Mapping and allelic sequencing of a long sterile lemma trait in rice

Bragantia, Campinas, v. 76, n. 2, p.229-237, 2017 AbstrAct: Some outer floral organs are unique in gramineous plants, like the sterile lemma and rudimentary glume in rice. However, their development mechanisms are still poorly understood. In this study, we used 4 mutants with long sterile lemma (LSL), named JF11, JF12, JF13 and JNY-7, to be crossed with Aijiaonante (AJNT) and Nipponbare (NIP), respectively. Genetic analysis indicated that LSL trait exhibited recessive heredity and was controlled by a common allele named sl-1(t). Using the method of bulk segregant analysis and linkage analysis between SSR markers and LSL trait based on F2 populations, the sl-1(t) gene was located at the interval between RM20903 and RM20948 on chromosome 7. The interval harbors a known G1 gene, which regulates the sterile PlAnt breeding Article

L. Jiang et al.

intrOdUctiOn
A flower is the vital reproductive organ determining quality of fruits and seed yiled in plants.Since the 1980s,   the mechanism of floral organ development has become one of hot spots in developmental biology by means of the floral organ mutants of model plants.The ABC model of plant flower development was put forward to elaborate the molecular mechanism of floral organ identity in eudicots based on their previous findings (Carpenter and Coen 1990;Bowman et al. 1991;Coen and Meyerowitz 1991;Coen and Carpenter 1993).Then, FBP7 and FBP11 genes, which regulated the identity and development of an ovule in petunia, were cloned in 1995 (Angenent et al. 1995;Colombo et al. 1995) and designated to an additional class D MADS box gene.Furthermore, the SEP1/2/3 genes were found as necessary for the development of petals, stamens, and carpels in Arabidopsis and were identified as a new class gene (E-class gene) of the floral quartet model (Pelaz et al. 2000;Honma and Goto 2001;Galimba et al. 2012).The findings of D-class and E-class enriched the ABC model and made the classical one extending to the ABCDE model.
It was also known that the divergence of monocot and dicot happened about 200 million years ago (Wolfe et al. 1989).However, the morphological characteristics and development process of monocot flower are distinct from those of dicot flower.For example, the lemma, palea, and lodicule around the stamen and pistil in rice floret are obviously different from the calyx and petal in dicot flower; in addition, there is a pair of sterile lemmas and rudimentary glumes remained outside each rice floret, respectively (Yoshida and Nagato 2011), which seems an unique floral organ in grass species.In some studies, it was believed that rice lodicule is similar to the petal in dicot flower (Kang et al. 1998;Ambrose et al. 2000) and the lemma and the palea amount to the calyx (Prasad et al. 2001).Besides, the sterile lemmas might be derived from the lemma (Prasad et al. 2001;Yoshida et al. 2009).Nevertheless, many questions still remain to be further studied, such as "the lemma and the palea are the same organ?","where do the sterile lemma and the rudimentary glume derive from?", and "how does every gene of floral organ work in perfect union?".In this study, we attempted to analyze the genetic characteristics of sterile lemma trait and cloned the target gene using 4 mutants with long sterile lemma in order to reveal the gene function and structure.

DNA extraction and linked SSR marker screening
DNA extraction referred to Cetyltrimethyl Ammonium Bromide (CTAB) extracting method (Wang and Fang 2003).The marker tightly linked to the target trait was followed the approach of Bulk Segregant Analysis (BSA) (Michelmore et al. 1991).In this study, DNA was extracted from individual F 2 plant.DNAs from 15 individuals with LSL trait were equally mixed and developed the mutant gene pool.Similarly, the wild type (WT) gene pool was developed as well.These 2 gene pools were subjected to screen the simple sequence repeats (SSR) markers.The polymorphic markers between the 2 gene pools might link tightly to the locus of LSL trait.

