Phenotypic variations in callus browning between DXCWR, the 2DIL introgression lines, and the recurrent parent GC2
In our previous study, we constructed a set of introgression lines (named 2DIL) using the O. rufipogon accession DXCWR as a donor and the elite indica cultivar GC2 (O. sativa) as the recurrent parent. DXCWR was found to be relatively resistant to browning, with a CBI of 0.22 (Zhang et al. 2020; Table 1). To identify QTLs involved in the regulation of callus browning in rice, we screened 129 introgression lines by inoculating mature seeds on unimproved NB medium (NB basal medium without additives, named NB1) for 30 days (Fig. 1). Meanwhile, based on the genotypes of the 129 introgression lines, we constructed the graphic genotype used the program GGT2.0 (Fig. 2). We divided the tendency for callus browning into five levels, compared to GC2, 0 level was recorded as no browning, 1 and 2 level was considered as medium browning, but 3 and 4 level was serious browning similar to GC2 (Fig. 1b–f). The recurrent parent GC2 was more susceptible to callus browning than DXCWR (Fig. 1a and Table 1). The callus browning rate (CBR), callus browning index (CBI), and the standard callus browning index (SCBI) of the 2DIL population varied significantly, ranging from 0.00 to 100%, 0.00 to 0.93, and − 1.00 to 0.40, respectively (Table 1 and Fig. 3).
Variance And Correlation Among The Three Callus Browning Indices
The three indices related to callus browning varied substantially among the introgression lines (Table 2). The ANOVA revealed that the highly significant variation in the three indices was due to a genotype effect (Table 2). The correlation among these indices was determined by calculating the correlation coefficients (r), revealing all indices were positively correlated: CBR with CBI (r = 0.895), CBR with SCBI (r = 0.828), and CBI with SCBI (r = 0.938) (Table 3). Consequently, CBR, CBI, and SCBI were all used for QTL analysis.
Identification Of Qtls For Callus Browning (Dup: Abstract ?)
A total of 30 putative QTLs closely related to the callus browning phenotype were identified, including 10 QTLs for CBR (qCBR1-1 ~ qCBR12-2), which explained between 3% and 11% of the phenotypic variation in this trait; 10 QTLs for the CBI (qCBI2-1 ~ qCBI12-2), explaining 3–8% of CBI variation; and 10 QTLs for SCBI (qSCBI2-1 ~ qCBI12-2), explaining 4–8% of the phenotypic variation in this trait (Table 4 and Fig. 4). These QTLs were located on chromosomes 1, 2, 3, 4, 8, 9, and 12 (Table 4 and Fig. 4). The QTLs linked to markers RM335 on chromosome 4, RM189 on chromosome 9, and RM7003 on chromosome 12 were found to be associated with all three indices. The QTLs accounted for 5–11% of the phenotypic variation, and the additive effect of the DXCWR alleles could alleviate callus browning (Table 4).
Screening Elite Introgression Lines With Low Callus Browning Level
We screened six introgression lines (2DIL18, 2DIL99, 2DIL101, 2DIL103, 2DIL110, and 2DIL112) exhibiting a significant reduction in callus browning compared with GC2 (Fig. 5). Genotype analysis showed that the six selected introgression lines possessed introgression fragments distributed across different chromosomes: 2DIL112 had an introgression fragment near marker RM71 on chromosome 2; 2DIL101 had an introgression fragment near marker RM114 on chromosome 3; 2DIL18 and 2DIL110 had introgression fragments near marker RM335 on chromosome 4; and 2DIL99, 2DIL103, and 2DIL110 had introgression fragments near markers RM328, OSR29, and RM189 on chromosome 9. These lines can be used not only as indica rice accession genetic transformation receptors, but can also be programmed as parent materials for the fine-mapping of callus browning.