Cotton (Gossypium spp.) is one of the most important cash crops, providing about 35% of the total fiber used worldwide
(USDA-ERS, 2013). Globally, 33.45 million hectares of cotton were planted in 2010/2011, with a total production of 116.33 million bales (USDA-FAS, 2013). Of more than 80 cotton-producing countries, China is the largest raw cotton producer and consumer, followed by India and the United States. These three countries together provide two-thirds of the world’s cotton (USDA-ERS, 2009).
Genetics, Breeding, and Marker-Assisted Selection for Verticillium Wilt Resistance
in Cotton
Jinfa Zhang,* Hui Fang, Huiping Zhou, Soum Sanogo, and Zhiying Ma
ABSTRACTVerticillium wilt (VW), caused by the soil-borne fungus Verticillium dahliae Kleb., is one of the most destructive diseases in Upland cotton (Gossypium hirsutum L.) production in the U.S. and worldwide. Development of VW-resistant cultivars remains the only economic option for controlling the disease. The objective of this review was to summarize the progress in screen-ing methods, resistance sources, and genetics, quantitative trait locus (QTL) mapping, marker-assisted selection (MAS) and breeding for VW resistance in cotton. Even though Gossypium barbadense L. carries high levels of resistance, its resistance has not been transferred into commercial Upland cultivars. Many Acala cot-ton cultivars developed in New Mexico and Cali-fornia between the 1940s and the 1990s, and some commercial transgenic cultivars are tol-erant or moderately resistant to VW. However, due to difficulties in achieving consistent and uniform inoculation and infection with V. dahliae, both qualitative and quantitative inheritance of VW resistance have been reported in numerous studies for resistant G. barbadense and Upland genotypes. Several QTL analyses have shown the existence of VW resistance QTLs on almost all the tetraploid cotton chromosomes; however, QTLs have most frequently been detected on c5, c7, c8, c11, c16, c17, c19, c21, c23, c24, and c26. This has led to MAS for progeny with favorable QTL alleles for VW resistance in several experi-ments. Phenotypic selection for VW resistance has been inefficient, while the effectiveness and efficiency of MAS remain to be validated. There is an urgent need for the development of better plant inoculation and screening methods, and for more molecular mapping studies to discern the genetic basis of VW resistance in cotton.
J. Zhang, H. Fang, and H. Zhou, Dep. of Plant and Environmental Sciences, New Mexico State Univ., Las Cruces, NM, USA; S. Sanogo, Dep. of Entomology, Plant Pathology and Weed Science, New Mexico State Univ., Las Cruces, NM, USA; and Z. Ma, North China Key Lab. for Crop Germplasm Resources of Education Ministry, Agricultural Univ. of Hebei, Baoding, Hebei, China. J. Zhang and H. Fang contribute equally. Received 17 Aug. 2013. *Corresponding author ( [email protected]).
Abbreviations: AB, advanced backcross; AFLP, amplified fragment length polymorphism; BIL, backcross inbred line; cM, centimorgan; D, defoliating; DDRT, differential display reverse transcription; ELS, extra-long staple; MAS, marker-assisted selection; ND, non-defoliating; PV, phenotypic variation; QTL, quantitative trait locus; RFLP, restriction fragment length polymorphism; RGA, resistance gene analog; RIL, recombinant inbred line; SNP, single nucleotide polymorphism; SSCP, single strand conformation polymorphism; SSR, simple sequence repeat; STS, sequence tagged site; USDA-ERS, United States Department of Agriculture-Economic Research Service; USDA-FAS, United States Department of Agriculture-Foreign Agricultural Service; VCG, vegetative compatibility group; VIGS, virus induced gene silencing; VW, Verticillium wilt.
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The genus Gossypium is in the family Malvaceae. It includes approximately 50 species, of which 45 are diploids (2n = 2x = 26) and five are allotetraploids (2n = 4x = 52) (Wendel and Cronn, 2003). Diploid species are grouped into eight genome groups (A to G and K) according to the pair-ing affinities of chromosomes (Stewart, 1994). The five allo-tetraploid species are assigned to (AD)1 through (AD)5 on the basis of their genome organizations (Wendel and Cronn, 2003). In the genus, four species [G. hirsutum L.-(AD)1, G. barbadense L.-(AD)2, G. herbaceum L.-A1, and G. arboreum L.-A2] were domesticated independently and cultivated. G. hirsutum, also known as Upland, short to medium staple, or Mexican cotton, is the predominant cultivated species, accounting for more than 97% of the annual cotton produc-tion in the U.S. from 17 southern states. The second major cultivated species is G. barbadense, also known as Sea-Island, extra-long staple (ELS), American Pima, or Egyptian cotton. Pima accounts for about 3% of the cotton in the U.S. and is only grown in southern Texas, New Mexico, Arizona, and California (USDA-ERS, 2013). The two cultivated cotton species originated from the same ancestor 1-2 million years ago, and are cross compatible (Wendel and Cronn, 2003). However, they differ in many morphological and agro-nomic traits. For example, Upland cotton is high yielding and widely adapted to different growing conditions or envi-ronments, while Pima cotton has superior extra-long, strong and fine fiber properties with much lower yield potential and narrower adaptation. Upland cotton is generally suscep-tible to Verticillium wilt (VW), while Pima cotton, as a spe-cies, is resistant or tolerant (Wilhelm et al., 1972a, 1974b).
VW, caused by the soil-borne fungus Verticillium dahl-iae Kleb., is one of the most destructive diseases through-out the U.S. Cotton Belt. VW was first reported in 1914 in Virginia (Carpenter, 1914), and occurs in all the cotton-growing regions of the world. The disease caused 0.5~3.5% cotton yield losses nationwide in the U.S. (Blasingame and Patel, 2011) and as high as 7.9% decrease in seed-cotton yield in other regions (Karademir et al., 2012). It significantly reduced fiber quality including 50% span length and micro-naire (Bell, 1992, 2001; Zhang et al., 2012c). Planting VW-resistant cultivars is the most cost-effective and economical control method (Bell, 1992, 2001). Numerous efforts have been made to improve Upland cotton resistance against VW, but little achievement has been made due to the scarcity of immune or highly resistant G. hirsutum germplasm, and to uncertainty about the genetic and molecular mechanisms of VW resistance in cotton (Cai et al., 2009; Zhang et al., 2012c). The objective of this review was to summarize the importance of VW-resistant cotton and research advances made on VW resistance in the past two decades. Emphasis was placed on the genetic basis of VW resistance and recent advances in breeding, mapping of VW resistance- related genes and quantitative trait loci (QTLs), and marker-assisted selection (MAS) for VW-resistant cotton.
VERTICILIUM DAHLIAE AND MANAGEMENT OF VERTICILIUM WILT IN COTTONVerticillium Wilt Symptoms and Causal PathogenVW symptoms are host-dependent and there are no par-ticular symptoms that belong to all plant species (Fradin and Thomma, 2006). Cotton plants affected by VW show symptoms that include irregular leaf chlorosis, necrosis, or wilting; discoloration of the stem vessels; petiole and leaf abscission; flower and boll abscission; and eventually death of the plant (Bell, 1992, 2001; Pegg and Brady, 2002). However, the symptoms are dependent on cotton geno-type and growth stage, pathotype and inoculum density of the pathogen, and environmental factors. Development of symptoms may be affected by environmental factors such as temperature, light intensity, photoperiod, ambient and soil moisture levels, irrigation, and cultural practices (Kara-demir et al., 2012; Robb, 2007). It is difficult to distinguish VW from Fusarium wilt (caused by Fusarium oxysporum f. sp. vasinfectum) in cotton based on field symptoms without the isolation of the causal agent.
