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Korean tomato cultivation area is growing as 5,270 ha in 2010 and 3,348 ha in 2001. And also seed market is steeply increasing from 3.5 billion won in 2001 to 14.9 billion won in 2011. However, the tomato breeding technique is now in its infant stage, In the crop breeding, the tomato crop used marker-assisted selection (MAS) at first time, and the MAS is being used most actively (Rick and Fobes, 1974; Tanksley and Rick 1980). As tomato is the global vegetable crop, the level of cultivar development in the advanced countries is high. However, the domestic tomato breeding is in its infant stage. In the aspect of investment scale, participant seed company and breeding manpower are also very limited. Thus, more than 90% of the domestic cultivars are foreign ones.

 Tomato yellow leaf curl virus (TYLCV) is a monopartite begomovirus that is transmitted via sweet potato whitefly, Bamisia tabaci Genn., that causes a devastating symptom in numerous tomato species (Cohen and Antignus, 1994). Severe losses in tomato (Solanum lycopersicum L.) production by TYLCV infection has been reported in many tropical and subtropical countries in Asia, Europe, and western hemisphere (Pico et al., 1996). Thus, it is being reinforced that the international quarantine of TYLCV-infected tomato fruit. After the first domestic infection of TYLCV at a farm in Tongyeong of Gyeongnam at 2008, now its outbreak is spread even into the central district such as Buyeo and Nonsan as well as the southland such as Tongyeong, Geoje, Jeju, Goseong, and Masan where are the main tomato cultivating area (Park et al., 2010).

 One of the most efficient methods to control plant diseases is to grow disease-resistant cultivars. Efforts to develop TYLCV resistant varieties have continued from the past decades, but cultivars or breeding lines available for excellent resistances to TYLCV and other begomoviruses are still limited (Ji et al., 2007c; Maxwell et al., 2006; Pico et al., 1999; Vidavsky et al., 1998).

 TYLCV is difficult to prevent by chemicals or other methods and the breeding of resistant lines is also very limited. However, it is urgent to breed a resistant cultivar, which has increased resistance through accumulation of individual TYLCV resistance gene Ty-1,2,3 (partial dominance).

 TYLCV-resistant cultivars and breeding lines that were confirmed to have the adaptability of growth in Korea are very limited, and the development of new cultivars with high levels of TYLCV resistance and commercially useful traits for domestic market is required. Rapid introgression of useful genes into an elite germplasm can be accomplished by backcrossing process assisted with the trait-linked molecular markers and the markers representing the genome background of recurrent parent (Frisch and Melchinger, 2001).

 The World Vegetable Center (AVRDC) has bred tomato cultivar from long times ago, and is especially accumulating Ty-1, 2, 3 genes in most of homogeneous cultivars. Even Ty-4 and Ty-5 are also being accumulated (Hanson, 2011). AVRDC uses two methods in selecting TYLCV resistant resource and breeding line. In selecting wild variety and resistant resource, the work is confirmed the resistant level in phenotype through direct inoculation by using medium as white fly, which can cause the infection of TYLCV. When the resistance is confirmed, the new cross combination is prepared through crossbreeding. In addition, there is selection by Ty-1, 2, 3, 4, 5 molecular markers. Thus, these genes can be easily accumulated into the breeding lines. Accordingly, the aim of this research was to examine whether the phenotypic resistance to TYLCV can be matched with the genetic resistance that was confirmed by MAS.

MATERIAL AND METHODS

Plant materials

 To breed tomato TYLCV resistant line, 11-TY5-AV (supersunroad) was used with comparison of disease susceptibility with 100 individuals in 3 lines of F₂-generation such as 11-TY1~3-AV, and 200 individuals in 4 lines of F₃-generation such as 11-TY4, 6, 7, 8-AV. The heterozygous F₂ plants for Ty-1, 3 introgression and F₃ lines were used for TYLCV evaluation. The cross combination and the level of disease resistance in the used materials are shown in Table 1. TYLCV resistance sources, GC 9 and GC 173 were used as one parent. And NIHHS inbred tomato lines, ‘Tamina’, ‘Marek’, ‘Sarang’, ‘Magic’, were used. GC 9 and GC 173 were evaluated for having resistance to TYLCV by the inoculation of Tomato yellow leaf curl Thailand virus (TYLCTHV) in AVRDC at December 2010 (Table 2).

