Phenotypic characterization of parental lines and development of mapping populations for stigma traits
In our previous study on pistil traits of wild rice species (Marathi et al. 2015), we suggested that OL possessed an ideal female organ structure (a long and exserted stigma phenotype) for increasing seed setting rate in hybrid seed production. Hence, in this study, we used an OL (IRGC110404) to develop mapping populations through crosses with O. sativa (IR64) and furthermore to identify the genetic loci controlling stigma traits. First, five pistil traits (stigma length, style length, stigma breadth, stigma area, and pistil length) were phenotyped from parents. As expected, the OL exhibited higher values for all five traits than those of IR64, especially for stigma length (OL: 2.39 mm, IR64: 1.31 mm) (Table 1). F1 plants were obtained through wide hybridization between IR64 and OL (IRGC110404). All five stigma phenotypes of F1 plants were the same as those of OL (α = 0.05) (Table 1). The results indicated that stigma traits (stigma length, style length, stigma breadth, stigma area, and pistil length) were controlled by dominance genetic factors of OL. Further, F1 plants generated 37 BC1F1 and 37 BC2F1 plants after backcrossing to the recurrent parent and the BC2F1 plants were self-pollinated to produce 357 BC2F2 plants. A total of 3,570 florets were collected and dissected for phenotyping of the key floral traits among the 357 BC2F2 segregating plants. The mean performance and the range of the trait values obtained from the mapping populations indicated segregation toward the cultivated parent for all the traits. The frequency distribution scores showed bell-shaped curves for each of the traits studied and partially skewed toward cultivated rice lines (Fig. 1). This result suggested that the mode of distribution of these traits was normal non-Mendelian segregation. The mean values of each trait were used for the linkage analysis for locating the loci influencing these key floral traits.
Table 1
Phenotypic characterization of parental lines and F1 (IR64 × OL) for stigma traits
Genotypes
|
Stigma length (mm)
|
Style length (mm)
|
Stigma breadth (mm)
|
Stigma area (mm2)
|
Pistil length (mm)
|
IR58025A
|
1.46b
|
1.01d
|
0.50d
|
0.44d
|
2.47c
|
IR58025B
|
1.44b
|
1.07c
|
0.47d
|
0.51d
|
2.51c
|
IR64
|
1.31c
|
1.05c
|
0.46d
|
0.53cd
|
2.35d
|
IR68897A
|
1.43b
|
0.87e
|
0.72b
|
0.67b
|
2.29d
|
IR68897B
|
1.51b
|
1.13b
|
0.63c
|
0.61bc
|
2.64b
|
OL (IRGC110404)
|
2.39a
|
1.29a
|
0.97a
|
1.13a
|
3.56a
|
F1(IR64 × OL)
|
2.46a
|
1.31a
|
0.92 a
|
1.15a
|
4.07a
|
SE(±)
|
0.15
|
0.02
|
0.02
|
0.04
|
0.05
|
abcdare LSD significant values at 0.05 level of significance |
A correlation study was carried out to assess the correlations among the floral traits spikelets with exserted stigma (%), stigma length (mm), pistil length (mm), and internal angle of stigma lobes. The highest significant correlation coefficient was noticed between stigma length and pistil length (0.95) at the 0.01 level of significance, followed by that between stigma length and stigma exsertion (0.86) and between stigma exsertion and pistil length (0.78). The positive significant correlation coefficients at a lower level of significance indicated that the traits stigma length, stigma exsertion, and pistil length were positively correlated. Hence, selection of anyone of these traits positively influences the selection of other traits (Table S1).