The genus Verticillium is comprised of several species which are capable of causing VW in different plant hosts (Barbara and Clewes, 2003; Klosterman et al., 2009; Robb, 2007; Zare et al., 2007). The two most economically destruc-tive species, V. dahliae and V. albo-atrum Reinke and Berth, cause billions of dollars of losses in annual crops worldwide (Fradin and Thomma, 2006; Pegg and Brady, 2002). Verticil-lium albo-atrum causes yield losses in hop and alfalfa, and it can cause wilt of cotton in the greenhouse at low temperatures (20–24°C). However, V. dahliae is the only causative agent of VW in cotton under field conditions (Bell, 1992, 2001; Pegg and Brady, 2002). It is a soil-borne vascular fungus that is capa-ble of infecting more than 400 plant species in several families (Lüders et al., 2008; Pegg and Brady, 2002), including some important crops (Bhat et al., 2003; du Toit et al., 2005) and weeds (Sanogo and Clary, 2003; Sanogo et al., 2009).
Disease Cycle of Verticillium Wilt and Diversity of V. dahliaeV. dahliae forms chains of black, thick-walled resting struc-tures, known as microsclerotia, which can survive in the soil for many years (Vallad et al., 2006; Wilhelm, 1955). The microsclerotia germinate to produce hyphae that invade the cortex directly through the root tips or wounds (Fitzell et al., 1980), and then grow in the xylem where they pro-duce many conidia. Due to structural and chemical changes resulting from the interaction between the fungus and the host plant, the vascular system becomes blocked, imped-ing water movement within the plant. As a result, leaves begin to wilt and develop chlorosis. As the affected plant senesces, the fungus produces microsclerotia which are
6–9 and high water content from rains or irrigation favor the development of VW in cotton (Bell, 2001).
Pathogenicity and Virulence of V. dahliaeOriginally, the mechanism of virulence of V. dahliae to plants was thought to be vascular occlusion resulting from blocking of the vascular bundle by V. dahliae mycelium and spores, and by the formation of tyloses, i.e., swellings produced by the growth of the xylem parenchyma cells into the lumen of the vessels by the host plant (El-Zik, 1985). More recent studies have shown that the effect of toxins secreted by V. dahliae is a more important mecha-nism for pathogenicity than the vascular occlusion (Buch-ner et al., 1989), and pathogenicity-related genes have been identified in V. dahliae (Maruthachalam et al., 2011). Cell wall-degrading enzymes (such as pectinolytic enzymes), protein-lipopolysaccharide complexes, and glycoproteins were found in crude extracts containing toxin complexes of V. dahliae with an important role in pathogenesis (Fradin and Thomma, 2006). Wang et al. (2004b) identified a pro-tein with elicitor activities. This protein, named VdNEP, is composed of 233 amino acids, and is a wilt-inducing factor that participates in the interactions of cotton with V. dahliae. Recent reports have demonstrated that post-tran-scriptional gene silencing controls the basal defense against Verticillium in Arabidopsis (Ellendorff et al., 2009).
Verticillium Wilt Control Strategies in CottonControl of VW is extremely difficult due to the persis-tence of the microsclerotia, the broad host range of the pathogen, scarcity of resistance in Upland cotton, and the unavailability of fungicides to kill the pathogens once they enter the xylem (Fradin and Thomma, 2006; Klosterman et al., 2009; Pegg and Brady, 2002). Methods to control VW include the use of soil fumigants such as chloropicrin (Wilhelm et al., 1972b), and nonchemical strategies such as crop rotations and soil amendments (Ebihara et al., 2010; Goicoechea, 2009). Biocontrol is a developing alternative to control VW, including the use of arbuscular mycorrhi-zal fungi (Goicoechea et al., 2010; Liu, 1995; Zhang et al., 2012b), endophytic fungi (Li et al., 2012; Shittu, 2010), antagonistic rhizobacteria (Berg et al., 2005), and nitrog-enous or organic soil amendments (Bailey and Lazarovits, 2003; Goicoechea, 2009; Huang et al., 2006). However, none of these strategies are efficient or effective when used alone. Chemical fumigants are expensive, and are gener-ally harmful to the environment and public health (Martin, 2003), thus their utilization is restricted. Crop rotation is rather inefficient as the microsclerotia of V. dahliae can remain viable for a long time in the soil, and the pathogen has a broad host range, requiring long-term rotations to reduce microslcerotia below crop-specific threshold levels (Fradin and Thomma, 2006). Biocontrol strategies are
released into the soil with the decomposition of plant mate-rials (Berlanger and Powelson, 2000). The severity of VW strongly depends on the virulence of the pathogen isolates, host genotype, and environmental factors. V. dahliae isolates infecting cotton can be classified into defoliating (D or P-1) and non-defoliating (ND or P-2) pathotypes, according to their ability to cause defoliation or no defoliation of green leaves from shoots and branches (Bell, 1992, 2001; Jiménez-Díaz et al., 2011; Pérez-Artés et al., 2000; Schnathorst and Mathre, 1966). The D pathotype can be lethal to the plant, whereas the ND pathotype causes only mild wilt and no defoliation, and affected plants can eventually show com-plete remission from these symptoms (Jiménez-Díaz et al., 2011). An intermediate virulent type was identified among 40 isolates of V. dahliae collected from the cotton-growing areas of Hebei province in China (Ma et al., 1997b).
Genetic diversity in V. dahliae has traditionally been studied by means of vegetative compatibility grouping (Daayf et al., 1995). Strains of V. dahliae are differentiated into subspecies groups on the basis of vegetative compat-ibility (Dobinson et al., 2000; Leslie, 1993). Six main veg-etative compatibility groups (VCGs), viz. VCG 1 to VCG 6, have been identified in V. dahliae from different hosts and geographic origins (Berbegal et al., 2011; Bhat et al., 2003; Klosterman et al., 2009). Isolates in a single VCG are considered to be a biologically distinct population because the pathogen has no known sexual reproduction cycle (Dobinson et al., 2000). Several VCGs are further divided into subgroups based on vigor and frequency of comple-mentation to tester isolates, such as VCG1A and VCG1B, VCG2A and VCG2B, and VCG4A and VCG4B (Berbegal et al., 2011). Studies of vegetative compatibility have shown that isolates from the D pathotype belong to VCG1A and are incompatible with strains of the ND pathotype, which form other subgroups (Bell, 2001; Daayf et al., 1995).
