Development and identification of Verticillium wilt-resistant upland cotton accessions by pyramiding QTL related to resistance

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1 Development and identification of Verticillium wilt-resistant upland cotton accessions by pyramiding QTL related to resistance GUO Xiu-hua, CAI Cai-ping, YUAN Dong-dong, ZHANG Ren-shan, XI Jing-long, GUO Wang-zhen State Key Laboratory of Crop Genetics & Germplasm Enhancement, Hybrid Cotton R & D Engineering Research Center, Ministry of Agriculture, Nanjing Agricultural University, Nanjing , China Abstract: Cotton Verticillium wilt is a serious soil-borne disease that leads to significant losses in fiber yield and quality worldwide. Currently, the most effective way to increase Verticillium wilt resistance is to develop new resistant cotton varieties. Lines 5026 and are two Verticillium wilt-resistant upland cotton accessions. We previously identified a total of 25 QTLs related to Verticillium wilt resistance from 5026 and by assembling segregating populations from hybridization with susceptible parents. In the current study, using 13 microsatellite markers flanking QTLs related to Verticillium wilt resistance, we developed 155 cotton inbred lines by pyramiding different QTLs related to Verticillium wilt resistance from a filial generation produced by crossing 5026 and By examining each allele s effect and performing multiple comparison analysis, we detected four elite QTLs/alleles (q-5/nau905-2, q-6/nau2754-2, q-8/nau and q-13/nau6598-1) significant for Verticillium wilt resistance, pyramiding these elite alleles increased the disease resistance of inbred lines. Furthermore, we selected 34 elite inbred lines, including five lines simultaneously performing elite fiber quality, high yield and resistance to V. dahliae, 14 lines with elite fiber quality and disease resistance, three lines with high yield and disease resistance, and 12 lines with resistance to V. dahliae. No correlation between Verticillium wilt resistance and fiber quality traits/yield and its components was detected in the 155 developed inbred lines. Our results provide candidate markers for disease resistance for use in MAS, as well as elite germplasms for improving important agronomic traits via modern cotton breeding. Keywords: upland cotton, Verticillium wilt-resistance, pyramiding QTL, germplasm enhancement 1 1. INTRODUCTION Cotton (Gossypium spp.) is the most important renewable textile fiber crop worldwide. Verticillium wilt is a serious soil-borne disease with a broad host range (Sal kova et al. 1965). The variation and differentiation of Verticillium dahliae strains have led to a scarcity of resistant cotton plants. Almost no suitable high-resistance variety of upland cotton (Gossypium hirsutum GUO Xiu-hua, @njau.edu.cn; Correspondence GUO Wang-zhen, Tel: , moelab@njau.edu.cn 1