Table 1. List of 7 tomato breeding lines and F₁ variety (susceptible check) in this study and their resistance to TYLCV (Ty1, Ty3).

Table 2. Results of TYLCTHV inoculation in resistance sources and susceptible check (2010_AVRDC).

Inoculated viral strain

 As the main begomovirus in Taiwan is Tomato leaf curl Taiwan virus (ToLCTWV), it showed resistance even as Ty-2 resistance gene. However, as TYLCTHV was first reported to Taiwan in 2005, the resistance wasn't indicated any more just with Ty-2 gene. It is the real situation that TYLCTHV is rapidly spread now in the whole area of Taiwan. Accordingly, TYLCV resistant line is bred by the accumulation of TY-1, 2, 3 genes. The most contagious virus, TYLCTHV, is used to screen resistant lines and selecting genetic resources.

Exposure and reading

 To evaluate resistance of tomato lines such as 11-TY1-AV, artificial inoculation room was used with white fly from April 13 to April 29. For better inoculation of TYLCV, tomato and cabbage plants were grown together to maintain enough population of white fly (Fig. 1). Exposure of white fly was carried out for 2 weeks. After then, agricultural chemical was sprinkled, resulting in having killed white fly. And, all tomato plants were taken out of the inoculation room. After 1 week from the inoculation, the infection symptom began to appear. An infection survey was carried out 5 times with the interval of one week.

Fig. 1. Image of tomato TYLCTHV inoculation (3 days after inoculation).

Inheritance analysis

 χ²(P > 0.05) test was carried out to examine the segregation ratio as 3:1, which is the theoretical segregation ratio in F₂ generation based on the phenotype through bioassay.

RESULTS AND DISCUSSION

 The main begomovirus in Taiwan is known as ToLCTWV and TYLCTHV. The ToLCTWV has become the most problematic virus from 2005. And it showed resistance just on Ty-2 gene within the plant (Tsai, 2011). However, the TYLCTHV is rapidly spreading now in the whole area of Taiwan. And, AVRDC is using TYLCTHV strain to select resistant germplasm and breeding lines. In TYLCV resistant breeding lines, all of Ty-1, 2, 3 genes is being accumulated.

 TYLCV is a disease symptom, which can be caused by a lot of plant viruses. So generally speaking, TYLCV is an aggregate of these viruses. In a long period, because of the geographic isolation, the different TYLCVs’ evaluations were based on the local conditions and formed a lot of different original ecotypes. However, today, due to globalization, the viruses carried by various vectors spread in the entire world and are recombined to the local species, which formed a lot of novel species and subspecies. All of these situations made TYLCV more and more complex (Chen et al., 2011). Kim et al. (2011) analyzed the whole base sequence and relationship among other country's strains to target 5 regions (Busan, Boseong, Hwaseong, Jeju, and Nonsan) with infection of TYLCV in Korea. As a result, genome in Busan and Boseong was 2,774 bp, thereby having been similar to strain in Japan. Genome in Jeju, Nonsan, and Hwaseong was 2,781 bp, thereby having been indicated to be similar to strain in China. TYLCTHV strain isn't reported now in Korea, but it seems to be introduced into Korea near soon. Even the pathogenicity of the strain is strong, but it is thought that that’s perfectly good material to screen the resistant germplasm or lines that we use in breeding project.

