a nucleic acid marker, wherein the nucleic acid marker specifically hybridizes with a nucleic acid molecule having a first nucleic acid sequence that is physically linked to a second nucleic acid sequence that is located on linkage group A2 of soybean A3244, wherein the second nucleic acid sequence is within 500 kb of a third nucleic acid sequence which specifically hybridizes with the nucleic acid sequence of SEQ ID NO: 7, complements thereof, or fragments thereof having at least 15 nucleotides; and, selecting the soybean plant based on the marker assisted selection.

The present invention includes and provides a substantially purified nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 8 23, 28 43, complements thereof, and fragments of either.

The present invention includes and provides a substantially purified first nucleic acid molecule with nucleic acid sequence which specifically hybridizes to a second nucleic acid molecule having a nucleic acid sequence selected from the group consisting of a complement of SEQ ID NOs: 5, 6, 8 23, 28 43.

The present invention includes and provides a substantially purified nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 7, 44 47, and 50 53, complements thereof, and fragments of either.

The present invention includes and provides a substantially purified first nucleic acid molecule with nucleic acid sequence which specifically hybridizes to a second nucleic acid molecule having a nucleic acid sequence selected from the group consisting of a complement of SEQ ID NOs: 50 53.

The present invention includes and provides a substantially purified protein or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1097, 1098, and 1100 1115 and fragments thereof.

The present invention includes and provides a substantially purified protein or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs 1099, and 1116 1119 and fragments thereof.

The present invention includes and provides a transformed plant having a nucleic acid molecule which comprises: (A) an exogenous promoter region which functions in a plant cell to cause the production of a mRNA molecule; (B) a structural nucleic acid molecule encoding a protein or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1097, 1100, 1098, 1101, 1102 1115; and (C) a 3' non-translated sequence that functions in the plant cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of the mRNA molecule.

The present invention includes and provides a transformed plant having a nucleic acid molecule which comprises: (A) an exogenous promoter region which functions in a plant cell to cause the production of a mRNA molecule; (B) a structural nucleic acid molecule encoding a protein or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1099, 1116 1119; and (C) a 3' non-translated sequence that functions in the plant cell to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of the mRNA molecule.

The present invention includes and provides a transgenic seed having a nucleic acid molecule which comprises: (A) an exogenous promoter region which functions to cause the production of a mRNA molecule; (B) a structural nucleic acid molecule encoding a protein or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1097, 1100, 1098, 1101, 1102 1115; and (C) a 3' non-translated sequence that functions to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of the mRNA molecule.

The present invention includes and provides a transgenic seed having a nucleic acid molecule which comprises: (A) an exogenous promoter region which functions to cause the production of a mRNA molecule; (B) a structural nucleic acid molecule encoding a protein or fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1099, 1116 1119; and (C) a 3' non-translated sequence that functions to cause termination of transcription and addition of polyadenylated ribonucleotides to a 3' end of the mRNA molecule.

DESCRIPTION OF THE FIGURES

FIG. 1 is an amino acid sequence alignment of the leucine rich repeat domain of rhg1.

FIG. 2 is an amino acid sequence alignment of the leucine rich repeat domain of Rhg4.

DESCRIPTION OF THE SEQUENCE LISTINGS

The following sequence listings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these sequences in combination with the detailed description presented herein.

SEQ ID NOs: 1 7 and 1097 1099 all refer to sequences from the line A3244.

SEQ ID NO: 1 is sequence ID 515002_region_G2 from line A3244, and is adjacent to the contig containing rhg1.

SEQ ID NO: 2 is sequence ID 240017_region_G3 from line A3244, and contains the rhg1, v.1 four exon gene at coding coordinates 45163 45314, 45450 45509, 46941 48763, 48975 49573. The amino acid translation for SEQ ID NO: 2 is SEQ ID NO: 1097.

SEQ ID NO: 3 is sequence ID 240017_region_G3 from line A3244, and contains the rhg1, v.2 two exon gene at coding coordinates 46798 48763 and 48975 49573. The amino acid translation for SEQ ID NO: 3 is SEQ ID NO: 1098.

SEQ ID NO: 4 is sequence ID 318013_region_A3 from line A3244, contains the Rhg4 gene at coding coordinates 111805 113968 and 114684 115204, and has an amino acid translation of SEQ ID NO: 1099.