2 L.) currently exists in China. No fungicides are available to cure cotton plants once they are infected with this disease, leading to severe losses in fiber quality and yield. In recent years, severe outbreaks of cotton Verticillium wilt disease have occurred for many reasons, such as the effects of global changes in the climate and the environment, the long-term continuous cropping of cotton and the frequent introduction of new cotton varieties from different regions. Between 2009 and 2010, more than 50% of the cotton growing area in China (5.0 to 6.6 million acres) was affected by Verticillium wilt disease (National Cotton Council of America - Disease Database) (Zhang et al. 2013). Therefore, there is an urgent need to breed new cotton varieties that are resistant to this disease. To date, many quantitative trait loci (QTL) related to Verticillium wilt disease have been tagged using family-based QTL mapping methods by assembling segregating populations of two cotton cultivars differing in Verticillium wilt resistance (Bolek et al. 2005; Yang et al. 2008; Wang et al. 2008; Jiang et al. 2009; Ning et al. 2013; Fang et al. 2013; Li et al. 2013; Zhang et al. 2014; Zhang et al. 2014) and by association mapping methods (Zhao et al. 2014). Zhao et al. (2014) reported the population characteristics, the extent of linkage disequilibrium (LD) and association mapping of Verticillium wilt disease resistance using 212 genome-wide marker loci in 158 elite cotton (G. hirsutum L.) lines from many regions of the world. Nevertheless, the currently available low-density genetic maps and temporary segregation populations make the accuracy and stability of the detected QTLs less than ideal. Few QTLs related to Verticillium wilt disease resistance are currently available for use in breeding by design, which has become a limiting factor for the application of molecular marker-assisted breeding (MAS) to cotton resistance breeding. To date, the development of Verticillium wilt-resistant cotton germplasms by QTL-MAS pyramiding technology has not been reported. We previously identified a large number of QTLs related to Verticillium wilt resistance from resistant accessions (5026 and 60182) by assembling segregating populations with susceptible parents. For example, Jiang et al. (2009) reported 15 major QTLs related to Verticillium wilt resistance from 60182, which are located on chromosomes A10, A11, D7, D9 and D10, using 229 (60182 Junmian 1) F 2:3 plant lines inoculated with different Verticillium dahliae isolates (BP2, VD8, T9 and mixed isolates), as well as the analysis of cotton lines at different developmental stages. By examining a (5026 Litai 8) RIL segregating population containing 169 lines, 10 QTLs related to Verticillium wilt resistance from 5026 were identified by respectively inoculating the lines with isolates BP2, VD8 and T9 at the seedling and mature stages (Yang et al. 2007). Based on these studies, in the current work, we employed SSR markers linked to the QTLs related to Verticillium wilt resistance to screen polymorphisms between 5026 and We then used the markers detecting polymorphic alleles between the two papents to pyramid QTLs for Verticillium wilt resistance by screening the filial generation developed from a single seed derived from a cross between 5026 and As a result, a set of accessions harboring different numbers of QTLs related to Verticillium wilt resistance was developed, and the markers linked to candidates for disease resistance via MAS were further evaluated. This study provides both markers and accession resources for the simultaneous improvement of yield, fiber quality and disease resistance via cotton molecular breeding. 2. RESULTS 2.1. Identification of cotton accessions harboring different QTLs related to Verticillium wilt 2

3 resistance In previous studies, we performed QTL screening of Verticillium wilt resistance based on diverse segregating populations assembled using the resistant accessions 5026 and and susceptible parents, respectively (Yang et al. 2007; Jiang et al. 2009). In this study, a total of 39 SSR primer pairs producing flanking markers of 25 resistant QTLs were used to screen loci polymorphisms between 5026 and Among these, 13 polymorphic loci (five loci from QTL-resistant donor parent 5026 and eight from 60182) were used to pyramid QTLs related to Verticillium wilt resistance. Based on the whole-genome scaffold sequence of the diploid cotton G. raimondii (Paterson et al. 2012), in silico analysis of 13 primer sequences was performed. The 13 primer sequences were anchored on the seven homoeologous chromosomes; the shortest distance between two markers located on the same chromosome was 1.17 Mb. Thus, 13 markers were used as unlinked or independent marker loci for further pyramiding QTL analysis. Information about the 13 markers and corresponding QTLs related to Verticillium wilt resistance is provided in Appendix A. In 2008, the 13 markers were selected to screen more than 1000 individuals in the ( ) F 2 segregating population. As a result, 51 genotypes with different resistance allele combinations were obtained, including 10 and 41 plants that were homozygous and heterozygous for the 13 marker loci, respectively. In 2009, the seeds from the 51 F 2 plants were planted at the Jiangpu experimental field, and the resulting plants were self-pollinated to produce F 2:3 plant lines. The identification of approximately 1300 F 2:3 plants using 13 primer pairs was further performed in the summer of that year. The results enabled us to isolate 25 more plant lines with homozygous genotypes. Other plant lines with homozygous genotypes were continuously self-pollinated over the next two years to confirm its genetic characteristics. In 2011, 155 inbred lines pyramiding different QTLs related to Verticillium wilt resistance were developed from the F 2:5 generation (Appendix B). 2.2.Verticillium wilt resistance performance In 2012 and 2013, we evaluated the disease resistance of the 155 inbred lines harboring different QTLs related to Verticillium wilt resistance and the two parents (5026 and 60182) by inoculating V. dahliae strain V991, a cotton defoliating virulent strain, in a growth chamber using the resistant/susceptible control accessions Hai7124 and TM-1, respectively, as the controls. In total, the relative disease index for each of the 155 inbred lines in 2013 was in agreement with that in Further, the data from 2013 was used for resistance analysis in detail. In summary, when the relative disease index (I R ) of TM-1 was set to 50, this index was for Hai 7124, which reached the level of disease resistance. The relative disease index of the two parents, 5026 and 60182, was and 29.67, respectively. The disease symptoms of the 155 inbred lines exhibited broad variation (Table 1). Among these values, the average I R was 29.96, ranging from 8.58 (V76) to (V151), with a 41.72% coefficient of variation (CV%). The resistance performance values were mainly in the resistant, tolerant and susceptible categories, accounting for 27.10, and 31.61% of the 155 inbred lines, respectively. No inbred line reached the level of immunity (I R =0), and only one inbred line exhibited high resistance (I R value of 8.58). In addition, 41 inbred lines were disease resistant, with an average I R of (ranging from to 19.83), while 64 inbred lines were disease tolerant, with an 3