Epidemiology of V. dahliaeA warm and humid climate favors the spread of V. dahl-iae (Bell and Presley, 1969). The optimum temperature for the D pathotype is 25–28°C, and the development of symptoms is slow if the temperature is lower than 25°C or higher than 30°C (Xu et al., 2012). Symptoms may disap-pear when temperatures exceed 35°C in mid-summer, and reappear and develop when temperatures decline (El-Zik, 1985; Bell 2001). Other factors affecting VW epidemiology include the pathotype of the pathogen, inoculum density in the soil (Bejarano-Alcazar et al., 1995; Pullman and Devay, 1982), host genotypes (Katsantonis et al., 2003), water status of the soil, soil type, soil pH, potassium and nitrogen availability to the growing plants, interactions with other pathogens, plant densities, and the movement of propagules by wind, water, machines, and animals (Paplomatas et al., 1992; Shittu, 2010). Silt loam or clay loam soils with pH of
limited by environmental factors or site-specific efficacy problems (Klosterman et al., 2009).
SCREENING FOR VERTICILIUM WILT RESISTANCE IN COTTONField EvaluationsFields heavily infested with V. dahliae can serve as long-term or permanent natural nurseries for screening and selecting cotton for VW resistance. In fact, screening for resistance in those fields allowed breeders and/or plant pathologists to identify lines that were later released as VW-resistant culti-vars in the U.S. (Bell, 1992). A field screening nursery can also be prepared by consecutively growing susceptible culti-vars to build up the inoculum, which can be supplemented with artificial inoculation or distribution of crop residues and gin trash from heavily infected cotton plants. Applica-tion of excess nitrogen fertilizer and frequent irrigation can further facilitate the development of VW in cotton. Since yield losses are closely correlated with leaf damage during boll development in midsummer that coincides with soil temperatures that promote the disease, VW severity ratings should be taken during the boll development stage (Bell, 1992). Therefore, early-maturing genotypes, due to heavy boll loading in this period, may appear to be more suscep-tible than late-maturing cotton which has more vegetative growth and less fruiting. Especially when VW resistance is evaluated at harvest, premature boll opening due to VW infection can be confounded with natural maturation of bolls in early-maturing genotypes. Thus, screening mature plants for VW resistance at harvest is not recommended (Bell, 1992).
Responses of cotton plants to infection by V. dahliae are affected by resistance genes, environmental factors, inoculum density, and their interactions. Under field con-ditions, high experimental errors for the development of VW symptoms make it very difficult to achieve a reliable rating of disease resistance due mainly to the difficulty of maintaining a relatively uniform distribution of the inoc-ulum at a high density level. Variations in environmen-tal factors and weather from season to season also affect screening results. Individual plants within a genotype, including susceptible types, may exhibit a wide range of apparent responses to VW, ranging from no symptom (i.e., escapes) to severe infection, even after artificial inoculation (Fang et al., 2013). As such, screening of VW resistance in the field should be performed on a population basis. It is not surprising that most genetic studies conducted in field conditions produced data suggestive of quantitative inheritance of VW resistance, regardless of cotton geno-types or pathogen strains used (e.g., Aguado et al., 2008; Verhalen et al., 1971). In breeding, progeny of single plant selections for VW resistance made in the field need to be tested in replicated field tests to confirm the resistance. In
germplasm screening and genetic studies, scoring multiple plants in each genotype for VW responses in two or more replications is necessary to reduce the experimental error and more reliably assess genotypic differences in VW resistance under field conditions. For example, Zhang et al. (2012c) and Fang et al. (2013) screened 100 plants for each genotype in two replications (50 plants each) for VW resistance in the field. In another study by Ning et al. (2013), 35 plants from each recombinant inbred line (RIL) were evaluated for their VW responses in a field inoculated with V. dahlia isolates.
Disease incidence, i.e., percentage of plants displaying VW symptoms, can be conveniently used to assess VW resistance, but it does not take into consideration disease severity (Allen et al., 2001). VW severity is usually rated on an arbitrary scale of 0-4 (Ning et al., 2013) or 0-5 (Devey and Roose, 1987; Zhang et al., 2012c), in which 0 =a healthy plant with no visible leaf symptoms; 1=mild to moderate leaf symptoms or <25% chlorotic/necrotic leaves; 2=severe leaf symptoms or 25-50% chlorotic/necrotic leaves, but little defoliation; 3=50-75% chlo-rotic/necrotic leaves or defoliation; 4= >75% chlorotic/necrotic leaves or up to 90% defoliation, some terminal dieback, plant often stunted; and 5=complete defoliation, with stems dying back or dead to ground level. An aver-age severity rating is then calculated for each genotype on a replication basis from the sum of rating × number of plants divided by the total number of plants evaluated (Zhang et al., 2012c; Fang et al., 2013). In China (e.g., Ning et al., 2013), the 0-4 scale is further converted to a relative disease index (%) on a 0-100% scale as the ratio between the average rating and the highest rating (i.e., 4).
Greenhouse EvaluationsAs stated above, cotton plants can be evaluated in a field with high V. dahliae populations built up over years natu-rally or by artificial inoculation. However, the pathogen populations and their distribution may not be uniform in the field. Furthermore, a large screening field is needed to grow plants to the boll development stage or maturity. Therefore, the field screening requires five to six months in a growing season. In some cases, crop, soil, and irriga-tion management may be a challenge in achieving reliable results in VW resistance screening. Due to the aforemen-tioned problems with field evaluations for VW resistance, artificial inoculation methods in facilities with a controlled environment such as greenhouses have been developed and often used to screen for VW resistance.
In the greenhouse assays, cotton plants with two to four true leaves are inoculated by root inoculation using root-dipping or root-ball techniques (Bell, 1992). With the root-dipping method, seedlings are first gently removed from a river sand soil, and roots are washed before being dipped in VW inoculum. The seedlings are
second inoculation (1 wk after the first) was implemented by Zhang et al. (2012c) and Fang et al. (2013).
Soil can be infested with V. dahliae grown on wheat bran and corn meal (1:1 w/w) in the ratio of 0.5–2% (w/w), or with microsclerotia (103 microsclerotia g-1 soil), but these soil infection methods are not as effective as the root-dipping method (Ma et al., 2004). In a few cases (e.g., Bolek et al., 2005a,b), a stem-puncture method (Bell, 1992) was used to inoculate cotton plants with spore suspension of V. dahliae (2-3 × 106 spores mL-1 per plant). This technique can be also employed in the field (Bell, 1992). However, this technique is not only labor intensive and time con-suming, but it also compromises the resistance mechanisms provided by the root. Thus, plant infection by V. dahliae is usually more severe from the stem-puncture method than that from the root inoculation technique.
Similar to field evaluations, VW disease severity can be rated on the same 0-4 or 0-5 scale 30-45 d after inoculation in the greenhouse (Fang et al., 2013; Ning et al., 2013; Zhang et al., 2012c). VW screening can thus be completed within a 2-mo period. Although the visual rating system is simple to use, VW severity ratings are subjective and in fact it is difficult to use in discerning two neighboring ratings. So a better and more subjective assessment method is needed. To further define genetic differences among genotypes in their responses to VW, Zhang et al. (2012c) used a modified scoring system based on yellowish or abscised cotyledons. Furthermore, the numbers of infected and defoliated leaves, and the total number of leaves can be counted on an individual plant basis to calculate mean percentages of infected leaves and defoliation for each genotype (Fang et al., 2014).
Other Inoculation and Evaluation MethodsThere are other evaluation methods, such as growing plants in hydroponics systems or in tissue culture, fol-lowed by inoculation with a conidial suspension of 107 spores mL-1 through root-dipping for 40 min (Peng et al., 2008). This method can further accelerate completion of VW resistance screening in 1 mo. Seedlings or callus tis-sues can be evaluated for VW reactions by growing them in a medium containing toxin from a crude extract of V. dahliae (Dai et al., 1989; Zhang et al., 2004). However, these techniques are not commonly used.
