Construction of SSR linkage maps and identification of QTL for resistance to root rot in sweetpotato (Ipomoea batatas (L.) Lam.) CURRENT

Background: Sweetpotato root rot is a devastating disease caused by Fusarium solani that causes significant yield losses of sweetpotato in China. There is currently no effective method to control the disease. The breeding of resistant varieties is the most effective and economic way to control the disease. To date, quantitative trait locus (QTL) for resistance to root rot have not been reported and the biological mechanisms of resistance remain unclear in sweetpotato. Thus, it is necessary and worthwhile to identify resistance loci to help develop disease-resistant varieties. Results: In this study, we constructed genetic linkage maps of sweetpotato using a mapping population consisting of 300 individuals derived from a cross between Jizishu 1 and Longshu 9 by simple sequence repeat (SSR) markers, and mapped seven QTLs for resistance to root rot. In total, 484 and 573 polymorphic SSR markers were grouped into 90 linkage groups for Jizishu 1 and Longshu 9, respectively. The total map distance for Jizishu 1 was 3,974.24 cM, with an average marker distance of 8.23 cM. The total map distance for Longshu 9 was 5,163.35 cM, with an average marker distance of 9.01 cM. Five QTLs ( qRRM_1 , qRRM_2 , qRRM_3, qRRM_4 , and qRRM_5 ) were located in five linkage groups of Jizishu 1 map explaining 52.6-57.0% of the variation. Two QTLs ( qRRF_1 and qRRF_2 ) were mapped on two linkage groups of Longshu 9 explaining 57.6% and 53.6% of the variation. 71.4 % of the QTLs had a positive effect on the variation. Three of the seven QTLs, qRRM_3 , qRRF_1 , and qRRF_2 , were colocalized with markers IES43-5mt, IES68-6fs**, and IES108-1fs, respectively. Conclusions: To our knowledge, this is the first report on the construction of a genetic linkage map for purple sweetpotato (Jizishu 1) and the identification of QTLs associated with resistance to root rot in sweetpotato

China. It directly affects sweetpotato production, resulting in yield losses and quality deterioration, and it is a long-term problem plaguing sweetpotato farmers. The disease can lead to yield losses of 10-20%, and even 100% in severely infected fields [3]. There are currently no effective methodologies to control sweetpotato root rot. The breeding of resistant varieties is the most effective and economic way to control the disease. Conventional breeding for root rot resistance in sweetpotato is complicated, with a long cycle length, and generally improves only single traits.
Combining molecular techniques with conventional breeding methods is an effective way to overcome the limitations of seasonal and environmental effects, species isolation, and linkage drag existing in conventional breeding. Root rot resistance loci have not been mapped in sweetpotato to date. One study identified a root rot susceptibility locus, but resistance loci were not successfully identified [4].
The construction of a genetic linkage map is important for quantitative trait locus (QTL) identification, gene cloning, comparative genomic research, and marker-assisted selection breeding. However, sweetpotato, as a highly heterozygous, generally self-incompatible, and outcrossing hexaploid species with a large number of small chromosomes (2n = 6x = 90), poses numerous challenges for genetic analysis and breeding [5]. As a result, the progress of molecular biology research on sweetpotato lags far behind that in other major crops. For constructing linkage maps in such heterozygous species, Grattapaglia and Sederoff developed a two-way pseudo-testcross strategy, in which linkage analysis is conducted for each parent separately, as dominant markers that are heterozygous in one parent and recessive homozygous in the other parent will segregate in the F 1 generation, resulting in the development of two parental linkage maps [6].
developed genetic linkage maps of sweetpotato cultivars 'Beauregard' and 'Tanzania' using 947 and resistance, and identified nine chromosome regions associated with root knot nematode resistance. In addition, 13 QTLs for dry matter rate, 12 QTLs for starch content, and eight QTLs for β-carotene content were identified [7,20,21]. Zhao  ultradense multilocus integrated genetic map and characterized the inheritance system in a sweetpotato full-sib family using a newly developed software, MAPpoly [12].
In the present study, we used a mapping population of 300 F 1 individuals derived from a cross between Jizishu 1 and Longshu 9 to construct linkage maps using SSR markers and to conduct QTL analysis for resistance to root rot in sweetpotato. The results of this study are expected to provide useful information for developing resistance to root rot based on major QTLs.

