NILs development
For development of the NILs, Jiafuzhan, an indica cultivar, was used as the recipient, and the restorer japonica cultivar Hui1586 was used as the donor. The F1 plants were generated from Jiafuzhan as the female and Hui1586 as the male, and the F1 plants were back-crossed with Jiafuzhan to produce the BC1F1 generation. These BC1F1 plants were then backcrossed with Jiafuzhan to produce BC2F1 plants. Using the same approach, 118 BC3F1 individuals were obtained, and these plants were self-pollinated to produce the BC3F2 lines. Based on their characteristics, we selected one or two individual plants from each line. As a result, 176 NILs were obtained (Fig. 1).
Identification and analysis of QTLs for plant height in the NILs
To evaluate the potential advantages of the NILs for QTL detection, the phenotypic variations in plant height were observed in 176 NILs, and NIL36 exhibited a lower plant height than Jiafuzhan. Further investigations and analyses showed that the plant height of Jiafuzhan was 116.22 cm, whereas that of Jiafuzhan NIL36 was 79.52 cm. The difference in plant height between these lines reached a very significant level (Fig. 2).
The phenotypic comparisons between NIL36 and Jiafuzhan are presented in Table 1. The results showed some significant differences in major agronomic traits, including the plant height, panicle length, spikelets per panicle and yield per plant, between NIL36 and Jiafuzhan. However, no significant difference in the number of effective panicles, seed setting rate, 1,000-grain weight, grain length or grain width was found (Table 1).
Table 1
Comparison of the main agronomic traits of Jiafuzhan, NIL36 and knockout lines
Traits
|
Jiafuzhan
|
NIL36
|
qPH-iaa30KO-
Line 1
|
qPH-iaa30KO-
Line 2
|
qPH-iaa30KO-
Line 3
|
Plant height (cm)
|
116.22 ± 2.26
|
79.52 ± 1.72**
|
80.22 ± 1.76**
|
81.02 ± 1.82**
|
80.35 ± 1.81**
|
Panicle length (cm)
|
27.12 ± 1.12
|
21.26 ± 1.08*
|
22.36 ± 1.24*
|
22.66 ± 1.12*
|
21.96 ± 1.32*
|
Number of effective panicles
|
10.54 ± 1.04
|
10.82 ± 1.08
|
10.42 ± 1.12
|
10.82 ± 1.18
|
10.72 ± 1.02
|
Spikelets per panicle
|
168.46 ± 4.86
|
125.86 ± 4.32**
|
128.76 ± 4.62**
|
130.12 ± 4.82**
|
122.86 ± 4.22**
|
Seed setting rate (%)
|
97.52 ± 1.26
|
98.22 ± 1.18
|
97.28 ± 1.28
|
97.38 ± 1.18
|
96.98 ± 1.18
|
1,000-grain weight (g)
|
23.32 ± 0.54
|
23.44 ± 0.42
|
23.12 ± 0.48
|
23.54 ± 0.53
|
23.64 ± 0.46
|
Grain length (mm)
|
10.95 ± 0.13
|
10.72 ± 0.16
|
10.80 ± 0.20
|
10.92 ± 0.214
|
10.88 ± 0.15
|
Grain width (mm)
|
2.68 ± 0.08
|
2.72 ± 0.05
|
2.70 ± 0.08
|
2.66 ± 0.09
|
2.68 ± 0.07
|
Yield per plant (g)
|
40.38 ± 1.02
|
31.35 ± 0.98**
|
30.17 ± 1.08**
|
32.33 ± 1.02**
|
31.16 ± 1.08**
|
*P < 0.05 and **P < 0.01 for the differences among Jiafuzhan, NIL36 and knockout lines. The data were derived from the trial performed at the Hainan experimental station in April 2019. |
Genetic analysis of NIL36 in terms of plant height
To determine whether NIL36 was controlled by a single gene, NIL36 was hybridized with Jiafuzhan. F1 hybridization showed the phenotype of Jiafuzhan, and the F2 population showed Mendelian segregation (Table 2). Segregation between the Jiafuzhan and NIL36 phenotypes fit a 3:1 segregation ratio in the two F2 populations (χ2 = 0.134 ~ 0.456, P > 0.5). The results showed that the NIL36 phenotype for plant height was controlled by a single recessive gene.