SEQ ID NO: 5 is sequence ID 240017_region_G3.sub.--8_mRNA, and comprises the two rhg1, v.2 exons from the coding sequence portion of SEQ ID NO: 3.

SEQ ID NO: 6 is sequence ID 240017_region_G3.sub.--8_cds, and comprises the four rhg1, v.1 exons from the coding sequence portion of SEQ ID NO: 2.

SEQ ID NO: 7 is sequence ID 318013_region_A3.sub.--17cds, and comprises the Rhg4 coding sequence portion from SEQ ID NO: 4.

SEQ ID NOs: 8 43 and 1100 1115 all refer to rhg1 sequences.

SEQ ID NO: 8 is sequence ID rhg1_A3244_amplicon from line A3244, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1891 3713, and 3925 4523, and has an amino acid translation of SEQ ID NO: 1100 and 1097.

SEQ ID NO: 9 is sequence ID rhg1_A3244 amplicon, contains two rhg1, v.2 exons at coding coordinates 1748 3713 and 3925 4523 and has an amino acid translation of SEQ ID NO: 1101 and 1098.

SEQ ID NO: 10 is sequence ID rhg1_peking_amplicon from the line peking, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1888 3710, and 3903 4501, and has an amino acid translation of SEQ ID NO: 1102.

SEQ ID NO: 11 is sequence ID rhg1_peking_amplicon, contains two rhg1, v.2 exons at coding coordinates 1745 3710 and 3903 4501, and has an amino acid translation of SEQ ID NO: 1103.

SEQ ID NO: 12 is sequence ID rhg1_toyosuzu_amplicon from the line toyosuzu, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1890 3712, and 3924 4522, and has an amino acid translation of SEQ ID NO: 1104.

SEQ ID NO: 13 is sequence ID rhg1_toyosuzu_amplicon, contains two rhg1, v.2 exons at coding coordinates 1747 3712 and 3924 4522, and has an amino acid translation of SEQ ID NO: 1105.

SEQ ID NO: 14 is sequence ID rhg1_will_amplicon from the line will, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1891 3713, and 3925 4523, and has an amino acid translation of SEQ ID NO: 1106.

SEQ ID NO: 15 is sequence ID rhg1_will_amplicon, contains two rhg1, v.2 exons at coding coordinates 1748 3713 and 3925 4523, and has an amino acid translation of SEQ ID NO: 1107.

SEQ ID NO: 16 is sequence ID rhg1_a2704_amplicon from the line A2704, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1891 3713, and 3925 4523, and has an amino acid translation of SEQ ID NO: 1108.

SEQ ID NO: 17 is sequence ID rhg1_a2704_amplicon, contains two rhg1, v.2 exons at coding coordinates 1748 3713 and 3925 4523, and has an amino acid translation of SEQ ID NO: 1109.

SEQ ID NO: 18 is sequence ID rhg1_noir_amplicon from the line noir, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1876 3698, and 3910 4508, and has an amino acid translation of SEQ ID NO: 1110.

SEQ ID NO: 19 is sequence ID rhg1_noir_amplicon, contains two rhg1, v.2 exons at coding coordinates 1733 3698 and 3910 4508, and has an amino acid translation of SEQ ID NO: 1111.

SEQ ID NO: 20 is sequence ID rhg1_lee_amplicon from the line lee, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1876 3698, and 3910 4508, and has an amino acid translation of SEQ ID NO: 1112.

SEQ ID NO: 21 is sequence ID rhg1_lee_amplicon, contains two rhg1, v.2 exons at coding coordinates 1733 3698 and 3910 4508, and has an amino acid translation of SEQ ID NO: 1113.

SEQ ID NO: 22 is sequence ID rhg1_pi200499_amplicon from the line PI200499, contains four rhg1, v.1 exons at coding coordinates 113 264, 400 459, 1876 3698, and 3910 4508, and has an amino acid translation of SEQ ID NO: 1114.

SEQ ID NO: 23 is sequence ID rhg1_pi200499_amplicon, contains two rhg1, v.2 exons at coding coordinates 1733 3698 and 3910 4508, and has an amino acid translation of SEQ ID NO: 1115.

