Improving cotton (Gossypium hirsutum L.) plant resistance to reniform nematodes by pyramiding Ren 1 and Ren 2

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1 Plant Breeding 130, (2011) doi: /j x Published This article is a US Government work and is in the public domain in the USA Improving cotton (Gossypium hirsutum L.) plant resistance to reniform nematodes by pyramiding Ren 1 and Ren 2 D AVID D. FANG 1 and S ALLIANA R. STETINA 2 1 Cotton Fiber Bioscience Research Unit, USDA-ARS-SRRC, 1100 Robert E. Lee Blvd, New Orleans, LA 70124, USA, david.fang@ars.usda.gov; 2 Crop Genetics Research Unit, USDA-ARS, 141 Experiment Station Road, Stoneville, MS 38776, USA With 4 tables Received April 28, 2011/Accepted June 11, 2011 Communicated by J. Jenkins Abstract Reniform nematode (Rotylenchulus reniformis) has become a major pest of cotton in the mid-south area of the United States. Resistance genes, Ren 1 and Ren 2 from Gossypium longicalyx and Gossypium aridum, respectively, have been identified and introduced into Upland cotton (Gossypium hirsutum). We developed an F 2 population of 184 progeny plants by crossing Ren 2 -containing plants with a LONREN-2 plant that had the resistance gene Ren 1. The F 2 plants were evaluated for their resistance to reniform nematodes in a growth chamber. Microsatellite markers BNL2662 and BNL3279 were analysed to assist the selection of proper parental plants and F 1 progeny and to study the segregation of the resistance genes in the F 2 population. Our results suggested that Ren 1 and Ren 2 were duplicate genes with Ren 1 residing on chromosome 11 (A subgenome) and Ren 2 on chromosome 21 (D subgenome). F 2 plants containing either Ren 1 or Ren 2 had significantly fewer nematodes than the susceptible Upland cotton genotype. No significant difference in nematode resistance was found between plants containing Ren 1 and those having Ren 2, indicating that these two genes may have similar resistance mechanisms. Plants containing both Ren 1 and Ren 2 appeared to have higher resistance than those with just one of the genes, and pyramiding these two genes may be a valuable tool to cotton breeders when managing this pest. Key words: cotton reniform nematode resistance microsatellite markers Gossypium aridum Gossypium longicalyx The reniform nematode (Rotylenchulus reniformis Linford & Oliveira) is an obligate plant parasite that feeds on roots. In recent years, it has been expanding its geographic distribution in the United States and has become a major pest of cotton (Robinson 2007). Currently, reniform nematode causes US cotton production losses estimated to exceed $100 million annually (Blasingame 2006). In Mississippi, Louisiana and Alabama, it has emerged as a very important pathogen that caused about 7% yield loss in 2008 (Blasingame et al. 2009). No sources of host-plant resistance have been found in cultivated Upland cotton (Gossypium hirsutum L.) despite extensive evaluation of more than 2000 G. hirsutum accessions by various researchers (Robinson et al. 1999, Usery et al. 2005, Weaver et al. 2007). To date, control of reniform nematodes has been largely limited to crop rotation and application of nematicides (Stetina et al. 2007). Breeding Upland cotton varieties with resistance to reniform nematodes will be a great advantage to the US cotton farmers and to the environment. The genus of cotton (Gossypium L.) consists of five allotetraploid (2n = 4x = 52) and 45 diploid (2n = 2x = 26) species (Fryxell 1992). The diploid species fall into eight cytological groups, or ÔgenomesÕ, designated A G and K based on the chromosome-pairing relationships (Endrizzi et al. 1985). To identify reniform nematode-resistant germplasm present in Gossypium species and related plants, Yik and Birchfield (1984) evaluated 200 accessions that included 37 Gossypium species and 29 related species in the Malvaceae family. They found that Gossypium longicalyx Hutchinson & Lee, a diploid F genome species native to Africa, failed to support egg production and was immune to reniform nematodes. Some accessions of Gossypium arboreum L. and Gossypium barbadense L. were highly resistant (egg production <10% of the susceptible check DP16) to the