4 average I R of (ranging from to 34.69). Finally, 49 inbred lines were susceptible to V. dahliae, with an average I R of (ranging from to 65.82). There were 70 inbred lines with better resistance levels than the two parents; their I R s ranged from 8.58 to The disease resistance data for the inbred lines are listed in Appendix B Exploration of elite alleles for Verticillium wilt resistance Multiple comparisons were conducted using the LSR method to study the relative disease index of different alleles per SSR marker. Among the 155 inbred lines harboring QTLs related to Verticillium wilt resistance, there were only two alleles per SSR marker (in both 5026 and 60182). In total, 26 alleles were obtained from the 13 SSR markers. Multiple comparisons analysis revealed four SSR loci (NAU905, NAU2754, NAU3053 and NAU6598) showing significant positive effects on the I R value (Table 2). By integrating the results of multiple comparisons with the alleles effects (a i ), four elite QTLs/alleles (q-5/nau905-2, q-6/nau2754-2, q-8/nau and q-13/nau6598-1) related to Verticillium wilt resistance were further elucidated. We examined the four elite alleles for Verticillium wilt resistance in 157 accessions, including 155 inbred lines and the two parents. Line 5026 has two elite QTLs/alleles (q-8/nau and q-13/nau6598-1), with an I R of Line also has two elite QTLs/alleles (q-5/nau905-2 and q-6/nau2754-2), with an I R of Among the 155 inbred lines harboring different QTLs, there were 13, 50, 43, 22 and 27 inbred lines containing four, three, two, one and zero elite alleles, respectively, and their average relative disease index were 20.58, 23.87, 28.25, and 40.22, respectively, with significant difference of I R value (P<0.01) when pyramiding different elite QTLs/alleles. This result implies that increasing the pyramiding of elite alleles decreases an accession s relative disease index and improves its disease resistance. The number of inbred lines containing 0 4 elite alleles and the average relative disease index are listed in Table Correlation analysis between fiber yield, quality and resistance We further evaluated the yield, yield components and fiber qualities of the 155 inbred lines harboring pyramided QTLs related to Verticillium wilt resistance over two consecutive years, including seven yield and yield component traits (lint percentage, seed index, lint index, boll weight, bolls/plant, seed-cotton yield and lint yield) and five fiber quality traits (fiber length, fiber strength, fiber fineness, fiber uniformity ratio and fiber elongation; Appendix C). Phenotypic characterization of agronomic traits showed that there were wide variations in the 12 traits investigated among the 155 inbred lines, with the highest CV% in lint yield observed in 2013 (LI: 48.04) and the lowest CV% in fiber uniformity ratio observed in 2012 (FU: 0.98). To further elucidate the relationship between fiber yield, quality and resistance, we performed correlation analysis of fiber quality traits, yield and its components and Verticillium wilt resistance in the 155 inbred lines based on the mean values for yield and fiber quality traits over two years, with two replications. No correlation between Verticillium wilt resistance and fiber quality traits/yield and its components was detected (Table 4). However, the negative genetic correlation between some fiber quality and lint yield traits were verified, which is consistent with the previous studies (Saha et al. 2011) Screening inbred lines pyramiding elite yield components, fiber quality and resistance traits 4