Table 2
Segregation of the F2 populations crossed with NIL36
Crosses
|
F1 phenotype
|
F2 population
|
χ2(3:1)
|
P
|
Normal type of Jiafuzhan
|
Normal type of NIL36
|
Total
plants
|
NIL36/Jiafuzhan
|
Normal type of Jiafuzhan
|
240
|
82
|
322
|
0.456*
|
0.5–0.75
|
Jiafuzhan/NIL36
|
Normal type of Jiafuzhan
|
286
|
90
|
376
|
0.134*
|
> 0.9
|
* The segregation ratio of normal to mutant plants was 3:1 at the 0.05 significance level. |
Linkage analysis of the QTL for plant height in NIL36
To identify the gene responsible for the NIL36 phenotype, we located the QTL for plant height in NIL36, and a total of 506 SSR markers from the rice molecular map were selected for polymorphism surveys between Hui1586 and Jiafuzhan[30]. Of these, 296 pairs exhibited polymorphism. Based on these 296 primer pairs, 45 recessive plants from the F2 population (NIL36/Jiafuzhan) were used for a linkage analysis between markers and the QTL. One of these SSR markers, RM3326 on chromosome 12, was found to be linked to the trait in the 45 F2 individuals.
Initial localization of the QTL for plant height
Published markers around RM3326 were used to initially locate the QTL. A genetic linkage analysis revealed that the QTL was located between the molecular markers RM2854 and RM235, which are located at a distance of 7.7 cM (Fig. 3-a).
To determine the location of the QTL within a smaller region, we identified 1264 recessive plants from the F2 population from Jiafuzhan/NIL36, and six polymorphic indel markers were screened from 18 newly developmental indels (Table 3). Indel markers from the open rice genome sequences were designed and tested to predict the likelihood of polymorphism between NIL36 and Jiafuzhan by comparing sequences from Nipponbare (http://rgp.dna.affrc.go.jp/) and the indica cultivar 93 − 11 (http://rice.genomics.org.cn/). The genotyping of all recombinant genes was performed using six polymorphic markers. The results showed that the QTL was located in the 295-kb region between the molecular markers Indel12-7 and Indel12-9 on chromosome 12 (Fig. 3-b and Table 3).
Table 3
Indel and SSR molecular markers used for the fine mapping of qPH-iaa30
Marker
|
Sequence of the forward primer
|
Sequence of the reverse primer
|
RM3326
|
CTCATCACCATCGTCACCAC
|
TCGTCGGGAGAGAGAGAGAG
|
RM2854
|
ATGAGAGAGAGAAAGAGAGT
|
AATGGAGAGAAAAAGTATTA
|
RM235
|
AGAAGCTAGGGCTAACGAAC
|
TCACCTGGTCAGCCTCTTTC
|
Indel12-1
|
CACCATGGACGATTTCTCTTCG
|
GATCGATGAGCAAGAAGGAGAGC
|
Indel12-4
|
GCGAGGTGTTGTGGACGATGG
|
ACACCTCCATCTTGGCCTTCTCG
|
Indel12-7
|
ATCATCGTCGTCATCCTCTCTCC
|
CGTCCAGTTCGTAGGCGTATAAGG
|
Indel12-9
|
ACGGTGGTGGTGGTGTTGTCG
|
TTAACCTTTGGCCGGGAGTGTGG
|
Indel12-12
|
GGTGTTGATTAAGCTGATCTCTCTCC
|
GATCAGCAACAAGCACCTCAGC
|
Indel12-15
|
TTGCTACTACCACAACAGGGTTCC
|
GCAGCCACAGCTTTGAATAGAGC
|
Indel12-20
|
CAAACAGGGTGAAAGAGAGA
|
CCTTTGCTACCTTGTGCTAC
|
Indel12-23
|
TAGAAGAGTGGGACAAGGAA
|
TGTTCATTTACATGCACCAT
|
Indel12-24
|
ACATCGATCCATTGCTAGTT
|
ACATCACGTGGTGGTTTATT
|
Indel12-26
|
TTCAGATACCAACACCTCCT
|
TTTTCCCTGACATTGGATAC
|
Indel12-29
|
TGCTGAACTAATCTGTGTGC
|
ATCTTTTCCTTGGGTTTCAT
|
Indel12-31
|
CATACACACAACAAATAGAA
|
CGCCAATCTTTAAATAGTTT
|
Indel12-33
|
ACACGTCTTTTCTGCAAGAT
|
GAACGAACATGAACGAGCTA
|
Indel12-36
|
TGGATGCATGGTAACTAATG
|
TGAATTGCTCTCCATGAAAT
|
Fine mapping of the QTL for plant height
For the fine mapping of the QTL, eight polymorphic indel markers were screened from 26 newly developed indels (Table 3). Recombinant screening with eight markers located in a more internal position within the target locus detected 13, 11, eight, six, two, one, two, and five recombinant plants, respectively (Fig. 3-c). Thus, the QTL was precisely located within the 31-kb region between the molecular markers Indel12-29 and Indel12-31.