Linkage map construction and localization of genomic regions associated with stigma traits
A mapping population consisting of 357 BC2F2 plants was genotyped by 164 polymorphic SSR and STS markers and a saturated linkage map was constructed (Fig. S2). The total length of the linkage map was 1,840 cM, with an average marker interval of 12.6 cM. The genotypic and phenotypic data of pistil traits were used to map the genomic regions conferring each floral trait. Composite interval mapping identified 14 QTLs in total on different chromosomes; 5, 3, 2, 1, and 3 QTLs for stigma length, style length, stigma breadth, stigma area, and pistil length, respectively (Table 2). Among the QTLs detected, the major QTL (qSTGL8.0) bordered by RM1109 and RM80 markers on the long arm of chromosome 8 showed the highest phenotypic variance of 35.40%, with 42.50 LOD for stigma length (Table 2; Fig. 2). For style length, three QTLs (qSTYL1-1, qSTYL5-2, and qSTYL8-1) were detected on chromosomes 1, 5, and 8, respectively. Among these QTLs, qSTYL1-1 showed the highest phenotypic variance (15.10%) and was identified between marker intervals RM319 and RM3640 on chromosome 1 with an LOD of 9.97. For stigma breadth, two QTLs (qSTGB1-1 and qSTGB3-1) were detected and QTL qSTGB1-1 showed the highest phenotypic variance (21.14%), with an LOD of 14.71. However, only one QTL (qSTGA8-2) was detected on the long arm of chromosome 8 for stigma area, with an LOD of 8.52 and phenotypic variance of 3.12%. Furthermore, for pistil length, three genomic regions (qPSTL1-1, qPSTL1-3, and qPSTL11-1) were identified and one of the QTLs, qPSTL11-1 on chromosome 11 with an LOD value of 5.63, explained 26.96% of the phenotypic variance (Fig. S3).
Table 2
List of floral trait QTLs detected in IR64 × OL BC2F2 mapping population by composite interval mapping at 0.01 level of confidence and 1000 permutations
Floral traits
|
Number of QTLs detected
|
QTL
|
Chromosome
|
Flanking marker (left)
|
Flanking marker (right)
|
LOD
|
Additive
|
Dominance
|
R2
|
Stigma length
|
5
|
qSTGL2-1
|
2
|
RM110
|
S02026
|
4.60
|
0.00
|
0.20
|
8.61
|
qSTGL5-1
|
5
|
RM421
|
RM7653
|
5.60
|
0.00
|
0.00
|
3.51
|
qSTGL8.0
|
8
|
RM1109
|
RM80
|
42.50
|
-0.10
|
0.10
|
35.40
|
qSTGL11-1
|
11
|
RM590
|
RM286
|
7.40
|
0.00
|
0.10
|
4.30
|
qSTGL11-2
|
11
|
RM120
|
RM229
|
5.70
|
-0.10
|
-0.10
|
6.80
|
Style length
|
3
|
qSTYL1-1
|
1
|
RM319
|
RM3640
|
9.97
|
0.10
|
0.00
|
15.10
|
qSTYL5-2
|
5
|
RM7653
|
RM6360
|
6.12
|
-0.08
|
0.01
|
10.21
|
qSTYL8-1
|
8
|
RM404
|
RM1109
|
4.58
|
0.06
|
-0.06
|
17.11
|
Stigma breadth
|
2
|
qSTGB1-1
|
1
|
RM403
|
RM319
|
14.71
|
-0.04
|
0.00
|
21.14
|
qSTGB3-1
|
3
|
RM3525
|
RM520
|
9.77
|
0.04
|
0.01
|
9.09
|
Stigma area
|
1
|
qSTGA8-2
|
8
|
RM80
|
RM502
|
8.52
|
0.04
|
0.03
|
3.12
|
Pistil length
|
3
|
qPSTL1-1
|
1
|
RM3604
|
RM3746
|
8.06
|
-0.13
|
-0.02
|
8.03
|
qPSTL1-3
|
1
|
RM3640
|
RM8134
|
8.59
|
0.15
|
0.05
|
9.00
|
qPSTL11-1
|
11
|
RM5997
|
RM254
|
5.63
|
0.25
|
-0.12
|
26.96
|
Fine mapping of the major QTL locus qSTGL8.0
To dissect the major-effect QTL qSTGL8.0 that was mapped between markers RM1109 and RM80 corresponding to 3.99 Mb size of the reference genome (IRGSP1.0), 21 InDel markers were newly designed based on a sequence comparison between OL and the reference genome within the flanking marker positions (Table S2). Of the 21 InDel markers, 14 showed polymorphism between IR64 and OL. These 14 markers were used to genotype a 357 BC2F2 mapping population. We carried out additional linkage analysis using the new genotypic data and previously collected phenotypic data and narrowed down the locus to 2.99 Mb size bordered by RM7356 and RM256 (Fig. 3). Further, the tightly linked markers for the long-stigma phenotype were identified through a marker validation experiment using 135 BC2F3 plants derived from the two BC2F2 plants (BC2F2-8 and BC2F2-51) with the same set of 14 markers. The marker PA08-18 was highly co-segregating with the phenotype (Fig. S4), suggesting that the causal gene for long stigma is located close to the PA08-18 marker. Hence, these markers were used for the introgression of locus qSTGL8.0 into maintainers and CMS lines (Fig. 3).