SEQ ID NO: 24 is sequence ID 240017_region_G3_forward.sub.--1, is a primer that hybridizes to coordinates 45051 45077 on contig 240017_region_G3 before the start codon, and can be used with SEQ ID NO: 25.

SEQ ID NO: 25 is sequence ID 240017_region_G3_reverse.sub.--1, is a primer that hybridizes to coordinates 47942 47918 on contig 240017 region G3, and can be used with SEQ ID NO: 24.

SEQ ID NO: 26 is sequence ID 240017_region_G3_forward.sub.--2, is a primer that hybridizes to coordinates 47808 47831 on contig 240017_region_G3, and can be used with SEQ ID NO: 27.

SEQ ID NO: 27 is sequence ID 240017_region_G3_reverse.sub.--2, is a primer that hybridizes to coordinates 49553 49531 of contig 240017_region_G3 prior to the stop codon, and can be used with SEQ ID NO: 26.

Primers given by SEQ ID NOs: 24 27 are used to create the amplicons of SEQ ID NOs: 8 23. The final 22 bases are added to the actual amplicons in order to simulate the rest of the gene to the stop codon, in order to allow complete translation.

SEQ ID NO: 28 is sequence ID rhg1_A3244_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 8.

SEQ ID NO: 29 is sequence ID rhg1_peking_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 10.

SEQ ID NO: 30 is sequence ID rhg1_toyosuzu_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 12.

SEQ ID NO: 31 is sequence ID rhg1_will_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 14.

SEQ ID NO: 32 is sequence ID rhg1_a2704_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 16.

SEQ ID NO: 33 is sequence ID rhg1_noir_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 18.

SEQ ID NO: 34 is sequence ID rhg1_lee_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 20.

SEQ ID NO: 35 is sequence ID rhg1_pi200499_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 22.

SEQ ID NO: 36 is sequence ID rhg1_A3244_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 9.

SEQ ID NO: 37 is sequence ID rhg1_peking_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 11.

SEQ ID NO: 38 is sequence ID rhg1_toyosuzu_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 13.

SEQ ID NO: 39 is sequence ID rhg1_will_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 15.

SEQ ID NO: 40 is sequence ID rhg1_a2704_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 17.

SEQ ID NO: 41 is sequence ID rhg1_noir_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 19.

SEQ ID NO: 42 is sequence ID rhg1_lee_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 21.

SEQ ID NO: 43 is sequence ID rhg1_pi200499_amplicon_cds.sub.--2, which is the coding sequence portion of SEQ ID NO: 23.

SEQ ID NOs: 44 53 and 1116 1119 all refer to Rhg4 sequences

SEQ ID NO: 44 is sequence ID rhg4_a3244_amplicon from the line A3244, contains Rhg4 at coding coordinates 79 2242 and 2958 3478, is made using SEQ ID NOs: 48 and 49, and has an amino acid translation of SEQ ID NO: 1116 and 1099.

SEQ ID NO: 45 is sequence ID rhg4_Minsoy_amplicon from the line Minsoy, contains Rhg4 at coding coordinates 79 2242 and 2958 3478, is made using SEQ ID NOs: 48 and 49, and has an amino acid translation of SEQ ID NO: 1117.

SEQ ID NO: 46 is sequence ID rhg4_Jack_amplicon from the line Jack, contains Rhg4 at coding coordinates 79 2242 and 2958 3478, is made using SEQ ID NO: 48 and 49, and has an amino acid translation of SEQ ID NO: 1118.

SEQ ID NO: 47 is sequence ID rhg4_peking_amplicon from the line Peking, contains Rhg4 at coding coordinates 79 2242 and 2958 3478, is made using SEQ ID NOs: 48 and 49, and has an amino acid translation of SEQ ID NO: 1119.

SEQ ID NO: 48 is sequence ID 318013_region_A3_forward, hybridizes to coordinates 111727 111756 of contig 318013_region_A3, and is a primer used with SEQ ID NO: 49 to create Rhg4 amplicons.

SEQ ID NO: 49 is sequence ID 318013_region_A3_reverse, hybridizes to coordinates 115206 115177 of contig 318013_region_A3, and is a primer used with SEQ ID NO: 48 to create Rhg4 amplicons.