reniform nematodes. None of the 100 G. hirsutum accessions evaluated were resistant. Scientists at various laboratories have been pursuing the introgression of reniform nematode resistance from G. longicalyx or G. arboreum into Upland cotton during the past decade (Sacks and Robinson 2007, Avila et al. 2007, Konan et al. 2007, Robinson et al. 2007, Dighe et al. 2009, Sacks and Robinson 2009, Mergeai et al. 2010). A research group in Texas successfully introduced the resistance from G. longicalyx into Upland cotton (Bell and Robinson 2004, Robinson et al. 2007). The resistance is controlled by one single dominant gene designated Ren lon that resides on chromosome 11 (an A-subgenome chromosome). In the present paper, we change Ren lon to Ren 1 for the reasons described in the Discussion section. Microsatellite (also called simple sequence repeat, SSR) markers were developed to tag this gene, and the closest marker BNL3279_114 was <1 cm away from the gene (Dighe et al. 2009). Two Upland cotton germplasm lines containing the resistance gene Ren 1 were released in 2007 under the names of LONREN-1 and LONREN-2. Concurrently, a research group in Belgium also successfully introduced the reniform nematode resistance from G. longicalyx into Upland cotton using different trispecies hybrids (Konan et al. 2007, Mergeai et al. 2010). In a parallel effort, USDA-ARS scientists in Mississippi have been working to introduce the resistance from the diploid A 2 genome species G. arboreum into Upland cotton. A trispecies hybrid was obtained from a cross between G. arboreum A2-190 (PI615699) and a hexaploid bridging line G371 (Sacks and Robinson 2007). A2-190 was reported as resistant to reniform nematodes (Stewart and Robbins 1995). G371 [2(G. hirsutum NC8 Gossypium aridum)] was originally developed by Belgian scientists in 1980s (Maréchal 1983) with wileyonlinelibrary.com

2 674 D. D. Fang and S. R. Stetina unknown resistance to reniform nematodes. Sacks and Robinson (2009) evaluated 27 S 2 progeny plants from the bridging line G371. They reported that all progeny plants were resistant and concluded that the parental line G371 was resistant. Although the authors could not determine the exact source of the resistance, they postulated that it came from G. aridum (Rose & Standley) Skovsted, a diploid D 4 genome species. Shortly after, we (Romano et al. 2009) further studied this resistance in the BC 1 F 1 and subsequent BC 1 F 2 progeny plants. We confirmed that the resistance was indeed from G. aridum. This resistance was possibly controlled by one single dominant gene designated Ren ari. For the same reasons described in the Discussion section, we change Ren ari to Ren 2 in the present paper. SSR markers BNL2662_90 and BNL3279_132 cosegregated completely with Ren 2. The Ren 2 was tentatively assigned to chromosome 21 (a D-subgenome chromosome) (Romano et al. 2009). However, further investigation was required to confirm this assignment. Because chromosomes 11 and 21 are homeologous chromosomes (Lacape et al. 2003, Guo et al. 2007, Yu et al. 2011), and SSR primer pair BNL3279 amplified fragments that were closely associated with both Ren 1 and Ren 2, we (Romano et al. 2009) suggested that Ren 1 and Ren 2 were likely located on homologous regions of chromosomes 11 and 21, respectively. However, to confirm this hypothesis, it would be necessary to observe the segregation of both Ren 1 and Ren 2 within the same population. Thus, we made crosses between plants containing Ren 2 and a LONREN-2 plant, combined these two resistance genes in one plant and analysed the segregation of reniform nematode resistance in an F 2 population. Through this research, we would like to answer following questions: Is Ren 1 allelic to Ren 2?DoRen 1 and Ren 2 reside on the same chromosome? Or are Ren 1 and Ren 2 duplicate genes that reside on different chromosomes? Is it possible to pyramid these two genes? Does a plant containing both genes have higher resistance than those with just one of the genes? We are herein reporting our research results. Materials and Methods Parental line development: Seeds of LONREN-2 were kindly provided by Dr. Bell, USDA-ARS, College Station, TX, USA. A LONREN-2 