Evaluation of the genetic effect of qSTGL8.0in commercial maintainer lines
To evaluate the genetic effect of qSTGL8.0 in different genotypes, the QTL was introgressed into two commercial maintainer (B) and CMS (A) lines, IR58025B/A and IR68897B/A, which had shorter stigma (1.43‒1.51 mm) and smaller size in other pistil traits than those of OL (Table 1). Line IR58025B was crossed with the OL (IRGC110404) and line IR68897B was crossed with another OL accession (IRGC92664) also exhibiting long stigma, and qSTGL8.0 was transferred into each maintainer background by the MABC method (Fig. S5). Briefly, the F1 plants were backcrossed to the corresponding B line and genotyping was conducted using 10 markers (PA08-03, PA08-05, PA08-06, PA08-09, PA08-11, PA08-12, PA08-16, PA08-17, PA08-18, and PA08-19) covering the qSTGL8.0 locus with 103 (IR58025B × OL-IRGC110404) and 98 (IR68897B × OL-IRGC92664) BC1F1 plants. Marker-trait association analysis revealed that the BC1F1 plants possessing OL alleles at qSTGL8.0 had long-exserted stigma phenotype while the plants of the maintainer lines had short-stigma phenotype in both backgrounds. The BC1F1 plants possessing the qSTGL8.0-OL allele were backcrossed and produced 29 and 14 BC2F1 seeds in IR58025B and IR68897B backgrounds, respectively. Further, the lines were advanced to the BC2F3 generation based on the genotypic data. A total of 158 BC2F3 plants having the homozygous OL allele at the qSTGL8.0 locus (59 in IR58025B and 99 in IR68897B backgrounds) were obtained and they showed significantly long-exserted stigma vis-à-vis the original recurrent parents as well as the BC2F3 segregants possessing the homozygous recurrent allele of qSTGL8.0 (Fig. S6). These results indicated that the genetic effect of theqSTGL8.0-OL allele was clearly seen in two different maintainer backgrounds in addition to IR64, and both the qSTGL8.0 alleles derived from two OL accessions (IRGC110404 and IRGC92664) are functional and increase stigma length in O. sativa. To examine the genome recovery status of the introgressed lines, background genotyping of the selected BC2F4 plants was carried out using high-density SNP markers (Illumina 7K SNP chip). The percent recovery of the recurrent parent genome was computed among the selected BC2F4 plants. The BC2F4 plants 91B-42 (IR58025B background) and 107B-12 (IR68897B background) showed maximum recovery of the recurrent parent genomes, 92.43% and 90.35%, respectively, carrying the homozygous OL alleles at the qSTGL8.0 locus (Table S3; Fig. 4). The stigma length of the lines 91B-42 and 107B-12 was 2.43 mm and 2.08 mm, respectively, while it was 1.30 mm for IR58025B and 1.36 mm for IR68897B (Table S3). Hence, these improved maintainer lines with maximum recurrent parent genome recovery were used as donor maintainer lines for the transfer of qSTGL8.0 to their corresponding CMS lines (Fig. S5).