SEQ ID NO: 50 is sequence ID rhg4_A3244_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 44.

SEQ ID NO: 51 is sequence ID rhg4_Minsoy_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 45.

SEQ ID NO: 52 is sequence ID rhg4_Jack_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 46.

SEQ ID NO: 53 is sequence ID rhg4_peking_amplicon_cds, which is the coding sequence portion of SEQ ID NO: 47.

SEQ ID NO: 1120 is sequence ID consensusLRR, which is a consensus sequence for the LRR repeats shown in FIGS. 1 and 2.

SEQ ID NO: 1121 is sequence ID rhg1LRR, which is the amino acid sequence of the LRR domain shown in FIG. 1.

SEQ ID NO: 1122 is sequence ID Rhg4LRR, which is the amino acid sequence of the LRR domain shown in FIG. 2.

SEQ ID NO: 1123 is sequence ID 240017_region_G3_forward.sub.--1_b, which is an alternate primer that hybridizes to coordinates 45046 45072 on contig 240017_region_G3 before the start codon, and which can be used with SEQ ID NO: 25.

Table 1 below provides further information on the sequences described herein.

In table 1, for all rows, "Seq Num" refers to the corresponding SEQ ID NO in the sequence listing.

For rows with SEQ ID NOs: 1 53 and 1120 1123"Seq ID" refers to the name of the SEQ ID NO given in the "Seq Num" column.

For rows with SEQ ID NOs: 2 4,8 23, and 44 47"Coding Sequence" refers to the coordinates of the coding portion of the SEQ ID NO given in the "Seq Num" column, and "AA" refers to the SEQ ID NO that is the amino acid translation of the SEQ ID NO given in the "Seq Num" column.

For rows with SEQ ID NOs: 24 27 and 1123, "Primer location on 240017_region_G3" refers to the coordinates of the 240017_region_G3 contig to which the SEQ ID NO given in the "Seq Num" column hybridizes.

For rows with SEQ ID NOs: 48 and 49, "Primer location on 318013region_A3" refers to the coordinates of the 318013_region_A3 contig to which the SEQ ID NO given in the "Seq Num" column hybridizes.

For rows with SEQ ID NOs: 54 400, "Seq ID" refers to the names of amplicon sequences. Within the Seq ID is the "_" (double length underscore) symbol. The name before this symbol refers to the name of the contig in which the amplicon is found, and the numbers after this symbol refer to the nucleotide location of the SSR on the contig.

For rows with SEQ ID NOs: 401 1096, "Seq ID" refers to the names of primer sequences used in PCR to generate the amplicon sequences in table 1. For these rows, the "Seq ID" name contains the same name as the amplicon that is generated by the pair of primers of which the SEQ ID NO referred to in the first column is a member. The "Seq ID" name also contains either "Forward" or "Reverse," which indicates the orientation of the primer. For these sequences, "location of primer on contig start" and "location of primer on contig end" refer, respectively, to the first and last base number of the contig on which the primer aligns.