Candidate genes in the 31-kb region
According to the available sequence annotation databases (http://rice.plantbiology.msu.edu/; http://www.tigr.org/), three annotated genes were located in the 31-kb region (Fig. 3-d), and all had a corresponding full-length cDNA. Among these genes, LOC_Os12g40860 encodes the leucine-rich repeat family protein, LOC_Os12g40880 encodes the uridine kinase family protein, and LOC_Os12g40890 is the auxin-responsive Aux/IAA gene family member OsIAA30.
Sequence analyses of the QTL for plant height
To identify the gene responsible for the observed phenotype, we then sequenced three genes of Jiafuzhan and NIL36. A deletion of only 1 bp (120:C) was found in LOC_Os12g40890 (Fig. 4), and no further difference was observed in the remaining two gene sequences. Thus, we hypothesized that LOC_Os12g40890 and OsIAA30 corresponded to the QTL for plant height in NIL36, and this gene was tentatively designated qPH-iaa30.
The analysis of the open reading frame (ORF) region showed that qPH-IAA30 had five exons. qPH-iaa30 exhibited a 1-bp deletion in the 120th base of the first exon, which resulted in the premature termination of OsIAA30 (Fig. 4).
qPH-iaa30 is responsible for the plant height phenotype of NIL36
To confirm that qPH-IAA30 confers a plant height phenotype, we examined whether the knockout of qPH-IAA30 in Jiafuzhan would lead to the NIL36 phenotype. One sequence-specific guide RNA (sgRNA) was designed to knock out qPH-IAA30 using the CRISPR/Cas9 gene editing system. A total of three plants from three independent events (OsIAA30KO-line1, OsIAA30KO-line2 and OsIAA30KO-line3) were obtained, and sequencing confirmed that these plants carry mostly insertions or deletions in the targeted sites (Supplementary Fig. 1).
We then investigated the plant height phenotype of these three homozygous lines at maturity and found that all three lines showed the NIL36 phenotype (Fig. 5 and Table 1). Therefore, the targeted mutation of qPH-IAA30 led to the NIL36 plant height phenotype, which indicated that the loss of function of OsIAA30 was responsible for this phenotype. Most importantly, OsIAA30KO-line1, OsIAA30KO-line2 and OsIAA30KO-line3 showed shorter panicle lengths, fewer spikelets per panicle and lower yields than Jiafuzhan (Fig. 5 and Table 1). Therefore, we hypothesized that the qPH-IAA30 gene not only affected the plant height but also regulated the panicle length, spikelets per panicle and yield in rice.
Comparative analysis of the hormone content between Jiafuzhan and OsIAA30KO-lines
To analyze whether qPH-IAA30 affects changes in the Aux/IAA levels, we measured the Aux/IAA content in Jiafuzhan (CK) and OsIAA30KO-line1. The results showed that the Aux/IAA content of OsIAA30KO-line1, OsIAA30KO-line2, OsIAA30KO-line3 and NIL36 was significantly lower than that of Jiafuzhan (CK) (Fig. 6).