5 In this study, one of our main purposes was to create some intermediate materials, resistance to V. dahlia with improving simultaneously fiber quality and yield, by pyramiding different QTLs related to Verticillium wilt resistance. Therefore, the 106 inbred lines (one with high resistance, 41 with disease resistance and 64 with disease tolerance) were further selected to evaluate their fiber quality traits, yield and yield components. With the average value of a target trait s phenotype taken as the threshold value, five inbred lines (V79, V52, V89, V90 and V51) that simultaneously exhibited elite fiber quality, high yield and resistance to Verticillium wilt (Table 5) were identified. We further compared the number of elite loci and disease resistance in five inbred lines and found that V51 and V52 contained all four elite loci, V79 contained three elite QTLs/loci (q-5/nau905-2, q-6/nau and q-8/nau3053-1) and V89 and V90 contained two elite QTLs/loci (q-5/nau905-2 and q-6/nau2754-2). These results indicate that several elite inbred lines with simultaneous improvements in fiber yield, fiber quality and resistance were obtained. Some of the inbred lines were resistant to Verticillium wilt but only exhibited one of the elite fiber quality or high-yield traits, such as the following: (1) Fourteen inbred lines had moderate yield performs but elite fiber quality and high Verticillium wilt resistance. Their I R values ranged from 8.58 to 19.11, with fiber length ranging from to mm, fiber strength from to cn/tex and fiber fineness from 3.54 to 4.99; (2) Three inbred lines had moderate fiber quality but high yield and Verticillium wilt resistance. Their I R values ranged from to 17.34, with lint percentages ranging from 36 to 39%, lint index weight from 6.38 to 7.58 g and boll weight from 5.36 to 6.01 g; (3) Twelve inbred lines only exhibited high Verticillium wilt resistance. Their I R values ranged from to 19.66; perhaps these lines could be used as donors in cotton disease resistance breeding. 3. DISCUSSION Verticillium wilt is a quantitative trait, and Verticillium wilt resistance is inherited based on a multigene model (Mert et al. 2005). Many QTLs related to Verticillium wilt resistance have been tagged in previous studies (Bolek et al. 2005; Yang et al. 2008; Wang et al. 2008; Jiang et al. 2009; Ning et al. 2013; Fang et al. 2013; Li et al. 2013; Zhang et al. 2014; Zhang et al. 2014; Zhao et al. 2014). Zhao et al. (2014) compared QTLs related to Verticillium wilt resistance identified in four different studies, revealing at least 60 different QTLs on 10 chromosomes or linkage groups. These QTLs, which were mapped in four different biparental populations, showed poor colocalization. Among these QTLs, which are useful? How can they be utilized? Answering these questions is critical for breeding disease-resistant cotton accessions. Based on previous studies using family-based QTL mapping methods by assembling segregating populations, we reasoned that the effect of an allele could be elucidated, and multiple comparisons could be performed, by analyzing the relationship between the alleles and Verticillium wilt resistance. In the current study, we detected four elite QTLs/alleles (q-5/nau905-2, q-6/nau2754-2, q-8/nau and q-13/nau6598-1) responsible for Verticillium wilt resistance by examining the allele s effect and performing multiple comparison analysis, and we isolated a population of inbred lines harboring multiple QTLs. Our results show that increasing the number of elite alleles in an inbred line increases its disease resistance. Based on the whole-genome scaffold sequence of the diploid cotton species G. raimondii (Paterson et al. 2012), we further mapped four markers to three homeologous chromosomes. As 5