TABLE-US-00001 TABLE 1 Seq Num Seq ID Coding Sequence AA No. 1 515O02_region_G2 2 240O17_region_G3 45163 45314, 45450 45509, 46941 48763, 48975 49573 1097 3 240O17_region_G3 46798 48763, 48975 49573 1098 4 318O13_region_A3 111805 113968, 114684 115204 1099 5 240O17_region_G3_8_mRNA 6 240O17_region_G3_8_cds 7 318O13_region_A3_17_cds 8 rhg1_A3244_amplicon 113 264, 400 459, 1891 3713, 3925 4523 1100 9 rhg1_A3244_amplicon 1748 3713, 3925 4523 1101 10 rhg1_peking_amplicon 113 264, 400 459, 1888 3710, 3903 4501 1102 11 rhg1_peking_amplicon 1745 3710, 3903 4501 1103 12 rhg1_toyosuzu_amplicon 113 264, 400 459, 1890 3712, 3924 4522 1104 13 rhg1_toyosuzu_amplicon 1747 3712, 3924 4522 1105 14 rhg1_will_amplicon 113 264, 400 459, 1891 3713, 3925 4523 1106 15 rhg1_will_amplicon 1748 3713, 3925 4523 1107 16 rhg1_a2704_amplicon 113 264, 400 459, 1891 3713, 3925 4523 1108 17 rhg1_a2704_amplicon 1748 3713, 3925 4523 1109 18 rhg1_noir_amplicon 113 264, 400 459, 1876 3698, 3910 4508 1110 19 rhg1_noir_amplicon 1733 3698, 3910 4508 1111 20 rhg1_lee_amplicon 113 264, 400 459, 1876 3698, 3910 4508 1112 21 rhg1_lee_amplicon 1733 3698, 3910 4508 1113 22 rhg1_pi200499_amplicon 113 264, 400 459, 1876 3698, 3910 4508 1114 23 rhg1_pi200499_amplicon 1733 3698, 3910 4508 1115 Primer location on 240O17_region_G3 24 240O17_region_G3_forward_1 45051 45077 25 240O17_region_G3_reverse_1 47942 47918 26 240O17_region_G3_forward_2 47808 47831 27 240O17_region_G3_reverse_2 49553 49531 28 rhg1_A3244_amplicon_cds 29 rhg1_peking_amplicon_cds 30 rhg1_toyosuzu_amplicon_cds 31 rhg1_will_amplicon_cds 32 rhg1_a2704_amplicon_cds 33 rhg1_noir_amplicon_cds 34 rhg1_lee_amplicon_cds 35 rhg1_pi200499_amplicon_cds 36 rhg1_A3244_amplicon_cds_2 37 rhg1_peking_amplicon_cds_2 38 rhg1_toyosuzu_amplicon_cds_2 39 rhg1_will_amplicon_cds_2 40 rhg1_a2704_amplicon_cds_2 41 rhg1_noir_amplicon_cds_2 42 rhg1_lee_amplicon_cds_2 43 rhg1_pi200499_amplicon_cds_2 Coding Sequence 44 rhg4_a3244_amplicon 79 2242, 2958 3478 1116 45 rhg4_Minsoy_amplicon 79 2242, 2958 3478 1117 46 rhg4_Jack_amplicon 79 2242, 2958 3478 1118 47 rhg4_peking_amplicon 79 2242, 2958 3478 1119 Primer location on 318O13_region_A3 48 318O13_region_A3_forward 111727 111756 49 318O13_region_A3_reverse 115206 115177 50 rhg4_A3244_amplicon_cds 51 rhg4_Minsoy_amplicon_cds 52 rhg4_Jack_amplicon_cds 53 rhg4_peking_amplicon_cds 54 240O17_region_G3_289711_11 55 240O17_region_G3_236585_14 56 240O17_region_G3_168772_13 57 240O17_region_G3_332420_21 58 240O17_region_G3_228126_18 59 240O17_region_G3_139723_11 60 240O17_region_G3_280585_14 61 240O17_region_G3_70509_14 62 240O17_region_G3_50537_17 63 240O17_region_G3_231556_17 64 240O17_region_G3_117057_11 