plant is homozygous at Ren 1 locus (Robinson et al. 2007). The trispecies hybrid, G. arboreum A2-190 G371 [2(G. hirsutum G. aridum)], was developed by Sacks and Robinson (2009). This hybrid was backcrossed to Upland cotton MD51ne in The pedigree, genomic constitution and resistance to reniform nematodes of the BC 1 F 1 progeny plants were reported by Sacks and Robinson (2009) and Romano et al. (2009). Twenty-six highly resistant BC 1 F 1 plants (<8% of egg production of the susceptible check DP16) were backcrossed to MD51ne in 2008 to obtain BC 2 F 1 seeds. All backcrosses were carried out in a greenhouse in Stoneville, MS. BC 2 F 1 and LONREN-2 plants were grown in a field in Stoneville in A total of 503 BC 2 F 1 plants were obtained. Young leaves were collected from all 503 BC 2 F 1 and 5 LONREN-2 plants when plants were about 30 days old. Then, they were analysed with SSR markers BNL2662 and BNL3279. BC 2 F 1 plants that had marker fragments BNL2662_90 and BNL3279_132 were assumed to be heterozygous at the Ren 2 locus. To avoid confusion with the F 2 population described below, we herein name these BC 2 F 1 plants used in making crosses as hirsutum aridum arboreum hirsutum (HAAH) parents. F 2 population development: Four HAAH parent plants were used as females to cross with a LONREN-2 plant in a field in F 1 seeds were planted in a greenhouse in Stoneville in November Young leaves were collected from each F 1 plant at about 30 days old. SSR markers BNL2662 and BNL3279 were analysed on these samples. Only F 1 plants that had SSR marker fragments associated with both Ren 1 and Ren 2 were kept. F 2 seeds produced from 4 F 1 plants were used in the present research. DNA isolation and SSR marker analysis: Young leaves from 503 BC 2 F 1, 5 LONREN-2, 41 F 1 and 184 F 2 plants were collected when plants were around 30 days old. Total DNA was extracted from fresh leaves using 2.0% hexadecyltrimethyl ammonium bromide according to Paterson et al. (1993). DNA was purified using Omega EZNA DNA isolation column (Omega Bio-Tek, Norcross, GA, USA). SSR markers BNL2662 and BNL3279 associated with either Ren 1 or Ren 2 were analysed (Dighe et al. 2009, Romano et al. 2009). Primer sequences are available at Cotton Marker Database ( Forward primers were fluorescent-labelled with 6-FAM (6-carboxyfluorescein) or HEX (4, 7, 2, 4, 5, 7-hexachloro-carboxyfluorescein). Primer oligos were purchased from Sigma Genosys (Woodlands, TX, USA). Multiplex PCR was performed according to Fang et al. (2010). Amplified PCR products were separated and measured on an automated capillary electrophoresis system ABI 3730 XL (Applied Biosystems Inc., Foster City, CA, USA). GeneScan TM -500 ROX TM (Applied Biosystems Inc.) was used as an internal DNA size standard. The output was analysed with GeneMapper 4.0 software (Applied Biosystems Inc.). SSR marker segregation among F 2 progeny plants was analysed using program JoinMap3.0 (Van Ooijen and Voorrips 2001) with logarithm of odds score 5.0. Chi-square tests were used to check the segregation of markers against appropriate expected ratios. Reniform nematode resistance assay: The reniform nematode resistance screening of the F 2 progeny plants was conducted in two experiments. The first experiment (99 F 2 plants) was conducted according to Romano et al. (2009) except that plants were grown in a growth chamber. Plants were randomly arranged in the chamber. The chamber was maintained at a constant 28 ± 1 C, with daily photoperiod of 14h provided by an equal mixture of fluorescent and incandescent lamps. Sixty days after inoculation, vermiform stages of reniform nematodes were extracted from 100 cm 3 of soil in each pot using elutriation and centrifugal rotation and counted as previously described (Romano et al. 2009). Based on the SSR marker information, a subset of 28 plants containing Ren 1, Ren 2, both genes or neither gene were kept and replanted in 18.9-l pots for future research. Eggs were extracted from roots of the plants that were not retained (Hussey and Barker 1973) and counted. The second experiment (85 F 2 plants) was conducted the same way as the first one except that plants were grown only 30 days after inoculation. The