Morphological and histological analysis of the del mutant at the flowering stage
The spikelet of the wild type contains one terminal fertile floret, one pair of sterile lemmas, and one pair of rudimentary glumes in rice (Fig. 1-A). The terminal fertile floret is bisexual and consists of one pistil and six stamens, as well as two lodicules and one pair of bract-like organs (lemma and palea) (Fig. 1-B and C). There are five and three vascular bundles in the lemma and palea, respectively. In addition, the lemma is composed of four cell layers: from the abaxial to the adaxial surface these are a silicified upper epidermis, sclerenchyma cells, parenchyma cells, and a vacuolated inner epidermis, and the palea is composed of two fused parts: the body of the palea (bop) and two marginal regions of the palea (mrp) (Fig. 1-D and E). The cellular structure of the bop is similar to that of the lemma, but the mrp displays a distinctive smooth epidermis. The lemma and palea are hooked together to protect the inner floral organs and grain (Fig. 1-F and G).
In comparison with the widle type, in the del mutant 36% of spikelets developed a slightly degenerated lemma (Fig.1-H and N), which was narrow and unable to hook with the palea, causing a cracked floret (Fig.1-H and J). The palea and inner floral organs developed normally (Fig.1-I, K and L). Inspection of paraffin sections revealed that the four-layer cell structure of the lemma persisted, but the number and volume of the cells were significantly lower in the mutant (Fig.1-M and N). In 53% of spikelets, a rod-like lemma formed (Fig.1-O and T), and in these spikelets, the lemma was extremely degenerated and transformed into an awn-like organ (Fig.1-O, Q and R). The morphology of the lemma was highly similar to that of the awn and contained only one midvein. The silicified cells that should have been present on the outside surface of the wild type were ectopic and found in the inner epidermis of the rod-like lemma. The internal cell structure was almost completely disordered and unrecognizable (Fig.1-T and U). The palea and inner floral organs in the mutant were normal (Fig.1-P, S and T), thus the del mutant showed degeneration of the lemma to differing degrees, which suggests that DEL plays an important role in lemma development.
The del mutant affects grain yield
The spike type of the del mutant was more erect and smaller than that of the wild type. Compared with the wild type, the plant height of the del mutant was increased by 6.55 cm, while the panicle length was reduced by 24.5% (Fig. 2-A, B, E and F). While the number of primary branches of del showed no significant difference, the number of secondary branches was reduced by 10.7% (Fig. 2-G and H). The rate of seed setting in del was reduced by 70% (Fig. 2-I). As well as impaired flower development, the mature grains of del were also affected.The glumes of the mature grains in del were still dehiscent, and the lemma was rod-like in the severe mutant (Fig. 2-C).The mature brown rice grains were shaped like water droplets, being narrow at the top and wide at the bottom, and the rod-like spikelets produced few grains (Fig. 2-D). The grain length and the brown-rice grain length of the del were reduced by 7.7% and 27.0%, respectively (Fig. 2-J and M). The grain width of the del was increased by 16.3%, but the brown-rice grain width of the del was reduced by 26.8% (Fig. 2-K and N). The 1000-grain weight and the 1000-grain brown-rice weight were also reduced by 71.3% and 83.0%, respectively (Fig. 2-L and O). The results reveal that the DEL gene plays an important role in spikelet development and affects rice grain yield.