Transfer of qSTGL8.0 from the improved maintainer lines to the corresponding CMS lines
The improved B lines were test-crossed with sterile CMS lines IR58025A and IR68897A to transfer the qSTGL8.0 locus, which could enhance out-crossing rate. Hence, during the 2014 dry season, 134 test crosses were made between the improved B lines and their corresponding CMS lines (IR58025A and IR68897A) to transfer qSTGL8.0 into a parental CMS line background. Based on pollen sterility and stigma traits, 19 F1 plants were selected and backcrossed with the respective improved IR58025B (Line 91B-42) and IR68897B (Line 107B-12) lines, and BC1F1 seeds were produced. A total of 101 BC1F1 plants underwent foreground selection for the qSTGL8.0 locus by using the flanking markers. The BC1F1 plants with long-exserted stigma possessing the homozygous OL allele at the qSTGL8.0 locus and being completely pollen sterile were selected and backcrossed again to develop BC2F1 plants and then BC3F1 plants (Fig. S5b). Background analysis of 91A-18 plants selected from BC3F1 progenies in the background of IR58025A showed as high as 92.21% recurrent parent genome recovery. Similarly, the improved CMS line 107A-35 derived from IR68897A showed as high as 94.48% recurrent parent genome recovery (Table S3; Fig. 4). The improved CMS lines 91A-18 and 107A-35 showed stigma lengths of 2.18 mm and 2.61 mm, respectively, which were significantly longer than those of their background parents, IR58025A and IR68897A (Table S3). These results suggested that the transfer of a single major QTL, qSTGL8.0, among the 14 QTLs detected in our study significantly increased stigma length in two different CMS lines and therefore the genetic effect of qSTGL8.0 was validated in all the backgrounds tested.
Phenotypic evaluation of parental and improved CMS lines
The improved CMS lines 91A-18 and 107A-35 along with the original parental CMS lines (IR58025A and IR68897A) were evaluated for agro-morphological traits and seed setting rate. The improved CMS lines showed similar trait performances for most of the traits studied (Table 3; Fig. 5). For the traits such as plant height and panicle number, the recurrent parent and improved CMS lines showed a similar performance as depicted by DMRT significance values. The CMS line IR58025A (82.67 cm) and its improved line 91A-18 (83.33 cm) were significantly taller than the other CMS line, IR68897A (74.67 cm), and its improved line, 107A-35 (72.88 cm). On the contrary, with 28.67 and 24.01 mean number of panicles, IR68897A and 107A-35 possess significantly more tillers than IR58025A (18.01) and its improved line, 91A-18 (16.10). However, plot yield and seed setting rate were significantly higher in the improved CMS lines than in both the recurrent CMS lines. All the CMS lines were pollinated with the respective B lines. Plot yield was 2076.11 kg ha− 1 and 2172.72 kg ha− 1 for the recurrent parents, IR58025A and IR68897, respectively, while it was 2431.92 kg ha− 1 and 2832.72 kg ha− 1 for 91A-18 and 107A-35, respectively. Similarly, the seed setting rate of the recurrent parents was 22.72% for IR58025A and 31.86% for IR68897A, whereas it was 69.36% for 91A-18 and 77.88% for 107A-35. This result clearly showed that there was an enhanced out-crossing rate of at least 2.50 times (245%) that of the recurrent parent IR58025A, whereas it was 3.05 times that of IR68897A. Nevertheless, the pollen sterility of all the CMS lines was higher than 99.90% consistently, indicating stable expression of male sterility across several seasons. These results suggested that a long-exserted stigma phenotype induced by qSTGL8.0-OL alleles significantly improved plot yield and seed setting rate in CMS backgrounds.
