Association of Root-Knot Nematode Resistance Genes with Simple Sequence Repeat Markers on Two Chromosomes in Cotton Pavathi Ynturi, Johnie N. Jenkins,* Jack C. McCarty, Jr., Osman A. Gutierrez, and Sukumar Saha ABSTRACT Breeding for root-knot nematode (RKN) [Meloidogyne incog- nita (Kofoid & White) Chitwood] resistance in cotton (Gossypium hirsutum L.) is hindered by intensive screening procedures. Identifi- cation of DNA markers associated with RKN resistance would pro- vide tools for marker-assisted selection (MAS). The objective of this study was to identify DNA markers associated with RKN resistance and associate these with chromosomes. Parents and an F 2 population from a cross of resistant near isoline (RNIL) 3 susceptible near iso- line (SNIL) were grown in a greenhouse, inoculated with RKN eggs, and scored for gall index, followed by genotyping with simple se- quence repeats (SSRs). The source of the resistance was from the Auburn 634 line. Genotype analysis was conducted on 86 F 2 plants with nine polymorphic SSR markers. Additive dominance model analysis showed that Brookhaven National Laboratory (BNL) SSR markers BNL 3661, 3644, 3545, and 1231 accounted for 21, 19, 12, and 11% of the variation in gall index, respectively. BNL 3661 and 1231 together accounted for 31% of the variation in gall index. BNL 3661 had significant additive and dominant genetic effects of 0.61 and 0.50, respectively. BNL 1231 had significant additive genetic effects of 0.51 and no dominant effects. BNL 3661, 3544, and 3645 were linked and these markers were located on the short arm of chromosome 14. BNL 1231 is located on the long arm of chromosome 11. The association of two different chromosomes with RKN resistance suggests at least two genes are involved in RKN gall score in the cross studied. T HE ROOT -KNOT NEMATODE is a serious pest of cotton. The nematode produces giant cells that act as met- abolic sinks, reducing the ability of the root system to provide nutrients and water to the shoot. Conventional breeding programs for developing RKN resistance are time-consuming, labor-intensive, and may require both greenhouse and field evaluations. Identity of DNA markers closely associated with RKN resistant gene(s) will expedite the breeding process by allowing marker- assisted selection (MAS). McPherson (1993) and McPherson et al. (2004) re- ported that a minimum of two genes control the high level of RKN resistance in M-315 RNR and one gene in moderately resistant M-78 RNR. Jenkins et al. (1995) data on postpenetration development of RKN in M-78 RNR and M-315 RNR also support the two-gene model. McPherson et al. (1995) reported that two major genes, one dominant and one additive gene controlled RKN resistance in Auburn 623 RNR-derived lines and named the dominant genes Mi 1 and the additive gene Mi 2 . Bezawada et al. (2003) reported in their study that a single recessive gene controlled the RKN resistance trait in moderately resistant Clevewilt 6-1. They also reported weak association with BNL 1421; however, this marker showed distorted segregation which they indicated may have caused a false linkage. Wang et al. (2006a) re- ported a single recessive gene in linkage group A03 was responsible for the resistance in Acala NemX. They fur- ther reported SSR marker CIR 316a to be 2.6 CM away from this recessive gene they call rkn1 and SSR marker BNL 1231 was 18.4 units away. Zhou (1999) and Zhou et al. (1999) reported that a single recessive gene con- trolled the resistance in moderately resistant commercial cotton cultivars, Acala NemX and Stoneville LA 887, and two major genes in the resistant cotton line M-240 RNR (Shepherd et al., 1989, 1996). The later was derived from the Auburn 634 source (Shepherd, 1982, 1983). Turcotte et al. (1963) reported that two homozygous recessive genes conditioned the resistance in the F 2 population in a cross of G. barbadense L. The objective of this research was to identify SSR markers associated with RKN genes and to assign these markers to specific chromosomes. MATERIALS AND METHODS A susceptible near isogenic line (SNIL), a resistant near isogenic line (RNIL), and an F 2 population of 86 plants were used in this study. The near isogenic lines were developed in our research program by crossing a highly resistant Auburn 634 RNR-derived line with susceptible cultivar Stoneville 213 (ST213) and backcrossing four times to ST213 while selecting resistant and susceptible sister lines. We used the modified methods of Shepherd (1979) to screen for RKN resistance in the greenhouse. Roots from RKN, race 3, infected tomato, and cotton plants were placed in 1.05% NaOCl solution and placed on a mechanical shaker for 3 min following the method of Hussey and Barker (1973) to obtain inoculum. Individual plants of parental lines (M8 susceptible check, M- 315 RNR resistant check) and F 2 were grown in 8.9 3 7.6 cm (diameter 3 depth) plastic pots placed in equidistant holes of 6-cm depth in the greenhouse beds. The pots and greenhouse beds were filled with screened, methyl bromide-fumigated Wickham sandy loam soil (fine-loamy, mixed, semiactive, ther- mic Typic Hapludults). Those pots were inoculated with ap- proximately 10 000 RKN eggs after planting a seed. The bed was covered for 7 d with sequential layers of brown paper and P. Ynturi, Dep. of Plant and Soil Sciences, Mississippi State Univ., Mississippi State, MS, 39762; J.N. Jenkins, J.C. McCarty, Jr., O.A. Gutierrez, and S. Saha, USDA-ARS, Crop Science Research Lab., Mississippi State, MS 39762. Mention of trade names or commercial products in this manuscript does not imply recommendations or en- dorsement by the USDA. Joint Contribution of USDA, ARS, and Mississippi State University. Journal paper No.J-10952 of Mississippi Agricultural and Forestry Experiment Station. Received 15 May 2006. *Corresponding author (jnjenkins@ars.usda.gov). Published in Crop Sci. 46:2670–2674 (2006). Crop Breeding & Genetics doi:10.2135/cropsci2006.05.0319 ª Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: BNL, Brookhaven National Laboratory; LOD, log likelihood ratio; MAS, marker-assisted selection; RKN, root-knot nematode; RNIL, resistant near isogenic line; SNIL, susceptible near isogenic line; ST213, Stoneville 213; SSR, simple sequence repeats. Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved. 2670 Published online November 21, 2006