Early morphological analysis of the del mutant
Using scanning electron microscopy, we examined spikelets of the wild type and del mutant during the early developmental stages. In the wild-type floret, the primordia of the lemma and palea began to develop during the spikelet 4 (Sp4) stage (Fig.3-A). At Sp4, no significant difference between spikelets of the wild type and the del mutant was observed (Fig.3-A, E, and I). In the wild-type floret, the lemma and palea primordia were formed and the palea was smaller than the lemma, the five spherical stamen primordia were formed synchronously during the Sp5 and Sp6 stages except for the primordium nearest the lemma, and the lodicule primordia were formed at a subsequent stage (Fig.3-B). In the del mutant, which was different from the wild type, the shape of the slightly degenerated lemma was normal (Fig.3-F), and the margin of the rod-like lemma was not developed and did not intersect with the palea primordium (Fig.3-J). During the Sp7 stage, the pistil primordium was initiated and the lemma and palea primordia were normally developed and semi-closed, enclosing the inner whorl organ primordia in the wild-type floret (Fig.3-C). In the del mutant, the slightly degenerated lemma primordium was noticeably shorter and narrower than the lemma primordium in the wild type (Fig.3-G), cell differentiation on each side of the rod-like lemma was slow, and development into a rod-like structure occurred gradually (Fig.3-K). During the Sp8 stage, the single floret of the wild-type spikelet underwent a further stage of development (Fig.3-D). In the del mutant, the slightly degenerated lemma was significantly narrowed, and the palea and lemma were unable to close to cover the inner floral organs (Fig.3-H). The rod-like lemma primordium was further elongated, but the flanks remained undeveloped, and the inner floral organs were completely bare. However, the inner floral organs were normal (Fig.3-L). Collectively, these observations reveal that the defects of the lemma in the del mutant arose during the early stages of spikelet development.
Map-based cloning of DEL
The F1 progeny was derived from the cross between 56S and the del mutant, and showed a wild-type phenotype. The segregation ratio of the normal to the mutant phenotype was 3:1 (1091 wild-type-like plants and 358 mutant-like plants; χ2 = 0.26 < χ20.05,1 = 3.84) in the F2 population. This result indicates that the del mutant phenotype was controlled by a single recessive gene.
A map-based cloning approach was used to fine-map the DEL gene. The 358 recessive individuals in the F2 population were used as a mapping population to localize the DEL gene. To screen for polymorphism between the parents, a total of 420 pairs of simple sequence repeat (SSR) and InDel primers evenly distributed in the rice genome were used (Table S1). Ninety InDel markers were selected and used to further screen two DNA pools prepared by mixing equal amounts of genomic DNA from either 10 wild-type-like F2 plants or 10 mutant-like F2 plants (Table S2). The DEL gene was localized between the InDel markers In1-18.68 and In1-20.376 on chromosome 1 (Fig. 4-A and B). To fine-map DEL, 30 pairs of InDel primers located between In1-18.68 and In1-20.376 were developed, of which In1-18.68, In1-18.79, In1-18.94, In1-19.08, and In1-20.38 exhibited polymorphism between the parents (Table S3). Among all the F2 individuals, these five markers detected separately 14, 8, 4, 12, and 23 recombinants (Fig. 4-C). These results show that DEL was located between the InDel markers of In1-18.94 and In1-19.08. The estimated physical distance between In1-18.94 and In1-19.08 was approximately 140 kb, and 15 annotated genes (RAPdb annotation) were included within this interval: four enzymes (Os01g0527600, Os01g0527700, Os01g0528800, Os01g0529800), five expressed protein (Os01g0528300, Os01g0528700, Os01g0529700, Os01g0530100, Os01g0530200), a transcription factor (Os01g0528000), two transposon protein (Os01g0527900, Os01g0529400), a non-protein coding transcript (Os01g0527801), a hypothetical protein (Os01g0530000), and a gene with no annotated information (Os01g0529101) (Fig. 4-D and Table S6).
Sequencing analysis showed that Os01g0527600 had a single-nucleotide substitution from T to A, which was an allele of OsRDR6. The substitution of the nucleotide substitution in the del mutant caused the amino acid mutation of Leu-34 to His-34.In order to confirm that the mutation of Os01g0527600caused the del mutant phenotypes, we cloned a fragment consisting of a 2144-bp upstream sequence from the start codon and 4890-bp coding region sequence of the Os01g0527600gene in the wild type into the pCAMBIA1301 vector with the green fluorescent protein (GUS). The recombinant plasmid was then introduced into the del mutant (Fig.4-F). Subsequently, a total of 13 complementary transgenic lines were obtained, and GUS staining showed that 7 lines of them appeared indigo blue. This indicated that these lines had correctly transformed and successfully transferred into exogenous target vectors. All the T0 positive plants were planted in the fields, and the spikelet phenotype of these 7 lines was restored to the wild-type phenotype (Fig.4-G). These results confirm that the Os01g0527600 gene is the DEL gene, and the phenotype of del is indeed caused by mutation of the Os01g0527600 gene.