65 240O17_region_G3_23092_13 66 240O17_region_G3_297741_14 67 240O17_region_G3_206502_14 68 240O17_region_G3_221223_13 69 240O17_region_G3_169084_14 70 240O17_region_G3_94891_14 71 240O17_region_G3_281852_61 72 240O17_region_G3_46583_12 73 240O17_region_G3_306835_13 74 240O17_region_G3_85471_12 75 240O17_region_G3_257208_12 76 240O17_region_G3_150390_17 77 240O17_region_G3_34697_75 78 240O17_region_G3_150374_13 79 240O17_region_G3_40513_22 80 240O17_region_G3_268602_14 81 240O17_region_G3_25357_13 82 240O17_region_G3_137548_13 83 240O17_region_G3_139131_13 84 240O17_region_G3_203855_12 85 240O17_region_G3_199049_15 86 240O17_region_G3_320907_12 87 240O17_region_G3_16407_17 88 240O17_region_G3_206516_17 89 240O17_region_G3_264495_13 90 240O17_region_G3_156785_13 91 240O17_region_G3_187129_12 92 240O17_region_G3_214106_13 93 240O17_region_G3_149013_12 94 240O17_region_G3_326352_16 95 240O17_region_G3_278962_12 96 240O17_region_G3_256930_13 97 240O17_region_G3_29646_14 98 240O17_region_G3_29618_13 99 240O17_region_G3_108561_14 100 240O17_region_G3_143975_14 101 240O17_region_G3_108431_20 102 240O17_region_G3_281764_11 103 240O17_region_G3_130058_15 104 240O17_region_G3_310590_52 105 240O17_region_G3_313405_14 106 240O17_region_G3_302190_13 107 240O17_region_G3_225343_17 108 240O17_region_G3_208823_14 109 240O17_region_G3_74285_11 110 240O17_region_G3_109052_16 111 240O17_region_G3_6395_12 112 240O17_region_G3_244905_16 113 240O17_region_G3_244956_13 114 240O17_region_G3_117220_13 115 240O17_region_G3_134707_14 116 240O17_region_G3_35078_13 117 240O17_region_G3_210506_16 118 240O17_region_G3_116961_26 119 240O17_region_G3_51073_13 120 240O17_region_G3_55291_15 121 240O17_region_G3_229651_18 122 240O17_region_G3_303308_19 123 240O17_region_G3_168373_20 124 240O17_region_G3_253333_17 125 240O17_region_G3_5791_13 126 240O17_region_G3_206841_19 127 240O17_region_G3_202827_12 128 240O17_region_G3_322656_13 129 240O17_region_G3_111841_14 130 240O17_region_G3_192719_13 131 240O17_region_G3_195630_17 132 240O17_region_G3_69999_13 133 240O17_region_G3_11176_13 134 240O17_region_G3_228643_13 135 240O17_region_G3_88478_19 136 240O17_region_G3_108950_13 137 240O17_region_G3_121054_14 138 240O17_region_G3_188337_14 139 240O17_region_G3_255944_21 140 240O17_region_G3_219518_14 141 240O17_region_G3_235601_15 142 240O17_region_G3_301529_13 143 240O17_region_G3_94795_14 144 240O17_region_G3_46703_23 145 240O17_region_G3_59616_14 146 240O17_region_G3_296933_15 147 240O17_region_G3_192428_17 148 240O17_region_G3_191490_14 149 240O17_region_G3_201115_11 150 240O17_region_G3_72882_15 151 240O17_region_G3_69514_13 152 240O17_region_G3_37699_47 153 