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 parents and the F1 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 progenies were deleted. In total, 300 progenies were used for genetic linkage map construction and QTL analysis.
Genomic DNA was extracted from fresh leaves of Jizishu 1, Longshu 9, and the 300 F1 individuals using the cetyltrimethylammonium bromide method [31, 32]. 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.
PCR was carried out using 20 μL reaction mixtures containing 1 μL of DNA (50 ng/μL), 0.6 μL of each primer (5 μM, Invitrogen, China), 6 μL of 2×Taq PCR StarMix with loading dye (for PAGE, GenStar, China), and 11.8 μL of ddH2O. Thermal cycles were as follows: initial denaturation at 95°C for 5 min, 35 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 40 s, final extension at 72°C for 5 min, and hold at 10°C. 400 pairs of SSR primers (Additional file 5: Table S3) were screened for polymorphism in the parents and ten progenies, and 8% acrylamide gels were used for electrophoresis detection. The polymorphic primers were used to characterise the F1 segregating population.
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 F1 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 5).
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 . 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 [5, 8]. According to the Chi-square analysis, 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-molecular-weight band in front (e.g., 05), (3) the type of marker, f, m, or ds (Table 5), (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  and the pseudo-testcross mapping strategy, as dominant markers that are heterozygous in one parent, and homozygous recessive in the other parent will segregate in the F1 generation, resulting in development of two parental linkage maps [34, 35]. Genotype codes for the 300 F1 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 were designated as ‘lm’, while offspring with a different band pattern were marked ‘ll’. When a band was present only in Longshu 9, progeny with the same band pattern as Longshu 9 were designated as ‘np’, otherwise, they were marked ‘nn’. When a band was present in both parents, however, segregated in the progeny, progeny with bands were marked ‘h-’, while those without bands were 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) single-dose 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 [5, 8]. 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.
Names of linkage groups are primarily composed of three parts: (1) the names of the corresponding parents, JZ1 (Jizishu 1) or L9 (Longshu 9); (2) a number between 1 and 15 (written as 01–15) indicating the sequence number of the homologous linkage group; (3) a number between 1 and 90 (written as 01–90) referring to the sequence number of the linkage group. For example, JZ1 (01.01) indicates that the linkage group belongs to the first linkage group on the Jizishu 1 map, and to the first homologous linkage group. JZ1 (00.66) indicates that the linkage group is the 66th linkage group on the Jizishu 1 map, and is not included in any homologous linkage group.
Identification of resistance to root rot
The two parents and the 300 F1 individuals were planted in the natural disease nursery of Xiong County, Hebei, China (39°06′43″N, 116°14′56″E), the main sweetpotato producing area, on May 16, 2016 and 2017. The experiment was completely randomly arranged, with ridge spacing of 0.85 m and plant spacing of 0.25 m. For each of the F1 individuals, five plants were planted, with three repeats. Each parent as the control were planted in each ridge with five. Forty days after planting, as well as in mid-October, the disease index of aboveground and underground was investigated and calculated, respectively.
The final disease index was determined according to the average value of disease index of aboveground and underground. Identification standards for aboveground sweetpotato resistance to root rot is as follows, ‘0’ the plants grow normally, no disease can be seen; ‘1’ the leaves are slightly yellowed, others are normal; ‘2’ the branches are few and short, the leaves are significantly yellowed, and the plant bud or flower; ‘3’ the plant is significantly dwarfed without branching, old leaves fall down from bottom to top; ‘4’ plant has died. Identification standards for underground sweetpotato resistance to root rot are as follows, ‘0’ the fibrous and tuberous roots are normal without any disease spots; ‘1’ a few fibrous roots have turned black (the number of diseased fibrous roots accounts for less than 10% of the total roots), and no disease spots are present on the underground stems, which have no significant effect on tuberous roots formation; ‘2’ a few fibrous roots turn black (the number of diseased fibrous roots accounts for 10–25% of the total roots), and a few diseased spots are apparent on underground stems and tuberous roots, which have a slight effect on tuberous roots formation; ‘3’ nearly half of the fibrous roots have turned black (the number of diseased fibrous roots accounts for 25.1–50.0% of the total roots), with numerous diseased spots are present on underground stems and tuberous roots, which have a significant effect on tuberous roots formation; ‘4’ most of the fibrous roots have turned black (the number of fibrous diseased roots accounts for more than 50% of the total roots), and many large diseased spots are present on the underground stem, without tuberous roots, or the plant has died.
[Please see the supplementary files section to view the equation.]
Where DI represents the disease index of aboveground and underground, respectively, A represents the number of plants at different levels (0, 1, 2, 3 or 4), B represents the corresponding level (0, 1, 2, 3 or 4), C represents the total number of investigated plants, and 4 represents the highest level (4).
Mapping of QTLs for root rot resistance
The frequency distribution of the 300 F1 individual disease index values in 2016 and 2017, as well as the means were determined using IBM SPSS Statistics 24.0 software. Genetic linkage map data, phenotypic data for each year, and the average values were used to map QTLs for root rot resistance using the MapQTL5.0 software . First, interval mapping analysis was used to determine the initial location of the QTL. Second, a multiple QTL model was used to precisely locate the QTL, in which the nearest marker associated with the QTL was selected as the cofactor. In this study, a LOD score of 3.0 was used as the typical threshold value to determine the location of the QTL. QTLs appearing at the same genomic location in the two environments and average data were considered stable QTLs. The linkage maps of QTL for the resistance to root rot were drawn by Map Chart 2.2 .