Marker data
In total, 400 SSR primer pairs were screened in the parents and 10 progenies. Among these, 155 primer pairs (Additional file 3: Table S1) were polymorphic between the two parents and were selected to analyse the F 1 population. Finally, 839 good-quality polymorphic markers were obtained, with an average of five markers per primer pair. In total, 506 polymorphic SSR markers were obtained for mapping Jizishu 1, including 217 simplex, 47 duplex, 8 triplex, and 234 double-simplex markers, and 567 polymorphic SSR markers were obtained for mapping Longshu 9, including 237 simplex, 76 duplex, 20 triplex, and 234 double-simplex markers. The percentage of simplex markers was 79.8% (217/(271+47+8)) and 71.8% (237/(237+76+20)) in Jizishu 1 and Longshu 9, respectively, which was in accordance with the theoretical values for an autohexaploid (75% simplex and 25% non-simplex) according to Chi-square test results, and could be used to construct a genetic map of the hexaploid sweetpotato [7,10,19].

Genetic linkage map construction
The single-dose markers were used to construct a framework map of each parent at a LOD score of  Table S2). The linkage map of Longshu 9 was composed of 573 polymorphic markers, of which 185, 217, 40, and 131 were simplex, duplex, triplex and double-simplex markers, respectively. The largest and smallest linkage groups contained 17 and 2 markers, respectively. The total map distance was 5,163.35 cM, with an average marker distance of 9.01 cM. The longest linkage group was 151.60 cM, the shortest linkage group was 4.07 cM, and the average linkage group length was 57.37 cM (Additional file 4: Table S3). There were 239 distorted markers (49.38%) and 250 distorted markers Double-simplex markers were used to detect the homology of the corresponding linkage groups in the two maps. Among them, 100 double-simplex markers revealed that 42 linkage groups in Jizishu 1 map had homologous linkage relationships with 40 linkage groups in Longshu 9 map (Additional file 6: Table S4). Homology between the two parental maps is an important criterion for consistency of the maps.

QTL analysis
The root rot disease index in the mapping population showed abnormal distributions in 2016 and 2017 ( Fig. 1). In the two years, the average disease index of the mapping population ranged from 3.2 to 100, with a population mean of 58.4. The average disease index of Jizishu 1 was 14.4, indicating high resistance to root rot, and the average disease index of Longshu 9 was 84.5, indicating high susceptibility. ANOVA showed that the disease index was significantly different between the two years (Table 1). Therefore, the disease index for each year and the average values were separately analysed for QTL mapping. In addition, transgressive segregation was observed, that is, some progenies showed a higher disease index and others showed a lower disease index than either parent.  Fig. 2). Among the five QTLs, only qRRM_4 had a negative effect on resistance to root rot, explaining 57.0% of the variation, whereas the remaining four QTLs exhibited a positive effect on resistance. Two QTLs, named qRRF_1 and qRRF_2, were located in two linkage groups of Longshu 9, L9 (00.64) and L9 (00.74), respectively (Fig. 3). qRRF_1 had a positive and qRRF_2 a negative effect on root rot resistance, explaining 57.6% and 53.6% of the variation, respectively ( Table 2). These results agree with the fact that Jizishu 1 is highly resistant, whereas Longshu 9 is highly susceptible to root rot. were colocalized with the markers IES43-5mt, IES68-6fs**, and IES108-1fs. qRRM_1, qRRM_2, qRRM_4, and qRRM_5 were closely linked to IES9-8mt*, IES356-2md, IES351-4md, and IES68-11ds**, respectively. These QTLs and their colocalized markers could be used for marker-assisted selection of resistance to root rot in sweetpotato.