resistance to reniform nematodes was measured by counting females attached to roots as previously described by Stetina and Young (2006). Briefly, roots were cut, washed, blot-dried and stained with red food colouring. Swollen females attached to the roots were counted using a stereomicroscope ( 20). Results were expressed as number of females per root system. According to Stetina and Young (2006), the assessment based on egg counts was equivalent to that based on female counts, and either life stage could be used to screen for resistance. However, it is worth to point out that the former method takes much longer time, but the results are more accurate. In both experiments, G. hirsutum DP444BGRR and G. barbadense TX110 were used as susceptible and resistant checks, respectively. Raw nematode count values were also transformed by xõ = log 10 (x + 1) to normalize the data. The mixed models procedure of SAS (SAS Institute, Cary, NC, USA) was used for analysis of variance. Results Segregation of SSR markers The genotypes of SSR markers BNL2662 and BNL3279 in parental lines, F 1 and 16 F 2 progeny are shown in Table 1. For the marker BNL2662, two fragments 82 and 90 bp segregated

3 Plant resistance to reniform nematodes by pyramiding Ren 1 and Ren Table 1: Genotypes of SSR markers BNL2662 and BNL3279 in parents, F 1 and 16 F 2 plants Samples BN- L2662 BNL Gossypium arboreum A Gossypium hirsutum MD51ne Gossypium aridum G LONREN HAAH (BC 2 F 1 ) parent F F F F F F F F F F F F F F F F F Markers associated with Ren 2. 2 Marker associated with Ren 1. HAAH, hirsutum aridum arboreum hirsutum; SSR, simple sequence repeat. Table 2: Allelic analysis of SSR marker fragments in an F 2 population Marker No. observed (No. expected) Expected ratio v 2 BNL bp 82 bp/90 bp 90 bp 1 : 2 : ns 41 (46) 105 (92) 38 (46) BNL bp 112 bp/114 bp 114 bp 1 : 2 : ns 41 (46) 94 (92) 49 (46) BNL bp 122 bp/132 bp 132 bp 1 : 2 : ns 41 (46) 105 (92) 38 (46) BNL bp 114 bp/132 bp 132 bp 1 : 2 : * 28 (46) 115 (92) 28 (46) *P = Showing non-allelic between alleles BNL3279_114 and BNL3279_132. SSR, simple sequence repeat. in the F 2 population, and they were allelic. The numbers of F 2 plants with 82, 82/90 and 90 bp fragments were 41, 105 and 38, respectively (Table 2). This fits the expected 1 : 2 : 1 ratio (v 2 = 3.77). The fragment 90 bp came from G. aridum, and the 82-bp fragment was contributed by Upland cotton. Neither Upland cotton nor LONREN-2 had the 90-bp fragment. Gossypium arboreum A2-190 was null at this locus. Gossypium arboreum is a diploid A genome species. This clearly indicates that BNL2662_82/90 locus does not reside on an A-subgenome chromosome; instead, it is on a D-subgenome chromosome. Because the marker allele BNL2662_90 completely co-segregated with the Ren 2 locus, the Ren 2 must reside on a D-subgenome chromosome. Primer pair BNL3279 amplified four fragments, i.e. 112, 114, 122 and 132 bp (Table 1). Fragment 114 bp came from Table 3: Independent assortment test for the loci BNL3279_112/114 and BNL3279_122/132 Alleles (bp) G. longicalyx and associated with Ren 1 (Dighe et al. 2009). Fragment 132 bp came from G. aridum and associated with Ren 2 (Romano et al. 2009). To test whether fragments 114 and 132 bp were allelic, we conducted allelic analysis in the F 2 progeny. The allelic analysis (Table 2) clearly showed that fragments 114 and 132 bp were not allelic (v 2 = 20.35). In addition, there were 13 F 2 plants that had neither 114- nor 132-bp allele, further indicating that fragments 114 and 132 bp did not belong to the same locus. We also conducted independent assortment test for the putative loci BNL3279_112/114 and BNL3279_122/132. Our results (Table 3) indicated that these two loci segregated independently among the progeny. Because 114 and 132-bp fragments closely linked to Ren 1 and Ren 2, respectively, we can conclude that Ren 1 is not allelic to Ren 2. BNL3279 marker fragment 112 bp is allelic to fragment 114 bp, while fragments 122 and 132 bp belong to the same locus (Table 2). Segregation analysis indicated that these two loci segregated independently. The 112/114 bp locus was previously mapped on chromosome 11 (Dighe et al. 2009). Although BNL3279_132 