RESISTANCE SOURCES AND INHERITANCE OF VERTICILLIUM WILT RESISTANCE IN COTTONResistance SourcesIt has long been widely accepted that breeding and utiliza-tion of resistant cultivars offers the best means for control-ling VW. Resistance sources for VW in cotton are very lim-ited, and no source of heritable immunity has been found
then transplanted back into the same soil. For the root-ball method, seedlings with the intact root balls are removed from the pots, and the root balls are sprayed with an inoculum suspension. The inoculated seedlings are then returned to the same pots or repotted in larger pots. The inoculum suspensions consist of spores or conidia and are used in the range of 1-4 × 107 spores per plant or 0.2-3 × 108 spores per pot with four to five plants (Ning et al., 2013; Fang et al., 2013). Soils used to grow cotton can be river sand, farm soil, or a commercial soil medium. With the above two methods, roots are wounded when seedlings or root balls are removed from the soil or pots, and this facilitates plant infection by V. dahliae. Seedlings can be also grown in bottomless plastic or paper pots con-taining silt loam or clay loam soils on a soil bed, and then removed for root wounding before spore suspension is applied to the roots ( Jian et al., 2001; Ma et al., 2004; Wang and Ma, 2002). To save time and effort, however, seedlings can be kept in pots during inoculation and root-wounding can be performed before or immediately after root inoculation, if needed (Zhang et al., 2012c). Root-wounding will disarm the first line of defenses against infection by V. dahliae in cotton, however, which will not reflect natural field resistance to VW. Zhang et al. (2012c) reported that Pima S-7 which is resistant to VW under natural field conditions, became susceptible when its roots were wounded during inoculation with V. dahliae in the greenhouse. Zhang et al. (2012c) and Fang et al. (2013) reported that inoculation of cotton plants was successfully achieved by simply pouring conidial suspensions directly onto the surface of potted soil without root wounding. VW symptoms are usually visible after 2 wk under the optimal temperature conditions for plant infection.
In greenhouse studies using artificial inoculations, most of the genetic studies were based on individual plants in early segregating populations, and results could not be repeated, even though Mendelian segregating ratios were obtained in some cases (Barrow, 1970a,b; Bell and Presley, 1969; Wilhelm et al., 1969; Wilhelm et al., 1972a, 1974b; Pan et al., 1994; Ma et al., 2000; Mert et al., 2005). There-fore, there is an urgent need to reduce experimental errors in VW evaluation methods and to evaluate VW resis-tance repeatedly on a replicated population basis, so reli-able phenotypic results can be obtained. Thus, permanent mapping populations and multiple replicated tests should be used to reduce experimental errors in evaluating VW resistance, and thereby provide more reliable phenotypic data for genetic studies and QTL mapping. Usually, one inoculation of conidial suspensions is applied for screening cotton plants for VW resistance. However, since V. dahliae is soil-borne, it is very difficult to achieve a uniform inocu-lum distribution in the field or artificial inoculation. To minimize escapes and achieve more uniform infections, a
in Upland cotton (Wilhelm et al., 1974b). Among the four cultivated cotton species, G. barbadense has the highest level of resistance against VW (Wilhelm et al., 1974b), but it is planted on a very limited area and the resistance traits have not been successfully transferred into commercial Upland cotton (Zhang et al., 2012c). Furthermore, the genetic diversity of Upland is very low because of intensive selec-tion for economically important traits during the domesti-cation and breeding process (Abdalla et al., 2001; Wendel et al., 1992). In a greenhouse study, Wilhelm et al. (1974b) found that the reaction to V. dahliae in G. barbadense ranged from near immunity to moderate susceptibility, while in G. hirsutum, reactions ranged from minimal resistance to high susceptibility. Zhang et al. (2012c) indicated that modern Pima cotton has higher levels of VW resistance than Upland cotton, if the root system was not wounded after inoculation. Therefore, breeding highly resistant or tolerant Upland cotton cultivars is possible through inter-specific breeding. In Upland cotton, intraspecific breeding has progressively increased the level of VW resistance in some cultivars since the 1970s (Bell, 1992, 2001; Zhang et al., 2012c). Many Acala cotton cultivars and some com-mercial cultivars, including transgenic ones from major seed companies in the United States, have displayed some levels of VW resistance (Zhang et al., 2012c). Zhou (2013) recently screened 367 obsolete U.S. Upland germplasm accessions released between the early 1900s and 2005, and identified 33 accessions with high levels of VW resistance. Zhou et al. (2014) further screened 223 recently released commercial cultivars and advanced breeding lines for VW resistance in the greenhouse, and identified six Upland cultivars, five advanced lines, two introgression lines from Upland × Pima, and four Pima cultivars with higher levels of resistance. In the Texas Southern High Plains, commer-cial cotton cultivars for VW resistance were routinely eval-uated under field conditions (Wheeler and Schuster 2006; Wheeler 2007; Wheeler and Woodward, 2009).
Inheritance of Verticillium Wilt ResistanceAlthough it is possible to cross G. barbadense with G. hir-sutum, the progenies suffer from wide segregations due to hybrid breakdown resulting from interspecific cross incompatibilities. To circumvent these problems, a better understanding of the molecular mechanism of resistance to VW is indispensable. However, there is no consensus on the genetic basis of resistance against VW in cotton. Stud-ies on inheritance of VW resistance in cotton have reported different inheritance patterns and most of the more recent genetic studies since the 2000s were from China.
Most studies indicate that the VW resistance of G. bar-badense is controlled by a dominant or partially dominant gene in F2 or BC1 of interspecific crosses between highly resistant G. barbadense and susceptible G. hirsutum (Bell and Presley, 1969; Wilhelm et al., 1969; Wilhelm et al., 1972a,
1974a; Pan et al., 1994; Ma et al., 2000; Du et al., 2004), and that the resistance genes from different sources of G. barbadense are allelic (Ma et al., 2000). The inheritance of resistance is more complicated for intraspecific hybrids within Upland cotton. The controversy lies in the nature of resistance, whether resistance is a qualitative trait con-trolled by one or two major genes, or a quantitative trait conditioned by minor polygenes, and whether the resis-tance is dominant or recessive. The controversy is related to different resistance sources and homozygosity of genes in resistance, virulence and inoculum levels of the patho-gen, evaluation methods, environmental factors (especially soil temperature and moisture), and plant maturity.