Discussion
When generating a genetic population, the genetic characteristics and differences among the parents should be thoroughly considered. Within a certain range, a higher level of polymorphism can be detected when the parents are distantly related and have greater genetic differences, and hence, the constructed map will be more accurate and more saturated. Jizishu 1 is a cultivar with purple skin, purple meat, and high starch content. Longshu 9 is a fresh-eating cultivar with red skin, yellow meat, and low starch content. The average disease index of Jizishu 1 was 14.4, indicating high resistance to root rot, whereas that of Longshu 9 was 84.5, indicating high susceptibility. The genetic variation between these two cultivars is high, and the cross was suitable for constructing a mapping population. As the difference in disease resistance was significant, the QTLs for root rot resistance could be located.  [24]. In addition to genetic factors, environmental factors and human intervention may cause distorted segregation. For example, the possibility of abnormal segregation appeared in population construction [25]. Sweetpotato is a highly heterozygous vegetative reproduction species, and its offspring is genetically unstable. An impure gene locus controlling a certain character may also cause distorted segregation in some lines [26].
Transgressive segregation was also observed for the root rot disease index in the mapping population. This may be due to the high heterozygosity of the parents, which results in the loss or accumulation of some favourable alleles in their offspring [21,27,28]. In sweetpotato breeding programs, this is commonly observed, especially in the hybrid offspring of parental materials with significant genetic differences.  [36]. Using composite interval mapping and inclusive composite interval mapping, two major QTLs and one minor QTL were validated which had significant effects in reducing stripe rust severity and explained 59.0-74.1% of the phenotype variation in disease response [37]. However, due to the limited number of markers on the genetic map, we need to increase the density of markers on the map in the future to accurately locate the genes against root rot.
Of the seven QTLs, five were mapped on the Jizishu 1 map, 80.0% of which had a positive effect, and two were located on the Longshu 9 map, one of which had a positive effect. These results agree with the fact that Jizishu 1 is substantially more resistant to root rot than Longshu 9. qRRM_3, qRRF_1, and qRRF_2 were colocalized with the corresponding markers IES43-5mt, IES68-6fs**, and IES108-1 fs.
The colocalized SSR markers identified in this study will be more useful for marker-assisted resistance breeding. These QTLs will have practical significance for gene cloning, and genome research of sweetpotato. Because of the lack of QTL mapping data for root rot resistance in sweetpotato in the literature, it is difficult to verify these QTLs. Therefore, the trait will be further studied and monitored in future studies to verify the loci identified in this research.

Conclusions
In this study, the first genetic linkage maps of purple sweetpotato (Jizishu 1) were constructed by SSR markers. To our knowledge, this is also the first report on the identification of QTLs associated with resistance to root rot in sweetpotato. These results will have practical significance for the fine mapping of root rot resistance genes and marker-assisted selection breeding for sweetpotato.

Plant materials
The mapping population was derived from a cross between the female parent Jizishu 1, a cultivar with resistance to root rot and high starch content that is popular in north China, and the male parent Longshu 9, a cultivar that is susceptible to root rot, has high yield and low starch content, and is popular in China. Both the parents and the F 1 generation were collected from the Institute of Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences (Shijiazhuang, China) and analysed for esterase isozymes, self-bred progeny was deleted. In total, 300 progenies were used for genetic linkage map construction and QTL analysis.

DNA extraction
Genomic DNA was extracted from fresh leaves of Jizishu 1, Longshu 9, and the 300 F 1 individuals using the cetyltrimethylammonium bromide method [38,39]. DNA concentrations and quality were determined using an ultraviolet spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, USA) and 1.0% agarose gel electrophoresis respectively. The DNA was diluted to 50 ng/µL.