Table 3
Performance of parental and improved CMS lines for yield and yield-associated traits
Genotypes
|
PH (cm)
|
PN
|
PL (cm)
|
PY (kg ha−1)
|
OC (%)
|
S (%)
|
IR58025A
|
82.67 ± 1.12b
|
18.01 ± 1.76a
|
24.50 ± 0.32b
|
2076.11 ± 7.38b
|
22.72 ± 3.93b
|
99.99
|
91A-18
|
83.33 ± 1.13b
|
16.10 ± 1.62a
|
26.10 ± 0.30a
|
2431.92 ± 7.06a
|
69.36 ± 3.60a
|
99.99
|
IR68897A
|
74.67 ± 1.05ab
|
28.67 ± 1.67b
|
21.33 ± 0.36c
|
2172.22 ± 7.71b
|
31.86 ± 3.72b
|
99.98
|
107A-35
|
72.88 ± 1.06a
|
24.01 ± 1.79b
|
28.09 ± 0.44a
|
2832.72 ± 7.75ac
|
77.88 ± 5.34a
|
99.99
|
abcare LSD significant values at 0.05 level of significance |
PH, plant height; PN, panicle number; PL, panicle length; PY, plot yield; OC, outcrossing rate; S, pollen sterility. Values are means ± SE. |
Assessment of stigma receptivity of parental and improved CMS lines
As stigma receptivity is the ability of the stigma to support viable and compatible pollen and is also one of the contributors for out-crossing rate, an experiment was conducted to determine the duration of stigma receptivity of the improved CMS lines possessing qSTGL8.0 and their original CMS lines, IR58025A and IR68897A. First, the stigma length of improved CMS lines and their recurrent parental CMS lines was characterized. Then, the same sets of lines were evaluated for studying the duration of stigma receptivity. As expected, the stigma length of improved CMS lines 91A-18-15 (2.62 mm) and 107A-35-43 (2.25 mm) was significantly higher than that of their background CMS lines, IR58025A (1.37 mm) and IR68897A (1.48 mm) (Table 4). Further, to study the duration of stigma receptivity, out-crossing rate (seed setting percentage) was considered as the measure of the duration of stigma receptivity from day one until the day when the lowest or no seed setting was computed. On the first day, the out-crossing rate of the improved A line (91A-18-15) was 90.35% whereas it was 40.24% in the background parent, IR58025A. The out-crossing rate for IR58025A was nil on the sixth day, whereas it was still 14.28% in the improved CMS line (91A-18-15). Similarly, for IR68897A, the out-crossing rate was nil on the sixth day, whereas it was 31.82% for improved CMS line 107A-35-43 (Fig. 6). These results indicated that stigma receptivity gradually decreased from the spikelet opening day in both recurrent and improved CMS lines; however, stigma receptivity was slightly longer in the improved CMS lines than in the original CMS lines.
Table 4
Stigma trait characters of the parental and improved CMS lines
Genotypes
|
Stigma length (mm)
|
Style length (mm)
|
Stigma breadth (mm)
|
Stigma area (mm2)
|
Pistil length (mm)
|
IR58025A
|
1.37b
|
1.04c
|
0.46d
|
0.38c
|
2.60b
|
91A-18-15
|
2.62a
|
1.22a
|
0.96a
|
0.99a
|
3.84a
|
IR68897A
|
1.48b
|
0.86b
|
0.70c
|
0.58bc
|
2.34b
|
107A-35-43
|
2.25a
|
1.50a
|
1.02a
|
1.19a
|
3.75a
|
IR64
|
1.31c
|
1.05c
|
0.46d
|
0.53bc
|
2.36b
|
OL
|
2.39a
|
1.29a
|
0.97a
|
1.13a
|
3.68a
|
SE(±)
|
0.11
|
0.06
|
0.03
|
0.07
|
0.06
|
abcdare LSD significant values at 0.05 level of significance |