6 shown in Appendix D, a total of 49 leucine-rich repeat receptor-like kinase (LRR-RLK) genes were identified within the 5 Mb intervals flanking each marker. LRR-containing proteins in plants, as well as those in virus, bacteria, archaea and eukaryotes, have diverse structures and functions (Dixon et al. 1998; Mondragon et al. 2005; Afzal et al. 2008), including LRR-RLKs, LRR-containing receptor-like proteins (LRR-RLPs), nucleotide binding site LRR (NBS-LRR) proteins, polygalacturonase-inhibiting proteins (PGIPs), etc. These proteins act as signal amplifiers in the presence of tissue damage, and they help establish symbiotic relationships and affect developmental processes (Matsushima et al. 2012). Of these proteins, plant LRR-RLKs regulate signaling during plant defense processes (Song et al. 2008; Wu et al. 2009; Xu et al. 2011). Xu et al. (2011) reported putative roles for LRR-RLKs in mediating early events in the response of cotton to V. dahliae based on RNA-Seq gene expression profiles. In the current study, we identified numerous LRR-RLKs near four elite alleles for Verticillium wilt resistance, further confirming their importance for improving disease resistance in cotton breeding via MAS. The two most common methods used in crop breeding are crossing and backcrossing. Backcrossing can be used to improve several traits/loci, and crossing can be used to create more variation in a population. Guo et al. (2005) reported that pyramiding two QTLs that control high fiber strength by MAS greatly improved the selection efficiency for cotton fiber strength via modified backcrossing/pyramiding breeding method. In the current study, through crossing and pyramiding multi-alleles related to Verticillium wilt resistance using MAS method, 155 inbred lines harboring different QTLs related to resistance were developed using 13 SSR markers. Association analysis showed that no correlations between fiber quality traits/yield and its components and 13 SSR loci, or between Verticillium wilt resistance and fiber quality traits/yield and its components, was detected. However, a series of accessions with Verticillium wilt resistance, including five inbred lines with elite fiber quality, high yields and resistance, 14 inbred lines with elite fiber quality and resistance, three inbred lines with high yields and resistance and 12 inbred lines with resistance to Verticillium wilt, were screened from the 155 inbred lines. Based on the result, we can not exclude that other QTLs related to Verticillium wilt resistance, fiber qualities, or yield components in donor parents, were not involved in this study, and need to be further explored. Nevertheless, these diverse accessions can be utilized for the improvement of multiple agronomic traits via modern cotton breeding. RNA-seq technologies have been applied to the study of the response of cotton to Verticillium dahliae infection (Xu et al. 2011; Sun et al. 2013). Numerous genes that are differentially expressed in the defense response have been identified, including genes involved in the phenylalanine metabolism pathway, the hydroxycinnamoyl transferase (HCT), and phenylpropanoid metabolism, etc. Recently, the whole-genome scaffold sequence of diploid cotton G. raimondii and G. arboreum was released by two different groups (Paterson et al. 2012; Wang et al. 2012; Li et al. 2014), which further lays the foundation for elite gene discovery of traits with interest at the whole-genome level. Based on the currently available gene and genome information, functional markers such as cssr and csnp markers will be developed, which will make cotton breeding via MAS more accurate, efficient and cost-effective in the future. 4. CONCLUSION The results provide candidate markers for improving disease resistance by MAS, and accession 6

7 resources for the simultaneous improvement of yield, fiber quality and disease resistance via cotton molecular breeding. 5. MATERIALS AND METHODS 5.1. Plant materials Lines 5026 and 60182, two upland cotton accessions resistant to Verticillium wilt, were bred and screened by cultivating them in Verticillium dahliae strains disease nursery with continuously evaluating the resistance for Verticillium wilt at least 7 yr during 1990s 2000s. TM-1 is a genetic standard line of Upland cotton. Hai7124 is a commercial Sea island Verticillium-resistant cultivar. All materials were continuously maintained by self-pollination for several years. In 2007, two Verticillium wilt-resistant upland cotton accessions, 5026 and 60182, were crossed at the Jiangpu experimental field of Nanjing Agricultural University, Nanjing, Jiangsu Province, China. In the winter of 2007, F 1 seeds were planted and self-pollinated in the Cotton Plantation of Nanjing Agricultural University, Hainan Island, to produce F 2 progeny. From 2008 to 2011, F 2 to F 5 seeds were planted at Jiangpu experimental field and self-pollinated to produce the next generation. Beginning in the F 2 generation, individuals pyramiding different QTLs related to Verticillium wilt resistance were selected, and inbred lines were identified in subsequent generations. The detailed construction process for accessions development by pyramiding different QTLs related to Verticillium wilt resistance is shown in Fig. 1. For Verticillium wilt resistance identification, TM-1 and Hai7124 were used as the susceptible control and the resistant control, respectively DNA extraction and genotype identification Cotton genomic DNA was isolated from the two parents (5026 and 60182), F 1 individuals and plant lines from the F 2 to F 5 segregating populations as described by Paterson et al. (1993). Simple-sequence repeat polymerase chain reaction (SSR-PCR) amplifications were performed using a Peltier Thermal Cycler-225 (MJ Research), and electrophoresis of the products was performed as described by Zhang et al. (2000). A total of 39 SSR markers flanking 25 QTLs related to Verticillium wilt resistance, which were detected in previous studies (Yang et al. 2007; Jiang et al. 2009), were utilized to screen polymorphisms between 5026 and As a result, 13 SSR polymorphic loci between two parents were confirmed and further used for pyramiding QTLs related to Verticillium wilt resistance. All SSR primer pair information can be downloaded from The maternal (5026), paternal (60182) and heterozygous (F 1 ) genotypes were scored as 1, 2 and 3, respectively, in the F 2 to F 5 populations Pathogen isolates and identification of resistant cotton accessions In the winter of 2012 and 2013, 155 inbred lines harboring pyramided QTLs related to Verticillium wilt resistance, the two parents (5026 and 60182) and the resistant/susceptible control materials (Hai7124 / TM-1) were cultured at 25/23 C (day/night) in a growth chamber with a 12-h light/12-h dark cycle for approximately 20 days before they were infiltrated with V. dahliae strain V991 spore suspensions (10 7 conidia ml -1 ). Each inbred line included plants with three repeats for resistance identification each year. V. dahliae strain V991, cotton defoliating virulent strain isolated from G. hirsutum, was used in this study. V991 was grown on solid potato sucrose agar at 25 C for 3 5 days and inoculated 7