Some researchers have concluded that resistance to VW is a qualitative trait conditioned by a single dominant gene in certain resistant Upland cotton genotypes based on early-segregating populations such as F2 and BC1 (Barrow, 1970a,b; Cai et al., 2000; Fang et al., 2003a,b; Mert et al., 2005; Qi et al., 2000; Wang et al., 1999, 2007b; Wilhelm et al., 1969, 1972a, 1974a, b; Wu et al., 2009). Others indi-cated that the genetic mechanism of VW resistance was quantitative rather than qualitative in the genotypes and populations studied (Bolek et al., 2005a; Gao et al., 2003; Ge et al., 2008; Liang et al., 2010; Roberts and Staten, 1972; Stith, 1969; Wang et al., 2004a, 2005, 2007b; Wu et al., 2010; Yang et al., 2008; Zan et al., 2008), conditioned by two or more dominant genes (Guo et al., 2008; Jiang et al., 2009; Lüders et al., 2008; Qi et al., 2000; Zhang et al., 2000). Guo et al. (2008) reported that the VW resistance in an introgressed Upland cotton with VW resistance was controlled by a dominant gene and two additive genes, and that the additive effect was more important. Individuals with both of the additive genes were resistant, while plants with only one of the additive genes showed tolerance. Individuals with neither of the two genes were susceptible. Similar results were obtained for the VW-resistant cultivar Zhongzhimian KV-3 in a cross with the susceptible cotton line KV9–1 (Wang et al., 2010). Jiang et al. (2009) crossed the resistant Upland cotton line 60182 with the suscep-tible Upland cultivar Junmian 1, and inoculated their F2 and F2:3 progenies with individual V. dahliae isolates BP2 (non-defoliating), VD8 (defoliating), T9 (defoliating), and a mixture of these isolates. They reported that VW resis-tance in their Upland cottons was controlled by two major genes with additive-dominance-epistatic effects, and that the resistance alleles of the two major genes were dominant.
Some researchers reported that the resistance to VW was also race or pathotype dependent. Based on a segrega-tion analysis, Pan et al. (1994) showed that the resistance in a G. barbadense × G. hirsutum cross was controlled by a major gene when inoculated with a single V. dahliae race or strain, while it was controlled by quantitative genes when inocu-lated with mixed races or strains. In Upland cotton, Mert et al. (2005) reported that the resistance to a defoliating
(D) pathotype was controlled by a single dominant gene; in contrast, the resistance to a non-defoliating (ND) pathot-ype was controlled by dominant alleles at two loci. Growth vigor, development, and maturity of cotton plants can affect their responses to infection by V. dahliae (Wilhelm et al., 1974b), even in plants that lack genetic resistance. Tempera-ture and the virulence and inoculum level of the pathogen affect plant responses to infection by V. dahliae and therefore results in genetic studies (Bell, 1992).
A quantitative genetic study was conducted on diallel crosses among 10 selected lines of Upland cotton by Verha-len et al. (1971). They found that resistance to VW in the resulting populations was a quantitative trait, and the addi-tive variance was larger than other parameters. Based on five cotton cultivars and their possible crosses from a half-diallel design, Aguado et al. (2008) detected both additive and dominance genetic variance components, but the addi-tive genetic variance component was more important.
In a few cases, resistance appeared to be controlled by recessive genes. Roberts and Staten (1972) investigated F2 and F3 populations from crosses between Upland cotton cultivars (8229 × Lankart 57, 8076 × Lankart 57, and 8861 × Lankart 57) in a heavily-infested field and found that resistance to VW was recessive. Heritability varied from 0 to 0.83 depending on exposure level, generation tested, and type of parental tolerance. Devey and Roose (1987) crossed the VW-resistant cultivar Acala SJC-1 with the susceptible lines S5971, Acala 4-42, and Deltapine 70, and seven gen-erations (P1, P2, F1, F2, F3, BC1P1, and BC1P2) of each cross were evaluated for a generation mean analysis. They found that more than one gene controlled resistance to VW, and that resistance was recessive.
MAPPING OF VERTICILLIUM WILT RESISTANCE QUANTITATIVE TRAIT LOCI AND CLONING OF VERTICILLIUM WILT RESISTANCE-RELATED GENESMapping of Verticillium Wilt Resistance QTLsMolecular markers are useful tools in understanding the mechanism of host resistance to VW through gene/QTL analysis and cloning and in breeding resistant cultivars through marker-assisted selection (MAS). Since the first linkage map was constructed from an interspecific G. hir-sutum × G. barbadense F2 population based on restriction fragment length polymorphic (RFLP) markers (Reinisch et al., 1994), many linkage maps in cotton have been con-structed for QTL mapping using various markers (Said et al., 2013). At present, resistance-related QTLs to VW have been detected in G. barbadense and G. hirsutum with vari-ous random markers. Using 11 linkage groups covering 531 cM from 35 SSR markers for an F2 mapping popula-tion derived from an interspecific cross of the highly resis-tant G. barbadense cultivar Pima S-7 and the susceptible
G. hirsutum cultivar Acala 44, Bolek et al. (2005b) iden-tified three QTLs on chromosome c11 with large effects on VW resistance. Wang et al. (2008) in China screened the susceptible Chinese cultivar Xinluzao1 (G. hirsutum), the resistant Egyptian cotton Hai 7124 (G. barbadense), and their F2:3 families for VW resistance in a VW-infected field. Based on a total of 430 SSR markers mapped onto 41 link-age groups with a total genetic distance of 3746 cM, nine QTLs explaining 10.6 to 28.8% of the phenotypic varia-tion (PV) for VW resistance were detected, six of which were located on the D sub-genome (c16, c24, and c26).
However, none of the above QTLs were detected by Yang et al. (2008) using 128 interspecific F2 individuals from the same VW-resistant G. barbadense line Hai 7124 and the susceptible Upland cultivar Junmian 1 and their BC1S2 families using Junmian 1 as the recurrent parent. Both F2 and BC1S2 families were inoculated with isolate BP2 (ND), while BC1S2 families were inoculated with iso-lates BP2 (ND), VD8 (D), and 592 (D) separately through root irrigation. Based on 36 linkage groups with 420 SSR, RGA (resistance gene analog) and DDRT (differential dis-play reverse transcription) markers covering 2727 cM in F2 and 35 linkage groups with 219 SSR markers covering 1773 cM in BC1, a total of 20 QTLs were detected for leaf and vascular traits on c5, c7, c8, c9, c19, c21, and c22 in the two segregating generations using the three V. dahliae isolates. Chromosome c5 carried 7 QTLs, while c8 and c18 each carried 3 QTLs, and c7, c21 and c22 each carried 2 QTLs. Two QTLs were consistent in different populations and four QTLs were detected at both the seedling and maturity stages. Using the same susceptible Upland culti-var, Junmian 1, to cross with the resistant Upland cotton line 60182, Jiang et al. (2009) evaluated its F2:3 population separately with three isolates of V. dahliae, including one non-defoliating pathotype and two defoliating pathotypes, and a mixture of the isolates. Based on a linkage map of 31 linkage groups with 139 SSR markers covering 1165 cM in F2, 41 VW resistance-related QTLs were detected. These QTLs were clustered on chromosome D7 (i.e., c16) with 16 QTLs and D9 (i.e., c23) with 25 QTLs, and each explained 7.5 to 33.4% of the phenotypic variation (PV).