allele was not firmly mapped before, Lacape et al. (2003), Nguyen et al. (2004) and Guo et al. (2007) all mapped BNL3279_122 allele on chromosome 21. Thus, based on the mapped SSR marker information and the segregation results, we determine that Ren 2 resides on chromosome 21. Ren 1 and Ren 2 are duplicate genes residing on two homeologous chromosomes and can be tagged using differentsized fragments amplified by the primer pair BNL3279. BNL2662_82/90 locus completely co-segregated with BNL3279_122/132 locus in the F 2 population. Thus, hereafter, we put our focus on the marker BNL3279. Because it was not possible to separate Ren 1 from Ren 2 based on the phenotypic data, we used BNL3279 marker data to predict the genotypes of the Ren 1 and Ren 2 for each progeny plant. Based on the marker data, we divided the F 2 plants into four groups (Table 3): (A) containing neither Ren 1 nor Ren 2 gene, total 13 plants; (B) having allele BNL3279_132 (Ren 2 ) but not BNL3279_114, total 28 plants; (C) having allele BNL3279_114 (Ren 1 ) but not BNL3279_132, total 28 plants; and (D) having both alleles BNL3279_114 and BNL3279_132 (Ren 1 and Ren 2 ), total 115 plants. Reniform nematode resistance No. observed (No. expected) bp 122/132 bp 132 bp (11.5) A 18 (23) B 10 (11.5) B 112/ (23) C 60 (46) D 15 (23) D (11.5) C 27 (23) D 13 (11.5) D ns 1 The capital letters A, B, C and D denote the groups described in the text and used in Table 4. Experiment 1 We separately counted vermiform nematodes and eggs in 100 cm 3 of soil per plant. Both counts provided equivalent information (Table 4). The average nematode counts of the susceptible check DP444BGRR were in 100 cm 3 of soil. The mean counts in the resistant check TX110 were 8910 or 46.69% of the susceptible check. The nematode counts of the v 2

4 676 D. D. Fang and S. R. Stetina Table 4: Vermiform nematode, egg and female counts in F 2 plant groups classified based on marker BNL3279 Experiment 1 Experiment 2 Nematodes in 100 cm 3 Soil Eggs per root system Females per root system Raw Log10 Raw Log10 Raw Log10 Groups Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Susceptible check DP444BGRR A A A A A A Group A (recessive) A A A A B A Resistant check TX B B B B C B Group B (BNL3279_132 Ren 2 only) BC BC BC BC C BC Group C (BNL3279_114 Ren 1 only) BC CD BC C C BC Group D (BNL3279_114 and BNL3279_132 Ren 1 and Ren 2 ) C D C C D C F P > F < < < < < < n Separation of means based on differences of least squares means at P = F 2 plants ranged from 0 to We further grouped F 2 plants according to the genotypes at the Ren 1 and Ren 2 loci predicted by SSR marker BNL3279. Plants without a resistance gene (group A) had similar nematode counts to the susceptible check. Plants with either Ren 1 or Ren 2 had significantly lower nematode counts (24.21% of the susceptible check). Although group B (Ren 2 only) plants had higher egg counts than group C (Ren 1 only), the difference was not statistically significant. In addition, the mean nematode counts of groups B and C were essentially the same. This result indicates that Ren 1 and Ren 2 may function in a similar way when conferring resistance to reniform nematodes, especially when controlling nematode reproduction. A further investigation into the resistance mechanisms of these genes is required to prove this. Based on the marker BNL3279, we could infer the heterozygosity of F 2 progeny plants at each resistance gene locus. Robinson et al. (2007) previously reported that Ren 1 functioned as a dominant resistant gene based on the phenotypic and marker analysis of 681 BC 1 and 241 S 1 progeny plants. In the present study, we did not observe significant difference between Ren 1 homozygotes (mean nematode counts 2997) and heterozygotes (mean counts 4372), which agreed with the statement made by Robinson et al. (2007). The mean nematode counts of the Ren 2 homozygous and heterozygous plants were 3795 and 5555, respectively. However, this difference was not statistically significant. This result suggests that Ren 2 may behave as a dominant gene. However, a larger population (>250 with presence of only Ren 2 gene) will be required to confirm this hypothesis. Experiment 2 In this experiment, we