240O17_region_G3_11301_29 154 240O17_region_G3_141875_12 155 240O17_region_G3_98090_18 156 240O17_region_G3_43298_35 157 240O17_region_G3_262094_11 158 240O17_region_G3_262079_15 159 240O17_region_G3_59090_12 160 240O17_region_G3_245723_13 161 240O17_region_G3_194628_54 162 240O17_region_G3_4566_16 163 240O17_region_G3_96209_14 164 240O17_region_G3_248715_17 165 240O17_region_G3_71410_40 166 240O17_region_G3_226519_13 167 240O17_region_G3_11282_19 168 240O17_region_G3_170504_12 169 240O17_region_G3_40864_14 170 240O17_region_G3_13529_14 171 240O17_region_G3_22858_14 172 240O17_region_G3_309211_13 173 240O17_region_G3_55568_26 174 240O17_region_G3_73238_16 175 240O17_region_G3_52488_19 176 318O13_region_A3_471518_14 177 318O13_region_A3_231599_23 178 318O13_region_A3_375912_13 179 318O13_region_A3_180013_12 180 318O13_region_A3_171606_14 181 318O13_region_A3_416256_13 182 318O13_region_A3_231395_15 183 318O13_region_A3_5502_47 184 318O13_region_A3_93061_14 185 318O13_region_A3_111684_19 186 318O13_region_A3_69328_14 187 318O13_region_A3_36529_17 188 318O13_region_A3_139128_12 189 318O13_region_A3_495674_13 190 318O13_region_A3_187577_13 191 318O13_region_A3_453036_14 192 318O13_region_A3_374041_13 193 318O13_region_A3_3412_11 194 318O13_region_A3_276495_28 195 318O13_region_A3_151839_17 196 318O13_region_A3_292912_12 197 318O13_region_A3_104560_12 198 318O13_region_A3_65193_11 199 318O13_region_A3_110573_70 200 318O13_region_A3_65117_12 201 318O13_region_A3_490837_16 202 318O13_region_A3_107448_11 203 318O13_region_A3_331_23 204 318O13_region_A3_193470_13 205 318O13_region_A3_183305_14 206 318O13_region_A3_55050_14 207 318O13_region_A3_224693_21 208 318O13_region_A3_207216_12 209 318O13_region_A3_4654_22 210 318O13_region_A3_408959_13 211 318O13_region_A3_132288_22 212 318O13_region_A3_292822_20 213 318O13_region_A3_311076_12 214 318O13_region_A3_509623_13 215 318O13_region_A3_190404_14 216 318O13_region_A3_164916_15 217 318O13_region_A3_21028_13 218 318O13_region_A3_208012_17 219 318O13_region_A3_484089_14 220 318O13_region_A3_332780_17 221 318O13_region_A3_480137_37 222 318O13_region_A3_441056_14 223 318O13_region_A3_77486_11 224 318O13_region_A3_272468_11 225 318O13_region_A3_425319_17 226 318O13_region_A3_413879_31 227 318O13_region_A3_80477_64 228 318O13_region_A3_277272_50 229 318O13_region_A3_509642_13 230 318O13_region_A3_321771_14 231 318O13_region_A3_26788_12 232 318O13_region_A3_262706_16 233 318O13_region_A3_243928_16 234 318O13_region_A3_23246_14 235 318O13_region_A3_165406_12 236 318O13_region_A3_486294_14 237 318O13_region_A3_46754_12 238 318O13_region_A3_381116_15 239 318O13_region_A3_350369_11 240 318O13_region_A3_138841_13