The above studies exclusively used F2, F2:3 or BC1S2 as populations for phenotyping and F2 or BC1F1 for genotyp-ing with markers, resulting in unrepeatable results due to the nature of populations used. Based on 52 linkage groups of 292 SSR, SSCP-SNP (single strand conformation poly-morphism-single nucleotide polymorphism), STS (sequence tagged site), cDNA-AFLP (amplified fragment length poly-morphism), and RGA-AFLP markers covering 1226 cM in a backcross inbred line (BIL) population evaluated for VW resistance in replicated tests in both greenhouse and field con-ditions, Fang et al. (2013) detected two VW resistance QTLs anchored by several RGA-AFLP markers on chromosomes c4 and c19. The BILs were derived from an interspecific cross
between the resistant Pima S-7 and the susceptible Upland Sure-Grow 747. RGA-AFLP markers, which were found to be widely distributed on the cotton genome, were also used to map root-knot nematode resistance (Niu et al., 2011). RGA-AFLP may provide candidate gene markers for disease resis-tance. In a RIL population of susceptible Upland TM-1 × resistant Upland NM 24016 with G. barbadense introgression was evaluated in two replicated tests under greenhouse condi-tions (Fang et al., 2014), 21 VW resistance associated QTLs were detected on 11 chromosomes including c8, c11, c13, c15, c17, c19, c20, c21, c24, c25, c26, and two linkage groups using 432 SSR, 414 SNP and 36 RGA-AFLP markers covering 25 chromosomes with 2267 cM (Gore et al., 2013). Chromo-somes c8, c21, c24, and c26 each carried two QTLs (detected in Test 1) on the same chromosomal regions. Two QTLs in the same test were detected on c19 but located in different regions. One QTL each on c19 and c26 was also detected in Test 2, while one QTL each in Test 1 and 2 was detected on c20. In a RIL population from susceptible Upland 86-1 × resistant Upland Acala Prema, Ning et al. (2013) recently detected seven VW resistance QTLs on A1 (c1), A3 (c3), A5 (c5), A7 (c7), D2 (c14), and D9 (c23) in the field, and five VW resistance QTLs on A9 (c9), D3 (c17), D9 (c23), and D11 (c21) in the greenhouse. Interestingly, the QTL anchored by SSR markers NAU3414 and NAU2954 on c23 was identified in both tests and was a main-effect QTL, which explained 62.8% of the PV in VW resistance under the field condi-tions. The authors suggested that the VW resistance in Acala Prema was introgressed into Upland cotton from G. thuberi Tod. or/and G. barbadense. However, the QTL on c23 did not share SSR markers with the VW resistance QTL cluster on c23 identified in the intraspecific F2:3 population involving another VW-resistant Upland line (Jiang et al., 2009).
Taken together, VW resistance QTLs have been detected on most of the 26 tetraploid cotton chromosomes (e.g., c1, c3, c4, c5, c7, c8, c9, c11, c13, c14, c15, c16, c17, c19, c20, c21, c22, c23, c24, c25, and c26), and VW resis-tance QTLs were more frequently detected on c5, c7, c8, c11, c16, c17, c19, c21, c23, c24, and c26. Through a single marker-trait association analysis, Zhou (2013) confirmed that all 26 chromosomes had SSR markers significantly correlated with VW resistance; however, more significant markers were from c5, c7, c9, c11, c13, c15, c16, c18, c19, c21, c23, c24, and c25. It is often difficult to reliably iden-tify VW resistance QTLs, because the contribution of each QTL to the phenotype may be relatively small and most quantitative traits such as VW resistance are very sensitive to environmental and developmental factors. The difficulty in accurately measuring cotton responses to VW results in confounding effects on VW resistance QTL mapping. Fur-thermore, low genome coverage of markers in these map-ping studies does not allow a genome-wide detection of QTLs with a high resolution.
Identification and Cloning of Verticillium Wilt Resistance-Related GenesBesides mapping QTLs conferring resistance to VW, exten-sive efforts have also been made on cloning of VW resis-tance-related genes. Recent advances in molecular char-acterization of plant resistance genes (R-genes) include the discovery and cloning of several VW R-genes or resistance-related genes in other crops. For example, R-genes Ve1 and Ve2 for VW have been cloned in tomato (Kawchuk et al., 2001). However, in cotton, no major race-specific resistance R-gene for VW or other diseases has been cloned, even though a few resistance-related genes (such as GbVe) have been cloned and characterized based on homologous gene analysis (Zhang et al., 2011b, 2012a). Zhang et al. (2011b) cloned and characterized a VW resistance-related gene from G. barbadense based on a homologous gene search. The gene, named GbVe, is 3819 base pairs (bp) in length, with an open reading frame of 3387 bp, encodes a 1128 amino acid (aa) leucine-rich repeat receptor-like protein precursor. GbVe shares 55.6% and 57.4% identities with Ve1 and Ve2 in tomato, respectively. The GbVe-overexpressing plants in Arabidopsis thaliana had an increased level of resistance to V. dahliae. Also from G. barbadense, Zhang et al. (2012a) cloned another Ve homologous gene Gbve1. While virus-induced silencing of the gene in resistant cotton compromised VW resistance, its overexpression in transformed A. thaliana and Upland cotton rendered resistance to both D and ND iso-lates of V. dahliae. The authors suggested that the Gbve1 gene is responsible for resistance to VW in Pima cotton and can be used for breeding resistant cultivars (Zhang et al., 2012a). However, the relationship between this gene or GbVe and VW resistance in Pima cotton has not been genetically estab-lished. Using an Agrobacterium-mediated virus induced gene silencing (VIGS) assay to silence GhNDR1 and GhMKK2 in several cotton cultivars with various genetic backgrounds, the susceptibility of cotton plants to V. dahliae was enhanced because a more severe wilting phenotype than the control plants was observed (Gao et al., 2011). The results showed that the GhNDR1 and GhMKK2 are required for VW resis-tance in cotton, but the direct relationship between the two genes and VW resistance genes is unknown. Most recently, a serine/threonine protein kinase gene GbSTK was cloned in VW-resistant G. barbadense, and its overexpression in Ara-bidopsis enhanced VW resistance (Zhang et al., 2013b).
In the past five years, many studies have attempted to address the molecular basis of VW resistance in cotton using transcriptomics (cDNA cloning and sequencing, RNA-Seq or microarray), proteomics, and transgenic approaches (e.g., Gao et al., 2011; Wang et al., 2011; Xu et al., 2011; Zhang et al. 2013a), resulting in identification of numerous differen-tially expressed genes during plant infection. However, the relationships between these genes and VW-resistant genes or QTLs remain to be established.
BREEDING FOR VERTICILLIUM WILT RESISTANCE IN COTTONBreeding for VW resistance in cotton is exemplified by the efforts from New Mexico and California in the United States (Smith et al., 1999; Staten, 1970; Zhang et al., 2005). The first successful example in breeding Upland cotton for VW resistance was through pedigree selections in the 1940s, when reselections from the genetically diverse Acala 1517 (released in 1939) led to the release of VW tolerant Acala 4-42 in Cali-fornia and Acala 1517WR in New Mexico in the mid-1940s. The first VW-resistant cultivar Acala 1517D was released in New Mexico in 1960 as a result of further selection from the heterozygous and heterogeneous Acala 1517 with signifi-cant G. barbadense introgression over years, which gave rise to Acala SJ-1 with improved VW resistance in California in 1967 when crossed with AxTE-1. Further pedigree selection from Acala SJ-1 gave rise to Acala SJ-2 in 1973.