only counted swollen females that were feeding on roots. The results are shown in Table 4. The susceptible check had more than 100 females per root system. Group A plants had an average of 33 females, lower than the susceptible check, but higher than the resistant check and the other three groups. Group B and group C plants had similar resistance. Plants with both genes had the lowest female counts. Based on female nematode counts, it appears that pyramiding Ren 1 and Ren 2 genes conferred higher resistance to reniform nematodes. Although our results did not show a statistically significant separation between group D and groups B and C with respect to 60-day nematode populations (Table 4), the fewest vermiform nematodes and eggs were recovered from plants with markers for both resistance genes. The mean nematode and egg counts of the group D plants were only 6.82% and 0.41% of the susceptible check, respectively. In total, these results suggest that pyramiding resistance genes may be valuable to cotton breeders when managing this pest. Discussion Dighe et al. (2009) named the reniform nematode resistance gene from G. longicalyx as Ren lon. Shortly after, Romano et al. (2009) named the resistance gene from G. aridum as Ren ari. According to the cotton genetic nomenclature rules (Kohel 1973), gene symbol superscripts are used to identify alleles, and subscripts loci. As reported in the present paper, Ren lon and Ren ari are two independent resistant loci and reside on different chromosomes. Thus, the name Ren ari seems to have been flawed in our original report (Romano et al. 2009). Herein, we rectify naming of these two loci and recommend to change Ren lon to Ren 1 and Ren ari to Ren 2. Yik and Birchfield (1984) classified G. longicalyx as immune to reniform nematodes based on the egg production. This immunity was confirmed by Stewart and Robbins (1995). Robinson et al. (2007) conducted more intensive evaluation using 28 G. longicalyx plants and further supported this conclusion. However, Robinson et al. (2007) also evaluated hundreds of Upland cotton introgression lines that contained this resistance trait. Their results clearly showed that a great majority of introgression lines (>95%) did not exhibit immunity to reniform nematodes but instead were highly resistant. We obtained similar results (Table 4). It seems that Ren 1 confers immunity to nematodes in its native G. longicalyx. However, the immunity got lost and became highly resistant after the Ren 1 was introduced into G. hirsutum. It remains unknown what causes this change. Gene duplication is a common phenomenon in allotetraploid cotton. Recent mapping research using SSR markers revealed many duplicate SSR loci in cotton (Nguyen et al. 2004, Guo et al. 2007, Yu et al. 2011). Our present research showed that BNL3279_112/114 and BNL3279_122/132 were

5 Plant resistance to reniform nematodes by pyramiding Ren 1 and Ren duplicate loci with the former being linked to Ren 1 on chromosome 11 and the latter associated with Ren 2 on chromosome 21. Considering their similar biological function, we believe that Ren 1 and Ren 2 are duplicate genes. Further sequencing the genes will provide more concrete evidence. The F genome species G. longicalyx was indigenous to Africa, while the D 4 genome species G. aridum was native to America (Wendel et al. 2009). However, both species contain a reniform nematode resistance gene at similar genomic regions, and both genes may confer resistance with a similar mechanism. It will be interesting to understand whether these two genes have significant sequence divergence or similarity and whether these two species arose from the same ancestor. Materials generated from this research may help us to answer these questions in the future. In addition, we are going to use plants retained from this experiment to study the nematodeõs responses to different genes. Successful introgression of reniform nematode resistance from G. longicalyx into Upland cotton was a significant milestone in the US cotton breeding. However, it has been a bitter struggle to breed an elite cotton variety containing Ren 1. Plants containing Ren 1 exhibited mild-to-severe stunting in the