Characterization and fine mapping of nonstop glumes 2 (nsg2) mutant in rice (Oryza sativa L.)

Abstract

In cereal crops, the grain number per panicle and the grain yield are greatly affected by the number of florets in a spikelet. In wild-type rice, a spikelet only produces one fertile floret and beneath the floret are a pair of sterile lemmas and a pair of rudimentary glumes. This study characterized a rice spikelet mutant nonstop glumes 2 (nsg2). In the nsg2 mutant, both the sterile lemmas and rudimentary glumes were elongated, and part of sterile lemma looked like a lemma in appearance, shape and size. Detailed histological analysis and qPCR analysis revealed that the sterile lemmas in the nsg2 mutant had homeotically transformed into lemma-like organs. This phenotype indicates that NSG2 is involved in the regulation of spikelet development and supports the long-held view that sterile lemmas were derived from the lemmas of the two lateral florets. This implies that the rice spikelet has the potential to be restored to the "three florets spikelet", which may have existed in its ancestors. Genetic analysis reveals that the nsg2 trait is controlled by a single recessive gene. The NSG2 gene was finally mapped between markers R-20 and R-39 on chromosome 7 with a physical region of 180 kb. The two MYB family factors LOC_Os07g44030 and LOC_Os07g44090 might be involved in the development of the spikelet and floral organ, and they were considered as candidate genes of NSG2. These results provide a foundation for map-based cloning and function analysis of NSG2, as well as evidence to support "three-florets spikelet" breeding in rice.

Keywords: gene mapping; nonstop glumes 2 (nsg2); rice (Oryza sativa); spikelet.

Figure 1. Phenotype and statistics of wild-type and nsg2 plants. (A): The whole plant of wild-type and nsg2; (B): wild-type spikelets and nsg2 spikelets with elongated/lemma-like sterile lemma; (C): phenotype statistics of normal spikelets (two rudimentary glumes are extremely degrade, two sterile lemmas are small and about 1/5 size of lemma, lemma is a little bigger than palea and hooked together with palea, enclosed the two lodicules, six stamens and a pistil) and abnormal spikelets (spikelets with one or more defects such as elongated rudimentary glume, elongated/lemma-like sterile lemma, degraded palea, decreased number of stamen) in wild-type and nsg2; (D): statistics of wild-type sterile lemma and sterile lemma of ngs2 spikelet (normal, elongated and lemma-like sterile lemma).

Figure 2. Phenotype of spikelets in the wild-type and nsg2. (A1–A3): Wild type spikelet; (A4): scanning electronic microscope analysis of spikelets in the wild type; (A5): epidermis of wild-type sterile lemma; (A6): epidermis of wild-type lemma; (A7–A9): histological analysis of spikelet in the wild type. A7: transverse section of wild spikelet; A8: magnified connection part of lemma and palea; A9: magnified wild-type sterile lemma; (B1–B3): nsg2 spikelet with degraded palea; (B4): scanning electronic microscope analysis of nsg2 spikelet with degraded palea; (B5): epidermis of elongated sterile lemma in nsg2; (B6–B8): histological analysis of nsg2 spikelet with degraded palea. B6: Transverse section of nsg2 spikelets with degraded palea and elongated sterile lemma; B7: magnified connection part of lemma and degraded palea; B8: magnified elongated sterile lemma; (C1–C3, D1–D3): nsg2 spikelet with lemma-like sterile lemma; (C4, D4): scanning electronic microscope analysis of nsg2 spikelet with lemma-like sterile lemma; (C5): epidermis of elongated sterile lemma in nsg2 spikelet with one lemma-like sterile lemma and one elongated sterile lemma; (C6, D5–D6): epidermis of lemma-like sterile lemma in nsg2; (C7–C9, D7–D9): histological analysis of nsg2 spikelet with lemma-like sterile lemma. C7, D7: transverse section of nsg2 spikelet with lemma-like sterile lemma; C8, D8: magnified connection part of lemma and palea; C9, D9: magnified lemma-like sterile lemma. le: lemma; pa: palea; dp: degraded palea; st: stamen; pi: pistil; rg: rudimentary glume; erg: elongated rudimentary glume; sl: sterile lemma; esl: elongated sterile lemma; lesl: lemma-like sterile lemma; black arrows represent the vascular bundles; red arrow represents elongated rachilla axis; red box with arrow in A7, B6, C7, D7 represents the unmagnified part of A8, B7, C8, D8. Bars=1,000 µm in A1–A6, B1–B5, C1–C6, D1–D6 and 250 µm in A7–A9, B7–B9, C7–C9, D7–D9.

Figure 3. Scanning electron micrographs of florets at early developmental stages in the wild type and nsg2. (A–D): Wild type spikelets; (E–H): nsg2 spikelets with degraded palea; (I–L): nsg2 spikelets with elongated lemma-like sterile lemma; fm: floral meristem; sl: sterile lemma; esl: elongated sterile lemma; lesl: lemma-like sterile lemma; le: lemma; pa: palea; dp: degraded palea; pi: pistil; *: stamens. Bars=100 µm.

Figure 4. qPCR analysis of OsMADS1, OsMADS6, OsMADS34 and DL in nsg2 and wild-type. sl: Sterile lemma; le: lemma; pa: palea; esl: elongated sterile lemma; lesl: lemma-like sterile lemma; dp: degraded palea. Error bars represent the standard deviation between three replicates in the qPCR data; * indicates a statistically significant difference (p<0.05); **indicates a statistically significant difference (p<0.01).

Figure 5. Linkage map among NSG2 and markers on chromosome 7 in rice. (A): Preliminary mapping of NSG2; (B): fine mapping of NSG2; (C): annotated genes in the mapping region.