Since the 1970s, cross breeding has become predomi-nant in breeding for VW resistance in cotton in both states. For example, VW-resistant Acala 1517V, 1517-70, 1517-75, and 1517-91 were all developed from cross breeding in New Mexico (Zhang et al., 2005). Acala 1517-70 was derived from a cross involving Hopcala with Tanguis (G. barbadense) introgression, which gave rise to Acala 1517-91 when crossed with NM 8874 which was derived from a cross involving 1517V. Several New Mexico VW-resistant lines were used in crosses to produce many VW-resistant Acala cultivars in California, such as Acala SJ-3 (released in 1974), Acala SJ-4 (released in 1975), Acala SJ-5 (released in 1977), SJC-1 (released in 1982), GC-510 (released in 1984), and Acala Maxxa (released in 1990). Many other commer-cial VW-resistant cultivars were released since the 1990s, such as Kings M-5, GC-356, Acala Prema, Acala Royale, and DL 6166. Some other cultivars such as Deltapine 20, Deltapine 50, Deltapine 51, DP Acala 90, Stoneville 495, Delcot 344, Paymaster 147, Paymaster 303, Paymaster 404, and Paymaster HS 26 have moderate resistance (Bell, 1992; Smith et al., 1999). Based on the breeding history for VW-resistant Acala cultivars, the VW resistance in Acala cotton may be derived from G. barbadense (Bell, 1992).
Since the mid-1990s, conventional cotton culti-vars including these VW-resistant ones in the U.S. have been rapidly replaced by transgenic insect-resistant and/or herbicide-tolerant cotton cultivars, most of which were developed through backcrossing breeding. Zhang et al. (2012c) evaluated 267 cultivars and germplasm lines in the greenhouse and 357 genotypes in the field for VW resis-tance. While confirming that many Acala cotton cultivars released in the past had moderate levels of VW resistance, some commercial transgenic cultivars developed by major seed companies in the U.S. also displayed similar levels of VW resistance. The results indicated that cotton breeding has made substantial advances in progressively increasing or maintaining the resistance level to VW in the U.S. in
the past 20 years (Bell, 1992; Zhang et al., 2012c). Most notably, the study by Zhang et al. (2012c) identified sev-eral backcross inbred lines that had similar or higher levels of VW resistance than the resistant Pima S-7 parent (G. barbadense), providing the first line of evidence that VW resistance in Pima cotton was successfully transferred into Upland cotton through backcrossing and selfing.
It should be noted that breeding for VW resistance has been actively pursued in some other countries, especially in China. However, publications are not accessible to the international community. Based on a review by Ma and Chen (1992), direct selections within cultivars under natu-ral VW-infected field conditions led to the development of several VW-tolerant cultivars (e.g., Zhong 8004, Zhong 7327, Zhong 3474, Liaomian 5, and Shan 1155) with toler-ance or moderate levels of VW resistance in China between 1950 and 1970. Since the 1980s, cross breeding has become the major method in breeding for VW resistance, as rep-resented by the release of CCRI 12 in 1987. CCRI 12 is high-yielding, highly resistant to Fusarium wilt and toler-ant to VW, and its commercialization accounted for more than 20% of the cotton acreage in China in 1990. In the 1990s, more VW-resistant cultivars such as Yumian 19, 86-6, BD18, Chuan 737, and Chuan 2802 were released (Ma et al., 1997a, 2002). The VW resistance in CCRI 12 and Chuan 2802 appeared to be controlled by different major genes (Cai et al., 2000; Wu et al., 2009). However, the resistance levels in the cultivars are still moderate and VW remains one of the most serious problems in cotton production in China.
MARKER-ASSISTED SELECTION FOR VERTICILLIUM WILT RESISTANCE IN COTTONDu et al. (2004) was likely the first to report that a SSR marker BNL 3556 on c8 or A02 was linked to a major VW resistance QTL which explained 50% of the pheno-typic variation for VW resistance based on a resistant G. barbadense × susceptible G. hirsutum F2 population. However, it appeared that this marker was later discounted, but a new SSR marker BNL3255–208 on the same chromosome was identified to be linked with the major VW resistance gene at a genetic distance of 13.7 cM based on screening of another G. barbadense × susceptible G. hirsutum F2/F2.3 population for VW resistance (Zhen et al., 2006). This marker was subsequently confirmed to be carried in 85–89% of the F2 or BC1F1 plants with VW resistance in interspecific crosses (Wang et al., 2007b; Li et al., 2011), and was successfully employed to transfer the VW resistance from G. barbadense into Upland cotton through MAS (Zhang, 2011).
Kong et al. (2010) tested 18 SSR markers associated with VW resistance in segregating populations of Upland cotton and found that three SSR (BNL1721, BNL2733, and BNL3452) on c8 were effective in selecting segregants for
VW resistance. In an F2.3 intraspecific Upland cotton pop-ulation between a resistant cultivar and a susceptible cul-tivar with G. barbadense germplasm introgression for fiber quality, Wang et al. (2007a) identified two VW resistance QTLs on c16 and reported that two SSR markers (NAU 751 and BNL195) each for the QTLs on c16 were effective in increasing VW resistance and the combined use of the two markers further enhanced VW resistance. In a more recent study (Li et al., 2013), 39 SSR markers that were pre-viously reported to be associated with VW resistance were screened for polymorphisms in progenies derived from a composite cross made between a mixture of pollen of 8 F1 hybrids and another 9 F1 hybrids and its reciprocal cross in Upland cotton. Of the 12 markers that were polymorphic, BNL3241 (on c17), NAU1225 (on c5 or c19), NAU1230 (on c5 or c19), JESPR153 (on c1 or c18), and BNL3031 (on c9 or c23) significantly increased VW resistance in lines carrying these markers, and the combination of JESPR153 and BNL3031 was superior to other marker combinations. Qi et al. (2012) reported that SSR markers NAU1225, NAU828, and NAU1269 (all on D5, i.e., c19) amplified specific bands to distinguish between VW-resistant and susceptible cultivars.
Even though numerous QTLs for VW resistance have been identified in cotton, markers associated with most of the VW resistance QTLs have not been confirmed or employed in MAS to select genotypes with VW resistance. It appears that chromosome c8 carries VW resistance, but SSR markers used for MAS differed between studies using interspecific (Wang et al., 2007b; Li et al., 2011) and intra-specific populations (Kong et al., 2010). The effectiveness of the two SSR markers on c16 (Wang et al., 2007a) and five other SSRs (Li et al., 2013) in MAS for VW resistance remains to be further validated.
ISSUES AND PERSPECTIVEGenetic PopulationsAs indicated above, there have been numerous reports in identifying, mapping, and characterizing QTLs respon-sible for VW resistance. Despite these successes, few if any examples have been reported to demonstrate that molecular marker techniques have led to the development of new cul-tivars with improved VW resistance in cotton. One of the important limiting factors is the lack of permanent mapping populations that can be repeatedly tested in multiple envi-ronments. As reviewed above, most of the studies on the inheritance and QTL mapping for VW resistance in cotton were based on F2, F2:3, or BC1 progenies, which could not be repeatedly evaluated. Compared with these early segre-gating mapping populations, a stable BIL or RIL population can be used in repeated experiments under different envi-ronmental conditions. There is more chance to identify recombination between linked markers using a BIL or RIL population, because the population has several occurences of
meiosis before it becomes inbred, resulting in more chance for linked genes to recombine. A BIL or RIL population is especially suitable for VW resistance studies in cotton. Since each BIL or RIL line can be repeatedly tested in multiple replicates and in multiple tests, and a number of plants in each line can be evaluated in each replicate, experimental errors will be greatly reduced for reliable phenotyping of quantitative traits, including VW resistance in cotton.