Marker recording
Specific bands were read from top to bottom according to the molecular weights in comparison with a standard DNA marker (50 bp DNA Ladder, Tiangen, China). Clear, high-quality and high-resolution bands with a size of 100-700 bp were selected to improve the recording accuracy and reliability.
Polymorphic markers were recorded as 1 or 0 according to their presence or absence, respectively, in the parents and the F 1 individuals, and vague or missing bands were recorded as 2. All polymorphic markers were divided into three categories (maternal, paternal, and double-simplex markers) according to their presence in the two parents (Table 3). Marker dosage was determined as the segregation ratio of markers (presence:absence) in the mapping population. Four cytological hypotheses proposed by Jones in 1967 were used to classify marker dosages without considering strict tetraploid isolation [40]. Based on the goodness-of-fit to the expected segregation ratios for all markers determined using the Chi-square test, we divided markers into four groups on the basis of their segregation ratios: (1) simplex or single-dose markers exist in one of the parents in the form of a single copy, and the segregation ratio in the progeny is 1:1 (presence:absence), (2) duplex or double-dose markers are present in one of the parents in the form of two copies, and the segregation ratio in the progeny is 4:1 for hexasomic, 5:1 for tetrasomic, or 3:1 for disomic or tetrasomic inheritance, (3) triplex or triple-dose markers are present in one of the parents in three copies, having a ratio of 19:1 (hexasomic), 11:1 (tetradisomic) or 7:1 (disomic); (4) double-simplex markers exist in both parents in a single-dose condition and segregate in a 3:1 ratio in the progeny [7,10]. According to the Chi-square test results, if the segregation ratio did not conform to Mendelian segregation, it was considered as a distorted marker.
Marker names were determined by considering the following four points: (1) the polymorphic primer names (e.g., IES87), (2) the corresponding specific band number, usually with a large-molecularweight band in front (e.g., 05), (3) the type of marker, f, m, or ds (Table 3), (4) the dosage of the marker, s, d, t, or ds, which represented simplex, duplex, triplex or double-simplex, respectively. For example, IES295-1fs represents the first polymorphic band from SSR primer IES295, and its marker type is a Longshu 9 single marker. For the distorted markers, * and ** as a suffix indicate significant differences at the 0.05 and 0.01 levels, respectively.

Linkage map construction
The genetic linkage map was constructed using the JoinMap 4.0 software [41] and the pseudotestcross mapping strategy [6,42]. Genotype codes of the 300 F 1 individual plants were recorded using the standard genotype analysis method in JoinMap 4.0. When a band was present only in Jizishu 1, progeny with the same band pattern as that of Jizishu 1 was marked 'lm', and offspring with a different band pattern was marked 'll'. When a band was present only in Longshu 9, progeny with the same band pattern as Longshu 9 was marked 'np', otherwise, it was marked 'nn'. When a band was present in both parents, but it was segregated in the progeny, progeny with bands was marked 'h-', progeny without bands was marked 'kk'. Only clear bands were recorded, missing and ambiguous bands were represented as '-'.
Using the outbreeder full-sib family analysis model, the map was constructed in two steps: (1) singledose markers were used to construct the framework map of each parent at a logarithm of odds (LOD) of 5.0, (2) duplex and triplex markers were inserted into the framework map to obtain the final genetic linkage map [7,10]. Linkage groups containing the same duplex or triplex markers were considered homologous and divided into corresponding homologous linkage groups in the same parental map. Linkage groups with the same double-simplex markers in the two maps were considered to be homologous.

Identification of resistance to root rot
The two parents and the 300 F 1 individuals were planted in the natural disease nursery of Xiong

Consent for publication
Not applicable.

Availability of data and material
All data generated or analyzed during this study are included in this published article and its supplementary information files.

Competing interests
The authors declare that they have no competing interest.   The QTLs for resistance to root rot identified in the Jizishu 1 linkage groups. QTLs were shown as vertical bars on the right side of the respective linkage groups. The QTL corresponding markers were indicated by underlined text.

Figure 3
The QTLs for resistance to root rot identified in the Longshu 9 linkage groups. QTLs were shown as vertical bars on the right side of the respective linkage groups. The QTL corresponding markers were indicated by underlined text.

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