241 318O13_region_A3_12158_14 242 318O13_region_A3_315368_13 243 318O13_region_A3_307549_13 244 318O13_region_A3_159857_14 245 318O13_region_A3_140551_15 246 318O13_region_A3_279869_11 247 318O13_region_A3_78292_35 248 318O13_region_A3_185019_12 249 318O13_region_A3_409164_13 250 318O13_region_A3_75392_14 251 318O13_region_A3_231320_12 252 318O13_region_A3_381102_14 253 318O13_region_A3_491826_15 254 318O13_region_A3_56365_21 255 318O13_region_A3_372628_15 256 318O13_region_A3_302609_11 257 318O13_region_A3_341804_11 258 318O13_region_A3_217037_11 259 318O13_region_A3_264929_68 260 318O13_region_A3_55499_12 261 318O13_region_A3_295634_14 262 318O13_region_A3_269358_15 263 318O13_region_A3_457009_24 264 318O13_region_A3_176598_14 265 318O13_region_A3_278266_12 266 318O13_region_A3_391810_12 267 318O13_region_A3_269485_15 268 318O13_region_A3_359247_17 269 318O13_region_A3_315094_13 270 318O13_region_A3_307823_13 271 318O13_region_A3_248588_15 272 318O13_region_A3_252426_85 273 318O13_region_A3_513314_16 274 318O13_region_A3_68183_14 275 318O13_region_A3_471191_13 276 318O13_region_A3_163547_18 277 318O13_region_A3_417867_15 278 318O13_region_A3_332465_14 279 318O13_region_A3_207697_14 280 318O13_region_A3_277229_43 281 318O13_region_A3_36366_11 282 318O13_region_A3_91970_12 283 318O13_region_A3_211533_11 284 318O13_region_A3_336301_11 285 318O13_region_A3_441603_14 286 318O13_region_A3_468354_15 287 318O13_region_A3_188983_18 288 318O13_region_A3_115502_17 289 318O13_region_A3_163006_13 290 318O13_region_A3_119283_14 291 318O13_region_A3_491126_11 292 318O13_region_A3_99512_21 293 318O13_region_A3_280291_17 294 318O13_region_A3_138443_19 295 318O13_region_A3_115973_14 296 318O13_region_A3_329977_14 297 318O13_region_A3_205203_14 298 318O13_region_A3_153114_12 299 318O13_region_A3_34581_13 300 318O13_region_A3_292577_19 301 318O13_region_A3_445391_20 302 318O13_region_A3_350540_17 303 318O13_region_A3_453879_15 304 318O13_region_A3_201246_13 305 318O13_region_A3_326020_13 306 318O13_region_A3_503801_14 307 318O13_region_A3_302400_52 308 318O13_region_A3_448857_15 309 318O13_region_A3_48364_14 310 318O13_region_A3_251804_48 311 318O13_region_A3_382583_13 312 318O13_region_A3_124737_14 313 318O13_region_A3_124766_13 314 318O13_region_A3_461351_16 315 318O13_region_A3_64953_19 316 318O13_region_A3_366586_13 317 318O13_region_A3_46190_15 318 318O13_region_A3_81016_11 319 318O13_region_A3_134426_14 320 318O13_region_A3_292724_14 321 318O13_region_A3_187096_17 322 318O13_region_A3_381693_13 323 318O13_region_A3_361286_33 324 318O13_region_A3_482668_14 325 318O13_region_A3_128002_12 326 318O13_region_A3_499270_14 327 318O13_region_A3_231650_12 328 318O13_region_A3_199851_13 329 318O13_region_A3_324629_13 330 318O13_region_A3_374190_19 331 318O13_region_A3_460603_13 332 318O13_region_A3_108681_14 333 318O13_region_A3_459791_47 334 318O13_region_A3_4257_20 335 318O13_region_A3_238810_14 336 318O13_region_A3_245817_14 337 318O13_region_A3_245956_14 338 318O13_region_A3_74148_14 339 318O13_region_A3_74089_15 340 318O13_region_A3_241686_12 341 318O13_region_A3_47476_12 342 318O13_region_A3_164550_12 343 318O13_region_A3_101255_15 344 515O02_region_G2_16189_11 345 515O02_region_G2_71925_13 346 515O02_region_G2_4707_12 347 515O02_region_G2_118904_18 348 515O02_region_G2_13655_17 349 515O02_region_G2_53900_13 350 515O02_region_G2_8079_14 351 515O02_region_G2_9969_28 352 515O02_region_G2_72308_77 353 515O02_region_G2_99475_19 354 515O02_region_G2_118615_18 355 515O02_region_G2_119001_46 356 515O02_region_G2_118958_43 357 515O02_region_G2_17197_13 358 515O02_region_G2_105163_29 359 515O02_region_G2_111335_13 360 515O02_region_G2_106396_13 361 515O02_region_G2_59229_17 362 515O02_region_G2_73795_20 363 515O02_region_G2_85664_20 364 515O02_region_G2_36921_17 365 515O02_region_G2_124150_19 366 515O02_region_G2_5089_14 367 515O02_region_G2_58221_15 368 515O02_region_G2_96139_14 369 515O02_region_G2_70595_13 370 515O02_region_G2_4340_15 371 515O02_region_G2_90417_11 372 515O02_region_G2_49711_17 373 515O02_region_G2_63053_13 374 515O02_region_G2_63076_14 375 515O02_region_G2_44442_12 376 515O02_region_G2_44422_19 377 515O02_region_G2_44158_19 378 515O02_region_G2_44141_17 379 515O02_region_G2_90762_17 380 515O02_region_G2_106241_14 381 515O02_region_G2_109676_12 382 515O02_region_G2_86242_14 383 515O02_region_G2_83109_12 384 515O02_region_G2_10461_15 385 515O02_region_G2_67608_15 386 515O02_region_G2_63275_46 387 515O02_region_G2_62405_14 388 515O02_region_G2_33563_12 389 515O02_region_G2_33146_14 390 515O02_region_G2_102179_29 391 515O02_region_G2_2646_15 392 515O02_region_G2_76652_24 393 515O02_region_G2_66280_14 394 515O02_region_G2_54768_13 395 515O02_region_G2_62580_14 396 515O02_region_G2_34598_55 397 515O02_region_G2_77680_13 398 515O02_region_G2_77693_12 399 515O02_region_G2_97392_14 400 515O02_region_G2_97359_15 location of primer location of primer on contig start on contig end 401 240O17_region_G3_289711_11_Forward_Primer 289637 289661 402 240O17_region_G3_289711_11_Reverse_Primer 289756 289732 403 240O17_region_G3_236585_14_Forward_Primer 236511 236535 404 240O17_region_G3_236585_14_Reverse_Primer 236638 236614 405 240O17_region_G3_168772_13_Forward_Primer 168683 168707 406 240O17_region_G3_168772_13_Reverse_Primer 168811 168786 407 240O17_region_G3_332420_21_Forwar