Moreover, most of the mapping populations devel-oped for VW resistance QTL analysis were based on inter-specific crosses between G. hirsutum and G. barbadense. Inbreeding after interspecific crossing results in hybrid breakdown, weakness and sterility, making it difficult to generate a large number of fertile segregants for mapping. This explains why there have been no reports in using RILs for mapping of quantitative traits on yield and VW resistance using the interspecific hybrids (Said et al., 2013). The use of advanced backcross QTL (AB-QTL) provides a strategy to overcome this problem by eliminating most of unwanted chromosome segments from an unadapted parent (Tanksley and Nelson, 1996). Since its invention, AB-QTL has been used in cotton to identify a number of QTLs for fiber traits (see Zhang and Percy, 2007 for a review). However, progenies in these advanced backcross populations were still segregating and not inbred, and could not be repeatedly tested. Therefore, backcross proge-nies should be further advanced to inbred lines by repeated self-pollination, resulting in backcross inbred lines (BILs). In BILs, not only very limited DNA fragments are trans-ferred from a donor parent to a recurrent parent through backcrossing, but they are homozygous inbred lines and can be tested in replicated tests across multiple environ-ments. Yu et al. (2012, 2013) have recently reported QTL mapping using such a BIL population of interspecific hybrid for seed quality, lint yield, and fiber quality traits in cotton. Fang et al. (2013) took advantage of the AB-QTL strategy using another BIL population for the first time to identify QTLs conferring resistance to VW in an interspe-cific Upland × Pima population.
Verticillium Wilt Resistance Evaluation TechniquesOne of the main reasons for the conflicting results in genetic and QTL studies is high experimental errors related to inoculation and plant infection. Ning et al. (2013) recently reported that an intra-Upland RIL population involving VW-resistant Acala Prema was evaluated for VW resis-tance under both field and greenhouse inoculation condi-tions and detected only one common VW-resistant QTL. Unexpectedly, another five QTLs were only detected in the field test, while another four QTLs were detected only in the greenhouse test. Similar results were obtained in our greenhouse study when an introgressed Upland RIL pop-ulation was evaluated in the greenhouse in two different
replicated tests (Fang et al., 2014), where four common QTLs were detected for different traits in one test and three QTLs were detected at different positions on three chro-mosomes in both tests. The results illustrate the persistence of the problem of achieving consistent results from the cur-rent inoculation and screening methods for evaluating VW resistance, even when multiple plants per replication for the same genotype in multiple replications and multiple tests were evaluated using double inoculations under controlled greenhouse conditions. This certainly calls for a better inoculation and screening method for evaluating VW resis-tance. Growing plants in the soil or potting soil may have been the major problem in preventing a uniform inocula-tion and infection. In genetic studies for VW resistance, it is worthwhile to try hydroponics systems where no soil is used and conidial suspensions can be added to the nutri-ent solution for certain period of time to ensure a uniform inoculation and infection. Plant growth stages and matu-rity will not confound the evaluation during the seedling stage. With a reliable VW evaluation method, the disputes in the genetic basis of VW resistance in different sources of resistance germplasm may be resolved. However, seedling resistance evaluated in a greenhouse or hydroponics systems will need to be verified under field conditions. Further-more, since resistance genes may be activated during differ-ent cotton growth stages, the relationship between seedling and mid to full mature plant resistance in the field or green-house should be studied.
Genetic Basis of Verticillium Wilt Resistance in CottonG. barbadense is known to carry high levels of VW resis-tance which, based on many studies, is conditioned by a major resistance gene and other genetic factors. How-ever, the major resistance gene has not been unequivocally located to any cotton chromosome using markers. Du et al. (2004), Zhen et al. (2006), Wang et al. (2007b), and Li et al. (2011) presented evidence that the major resistance gene is located on chromosome c8 based on the linkage between the resistance and a SSR marker, which was sup-ported by the positive results from MAS using three dif-ferent SSR markers from the same chromosome (Kong et al., 2010). In a RIL population involving an introgression Upland line from G. barbadense, a common VW resistance QTL on c8 was consistently detected for two VW resis-tance traits in one replicated greenhouse test in our study (Fang et al., 2014). However, this gene or QTL was not detected in two early interspecific segregating populations (Wang et al., 2008; Yang et al., 2008) and a BIL population (Fang et al., 2013) due to the low genome coverage with markers. Therefore, it is reasonable to speculate that the major VW-resistant gene in G. barbadense may reside on an A-subgenome chromosome c8, even though many other VW-resistant QTLs were detected on other chromosomes.
However, this speculation is not supported by Jiang et al. (2009) and Ning et al. (2013) using intra-Upland cotton crosses where no QTL was detected on c8. Many Acala cotton and current cultivars are resistant to VW even though the resistance levels are not as high as in G. barbadense. There are lines of evidence that the resistance in Acala is likely derived from G. barbadense. In a Chinese VW-resistant cultivar, the VW-resistant QTLs were clus-tered on two D-subgenome chromosomes c16 and c23 (Jiang et al., 2009), while a VW-resistant QTL on c23 was detected in the resistant U.S. cultivar Acala Prema in the field (with a major effect) and greenhouse (with a lower effect). Wang et al. (2008) also reported the detection of a VW-resistant QTL on c16.
Considering that major VW-resistant genes were implicated in segregation analyses in several VW-resistant Chinese Upland genotypes such as CCRI 12 and Chuan 2802, which did not have apparent G. barbadense germplasm introgression, it is likely that the genetic bases for VW resis-tance between resistant Upland cotton and G. barbadense are different. Whether the resistance genes or QTLs in Acala or Upland were from G. barbadense, only allelic tests among these different resistant sources and further QTL mapping using more markers and more reliable VW inoculation and methods will provide answers.
Breeding Cotton for Verticillium Wilt ResistanceDue to the problems in VW evaluation, selection for VW resistance has been extremely inefficient and ineffective. MAS may facilitate the selection process; however, vali-dation of markers to be used in MAS remains an issue. Since VW resistance QTLs have been detected in almost all the 26 chromosomes (except for c2, c6, c10, c12, and c18) with lower genome coverage by markers, it is difficult to choose QTLs and closed linked anchoring markers for MAS. Only when better VW inoculation and screening methods are developed and resistance QTLs and genes are better delineated using more markers, will MAS become more efficient and effective in breeding for VW resistance than conventional phenotypic selection. Furthermore, selection for VW resistance will need to be accompanied by simultaneous selections for high yield, better fiber qual-ity, and other desirable traits. The task is even more chal-lenging since VW resistance appears to be negatively asso-ciated with lint yield. The use of markers for different traits on different chromosomes or regions will likely break this deleterious association or linkage. A VW-resistant cultivar should have comparable yield and fiber quality to suscep-tible cultivars under VW-free field conditions.
AknowledgmentsThe project was funded in part by USDA-ARS, Cotton Incor-porated, and the New Mexico Agricultural Experiment Sta-
tion. We thank Dr. Jack C. McCarty, Jr., USDA-ARS, Mis-sissippi and Dr. David Walker, USDA-ARS, Illinois, for their critical review of the manuscript.
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