presence of high populations of nematodes and usually had lower, sometimes much lower, yield (Nichols et al. 2010). It is not clear whether this yield drag is attributable to the pleiotropic effect of Ren 1 or to the G. longicalyx genomic block around the Ren 1 locus. Of the two released LONREN varieties, LONREN-2 carries much shorter G. longicalyx genome than LONREN-1. However, LONREN-2 still contains at least 5 cm G. longicalyx genome in terms of genetic distance (Dighe et al. 2009). The physical distance of allotetraploid cotton is estimated between 2250 and 2700 Mb (Rong et al. 2004), and the genetic distance is about 3400 cm (Guo et al. 2007). This translates to about 735 kb/cm although the ratios of physical and genetic distances can vary greatly from one genomic region to another. Thus, a minimum 3.68 Mb of G. longicalyx genome is present in LONREN-2. More breeding will be required to reduce this G. longicalyx genomic block if the yield drag is attributable to the deleterious linkage. The identification of Ren 2 from G. aridum and successful introgression into Upland cotton provide an alternative to cotton breeders when managing reniform nematodes. At this time, it remains unknown to us whether Ren 2 would induce the same severe stunting of the plants in the presence of high population of nematodes, as observed in Ren 1 materials in a field situation. In the present research, we measured root weight of progeny plants. Root fresh weights ranged from 1.81 to 4.16 g, but significant differences between neither four groups of the progeny plants nor progeny plants and checks were detected. We planted early generation (BC 3 F 1 )ofren 2 materials in a field in 2011, which may provide us some preliminary information about the effects of Ren 2 on the agronomic performance. Robinson et al. (2007) reported that Ren 1 behaved as a dominant resistance gene. Our results obtained from a very small population agreed with this suggestion. However, it is worth to point out that the Ren 1 homozygotes had lower nematode counts than heterozygotes though the difference was not statistically significant. Our results also indicated that Ren 2 might function as a dominant gene. However, it is also possible that Ren 2 exhibited incomplete dominance as Ren 2 homozygotes had lower nematode counts than heterozygotes. To draw concrete conclusion, it will be desirable to use a population with more than 250 progeny and containing only Ren 2 gene. We are planning to conduct this research in future. Molecular markers have been used to assist cotton breeding (Dighe et al. 2009, Fang et al. 2010). In the present research, we did not assay the resistance of parents and F 1 plants owing to technical difficulties (in field planting) and large number of plants. Instead, we used molecular markers to tag the resistance genes at each generation and selected plants purely based on marker information. This practice enabled us to use a few plants when making crosses and to discard unwanted plants at early stages, thus saving precious greenhouse space and reducing experiment costs. The nematode assay results of the F 2 progeny clearly demonstrated our success in applying molecular markers in genetic study and breeding. Conclusion The reniform nematode resistance genes Ren 1 and Ren 2 were duplicate genes with Ren 1 residing on chromosome 11 and Ren 2 on chromosome 21. Upland cotton plants containing either Ren 1 or Ren 2 had significantly fewer nematodes than the susceptible genotype. There was no significant difference in nematode resistance between plants containing Ren 1 and those having Ren 2. Plants containing both genes appeared to have higher resistance than those having only one gene. Pyramiding Ren 1 and Ren 2 may be very valuable to cotton breeders when managing reniform nematodes. Acknowledgements The authors thank K. Jordan, M. Gafford, R. Jordan and S. Simpson for their technical assistance. We also greatly thank two anonymous reviewers who made excellent suggestions for revising the manuscript. This research was funded by the United States Department of Agriculture, Agricultural Research Service, CRIS project number D. 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