8 in Czapek s liquid medium with shaking at 25 C for 5 8 days. The conidia were collected and diluted to a concentration of 10 7 conidia/ml for use in inoculation. Cotton plants were grown to the two true-leaf stage and dip-infected with spore suspensions. After disease symptoms appeared, the incidence rate of disease was surveyed every 7 d, and continuous investigations for three times. The severity of disease symptoms was recorded based on an index ranging from 0 (healthy plant) to 4 (dead plant) (Shi et al. 1993). Plant disease index (DI) was calculated using the following formula: Disease index=[ (Ni i)/(n 4)] 100, where i represents the disease level (0 4), Ni indicates the number of plants exhibiting reaction i, and N means the total number of plants (Fang 1998). To overcome differences in disease severity under different ecological conditions or in different processing batches, the disease index value was converted into the relative disease index (I R ) based on the disease index correction coefficient K. K=50/disease index of the susceptible control material (TM-1). Host reaction type divided into the following five classes: I R =0, <10, 10~20, 20~35, and >35 were considered as immunity, high resistance, resistance, tolerance and susceptible, respectively (Sun et al. 1997) Characterization of agronomic traits of cotton accessions In both 2012 and 2013, the 155 inbred lines harboring QTLs related to Verticillium wilt resistance were planted at the Jiangpu experimental station of Nanjing Agricultural University, Nanjing, Jiangsu Province, China and grown using normal field practices. A randomized complete block design with two replications was used in the field trials. Five plants in the middle of each row were selected for the evaluation of fiber quality traits, yield and its components. Yield and its components included lint percentage (LP), seed index (SI), lint index (LI), boll weight (BW), bolls/plant (BN), seed-cotton yield (SY) and lint yield (LY). Fiber quality traits included fiber length (FL), fiber strength (FS), fiber fineness (FF), fiber uniformity ratio (FUR) and fiber elongation (FE). Fiber samples were tested at the Supervision, Inspection and Test Center of Cotton Quality, Ministry of Agriculture in China using three biological replicates Data analysis An allele s effect (a i ) on Verticillium wilt resistance was evaluated by comparing the average I R of the specific allele/average I R for all 155 inbred lines, following the methods reported previously (Breseghello et al. 2006; Cai et al. 2014). Multiple comparisons were conducted using the LSR (the least significant range) method to study the relative disease index of different marker alleles. Correlation analysis between the values for fiber quality traits, yield and its components and Verticillium wilt resistance in the 155 inbred lines was performed. The multiple comparisons and correlation analysis were estimated using SPSS18.0 ( Acknowledgements This program was financially supported in part by the National Natural Science Foundation of China ( ), the National High Technology Research and Development Program of China (863 Program) (2012AA101108), the Jiangsu Agriculture Science and Technology Innovation Fund (cx(13)3059), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions ( ) and JCIC-MCP (No. 10). References Afzal A J, Wood A J, Lightfoot D A Plant receptor-like serine threonine kinases: roles in signaling and plant defense. Molecular Plant-Microbe Interactions, 21,