Spatiotemporal expression pattern of DEL
In order to clarify the functions of the DEL gene, we used RT-qPCR and in situ hybridization to test for DEL expression in the wild type. RT-qPCR analysis showed that DEL was constitutively expressed in whole organs, including root, stem, blade, sheath, and panicles. The lowest expression was found in the stem, and the highest expression was found in the blade. The expression level of the sheath was slightly higher than that of the stem, and the expression levels of the root and panicle were similar (Fig.5-A). In the spikelet, DEL was expressed in the lemma, palea, pistil, stamen, and lodicule (Fig.5-B). Later, in situ hybridization was used to detect the expression pattern of DEL, and a strong signal was shown in the rice spikelet (Fig.5-C–F). At the Sp3 stage, the DEL gene was highly expressed in the rudimentary glume, the sterile lemma, and the floral meristem (Fig.5-C). At the Sp4 stage, a strong DEL signal was detected in the lemma and palea primordia and floral meristem (Fig.5-D). At Sp5-Sp8, DEL was expressed in the lemma, palea, stamen, pistil, and lodicule (Fig.5-E–F). These results indicate that DEL was mainly expressed in the spikelet and florets, and was highly expressed throughout the period of plant growth, which implies its role in regulating the development of the lemma. Because del showed a phenotype of lemma degeneration to differing degrees, we also explored the expression of DEL in the wild-type glume. The results show that DEL was highly expressed in the whole of the lemma and palea, especially in their eight vascular bundles (Fig. 5-F).
Expression analysis of floral organ identity genes in the del mutant and wild type
Given that the del mutant showed an abnormal phenotype of floral organs, the expression patterns of known genes associated with floral organ identity were measured. DL (which is expressed in the middle of lemma)expression was 150% higher in the slightly degenerated lemma of the del mutant than in the lemma of the wild type, but the expression level of DL in the rod-like lemma was greatly reduced, with only a basal level similar to that of the wild-type palea (Fig.6-A). Moreover, the expression level of OsMADS1 in both the slightly degenerated and the rod-like lemma was higher than that of the wild-type lemma, by a factor of 2 and 12, respectively. The expression level of OsMADS1 in the del palea was only twice as high as that of the wild type (Fig.6-B). OsMADS14 expression was similar to OsMADS1, and the expression of the slightly degenerated and the rod-like lemma was much higher than that of the wild-type lemma. There was no significant difference between the expression of the palea in the wild type and del (Fig.6-C). For the expression level of OsMADS15, there was little difference between the rod-like and the wild-type lemma, but the expression level of the slightly degenerated lemma was 2.5 times that of the wild-type lemma, and the expression level of the palea in del was 1.5 times that of the wild-type palea (Fig.6-D). There was no difference in expression between the two types of del lemma and the wild-type lemma, and the expression of the palea in the del palea was about 70% higher than that of the wild-type palea (Fig.6-E). These results suggest that the slightly degenerated lemma retained the identity of the lemma, and degeneration of the rod-like lemma in the del mutant may be caused by reduced expressionof DL.OsMADS6 (which is an mrp identity gene) was expressed in the palea of the wild type and del mutant, which indicates that the palea identity in the del mutant was normal (Fig.6-E). In addition, we also assessed the expression of OsMADS2, OsMADS3, and OsMADS4 (the stamen and pistil genes). Compared with the wild type, the expression of OsMADS2 and OsMADS3 was reduced by a factor of 2 and 4 in the pistil of del, and was increased by a factor of 111 and 59 in the stamens of del (Fig.6-F and G). The expression level of OsMADS4 was slightly higher in the del than in the wild-type pistil, but was 50 times higher in the del than in the wild-type stamen, which indicates that the stamen and pistil of the del mutant showed normal identities (Fig.6-H). Collectively, these results indicate that the del mutation plays a critical role in the regulation of DL for lemma identity.