 Susceptibility evaluation (11-TY5-AV) and 3 lines of F₃-generation & 4 lines of F₂-generation were inoculated by using TYLCTHV strain. The individuals based on TYLCV infection investigation were divided into diseases indexes 0, 1, 2, 3, 4 (Fig. 2). The susceptible symptom could be confirmed from one week after inoculation. After 10 days of inoculation, 83% of susceptibility control showed the infected symptom. After 13 days, 100% showed the susceptible symptom. Twenty-three individuals were infected out of 48 in 11-TY4-AV. Fourteen individuals were infected out of 42 in 11-TY6-AV. Fourteen individuals were infected out of 48 in 11-TY7-AV. Fifteen individuals were infected out of 48 in 11-TY8-AV (Table 4).

Fig. 2. Tomato TYLCV diseases index. 0, healthy - 4, severe symptom.

Table 3. Sown number, germination number, and inoculation date by tomato TYLCV breeding lines.

Table 4. Date-based infected-plant number after inoculation of TYLCTHV by tomato TYLCV breeding lines.

 Three lines of F₃ 11-TY1, 2, 3-AV were indicated to be different in resistance level depending on line in the face of homo resistance in Ty-3 and Ty-1 locus. These lines used equally GC 9, which is the resistant resource to TYLCV, as paternal, and used mutually different line, which is susceptible to TYLCV, as maternal. It could be confirmed that resistance level is indicated differently depending on using which thing as maternal even if using the same resource, which is resistant to TYLCV, as paternal. This is consistent even with the result that a little more infection symptoms appear given Ty-3 locus with hetero resistance and Ty-2 with susceptible than Ty-3 locus with homo resistance and Ty-2 locus with susceptible when comparing the infection degree of TYLCV according to the combination of Ty-3 and Ty-2 locus, which was reported by Mejia et al. (2011).

 Seeing Fig. 3, ANT 22 (left tray in each Figure) and 11-TY5-AV (right tray in each Figure), which were used as the comparison of susceptibility with the image when 18 days passed after inoculation, were infected all individuals with TYLCV, thereby having been nearly stopped growth. However, the breeding lines (central tray in each photo) could be confirmed that the plants are growing healthily except individuals with susceptibility. As a result of surveying the genetic segregation ratio in phenotype by setting for resistance when the infected index is 0~1 and for susceptibility when the infected index is 3 ~ 4, all of F₂ 4 lines showed the normal genetic segregation ratio (Table 5).

Fig. 3. Image of tomato TYLCTHV inoculation by each line (18 days after inoculation). Comparison of susceptibility in left (ANT 22) and right (11-TY5-AV) of each photo.

Table 5. Segregation of phenotype by F2 generation in tomato TYLCV breeding lines.

적 요

 토마토 TYLCV는 병이 발생되면 약제나 다른 방법으로 방제가 어려우므로 내병성 품종의 개발이 반드시 필요하다. 특히, TYLCV 저항성 유전자 Ty-1,2,3(부분우성)을 한 개체 내로 집적을 시켜서 저항성이 증대된 품종의 개발이 필요하다. 토마토 TYLCV 저항성 11-TY1-AV 등 7계통을 대상으로 보독인 담배가루이를 이용하여 표현형을 조사하였다. 이병성 대비(11-TY5-AV; 슈퍼썬로드)와 F₃ 세대 3계통 및 F₂ 세대 4계통을 TYLCTHV 균주를 이용하여 접종하였다. 접종 13일 후에 이병성 대비가 100% 이병증상을 나타내었으며, 이때 11-TY4-AV는 48개체 중에서 23개체가 이병되었으며, 11-TY6-AV는 42개체 중에서 14개체, 11-TY7-AV는 48개체 중에서 14개체, 11-TY8-AV는 48개체 중에서 15개체가 이병 되었다. F₂ 세대 4 집단 모두 p>0.05 수준에서 TYLCV 접종 후 표현형이 정상적인 유전분리비(3:1)를 보이는 것을 확인 하였다. 또, TYLCV에 저항성인 동일한 자원을 부계로 사용하더라도 모계로 어떤 것을 사용 하느냐에 따라서 저항성 정도가 다르게 나타나는 것을 확인할 수 있었다.