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11 Assembled at Jiangpu experimental station in 2007 F 1 Selfed at Cotton Plantation/Hainan in the winter of 2007 F 2 Selfed at Jiangpu experimental station from 2008 to 2011, and MAS F inbred lines Planted at Jiangpu experimental station in 2012 and 2013, and finished fiber quality traits, yield and its components evaluation Planted in growth chamber in the winter of 2012 and 2013 for Verticillium wilt resistance identification Fig. 1 Flow chart for the development of 155 accessions harboring different QTLs related to Verticillium wilt resistance. 11

12 Table 1 Disease reaction distribution of the 155 inbred lines harboring QTLs related to Verticillium wilt resistance Disease reaction type I R range No. materials Mean% (range %) Std Dev CV% High resistance (HR) < ( ) \ \ Resistance (R) ( ) Tolerance (T) ( ) Susceptible (S) > ( ) Table 2 Allelic effects of 13 SSR loci and exploration of elite QTLs/alleles The allele in 5026 The allele in Marker QTL name 1) Homoelogo No. of Average Effect No. of Average Effect us Chr. 2) materials I R (a i ) materials I R (a i ) NAU4045 q-1 A1/D NAU2741 q-2 A1/D NAU1225 q-3 A5/D NAU3569 q-4 A5/D NAU905 ** q-5 A6/D NAU2754 ** q-6 A6/D NAU1043 q-7 A7/D NAU3053 * q-8 A7/D BNL3031 q-9 A9/D NAU5508 q-10 A9/D NAU2508 q-11 A10/D

13 MUCS219 q-12 A11/D NAU6598 ** q-13 A11/D ) The abbreviation for 13 detected QTLs. The complete information for each QTL can be found in Appendix A. 2) Homoelogous chromosome numbers refer to our newly updated interspecific genetic map of allotetraploid cultivated cotton species (Zhao et al. 2012) * and **, significant at P<0.05 and P<0.01, respectively. Table 3 Average relative disease index of inbred lines harboring different elite alleles No. of elite alleles No. of materials Mean of I R Std Dev CV% 5% difference 1% difference a A a A b B bc BC c C Differences among the number of elite QTLs/alleles and the I R were verified by the LSD analysis. capital letter show significant difference at 0.05 and 0.01 level, respectively. The lowercase letter and 13

14 Table 4 Correlation analysis between fiber quality traits, yield and its components and Verticillium wilt resistance Trait LP SI LI BW BN SY LY FL FS FF FUR FE I R LP 1 SI ** 1 LI ** ** 1 BW ** BN SY ** 1 LY * ** ** 1 FL ** ** ** FS ** * ** 1 FF * ** ** ** 1 FUR ** ** ** ** 1 FE ** * ** ** ** ** 1 I R LP (lint percentage), SI (seed index), LI (lint index), BW (boll weight), BN (bolls/plant), SY (seed-cotton yield), LY (lint yield), FL (fiber length), FS (fiber strength), FF (fiber fineness), FUR (fiber uniformity ratio), FE (fiber elongation), I R (relative disease index). *, ** Significant at P < 0.05 and P < 0.01, respectively. 14

15 Table 5 Characterization of elite accessions with high yield, good fiber quality and tolerance to Verticillium wilt 1) Inbred line Elite loci 2) LP LI(g) BW(g) FL(mm) FS(cN/tex) FF I R ± ± ± ± ± ± ± ± ± ± ± ± ± ±1.44 V ± ± ± ± ± ± ±1.74 V ± ± ± ± ± ± ±0.32 V ± ± ± ± ± ± ±0.26 V ± ± ± ± ± ± ±3.09 V ± ± ± ± ± ± ±2.25 1) LP (lint percentage), LI (lint index), BW (boll weight), FL (fiber length), FS (fiber strength), FF (fiber fineness), I R (relative disease index). 2) Indicates elite loci NAU905-2, NAU2754-2, NAU and NAU6598-1, sequentially. 15