Genotyping and mapping
The SSR markers linked tightly to LSL trait were used to genotype 700 individuals with LSL in the F 2 population derived from the JF13/AJNT crossing.Linkage analysis between genotype and phenotype was conducted by MAPMAKER/EXP 3.0 (Lander et al. 1987;Lincoln et al. 1992), and the linkage map was drawn by MapChart 2.2 (Voorrips 2002).

SSR markers and specific primers
Sequences of 522 SSR markers used in this research were downloaded from the GRAMENE website (http:// www.gramene.org/microsat/ssr.html).According to the NIP sequence at the 2 flanks of the target locus, 72 SSR markers were designed by Preimer 5.0 and synthesized by Life Technologies Corporation.
A pair of primers, XMsl-7 (For ward primer : GAATGGAGGGTTGGGTCACT; XMsl-7, and Reverse primer: CGAAGCAACGGAACGAACAC), was designed and synthesized to amplify the Open Reading Frame (ORF), its upstream and downstream sequences of G1.
A set of 137 SSR markers with polymorphism between JF11 and AJNT was used to test the genotype of the DNA pools of wild type and mutant.Two markers with polymorphism on the short arm of chromosome 7, RM51 and RM295, were further applied to validate the genotype of 200 F 2 LSL individuals.Linkage analysis between molecular marker and phenotype trait showed that the locus of sl-1(t) was mapped on the inner side of RM51 and RM295, and the genetic distances from the target locus to RM51 and RM295 were 13.5 and 12.7 cM, respectively.In addition, another 6 SSR markers on chromosome 7, RM20828, RM20852, RM20903, RM20948, RM21004, and RM21035, were used to fine map the sl-1(t), through genotyping 700 mutants of F 2 plants.The sl-1(t) was narrowed in the interval between RM20903 and RM20948, and the genetic distances between sl-1(t) and the 2 markers were 3.8 and 4.9 cM, respectively (Figure 2).The G1 gene (LOC_Os07g04670) with a 831-base length ORF was deemed to confer the sterile lemma trait (Yoshida et al. 2009).In the present research, sl-1(t) was located in the interval between RM20903 and RM20948, which harbored G1.So we speculated that the SL-1(t) might be allelic to G1.
The PCR product with 1,873 bases included an 831base ORF, an 874-base upstream sequence, and a 168-base downstream sequence of G1.Allelic sequencing showed that the homologous G1 sequences of JF11, JF12 and JF13 mutants had a same deletion with 11 bases (GACGGCGCCGC) in the exon (Figure 3a), which directly led to the event of frame-shifting mutation, while JNY-7 had a base substitution which made an arginine convert into a histidine at the site of the 117th amino residue (Figures 3b,c).The results indicated that the mutation of G1 should be able to generate the phenotype of long sterile lemma of the 4 mutants in this study.
Rice sterile lemma was a characteristic floral organ that was considered to be a vestigial floret in rice (Yoshida et al. 2009).Some genes related to the sterile lemma have been mapped or cloned in rice.G1 was cloned by the map-based cloning method (Yoshida et al. 2009).Osleg was mapped in a 207-kb region on the short arm of chromosome 7, which was close to G1 (Chen et al. 2010).In the present study, it was believed that sl-1(t) gene should be allelic to G1 based on the locus location and allelic sequencing.It was very obvious that G1 would be a key gene for further research on rice sterile lemma.
G1 has only 1 exon with 831 bp, which codes a ALOG domain, a distinct N-terminal DNA-binding domain with an additional zinc ribbon (Figure 4) (Yoshida et al. 2009;Iyer and Aravind 2012).The g1-4 mutant has a 68-base deletion in the downstream of ALOG domain; g1-2 and g1-3 have a same base transversion in the zinc ribbon insert region and a transition in the third helix of the ALOG domain, respectively (Yoshida et al. 2009).In this research, the mutant locus, an 11-base deletion near to the mutant site of g1-4, happened downstream the ALOG domain (Figure 4).In the G1 gene of JNY-7, a base transition happened at the base No. 344 in the third helix of the ALOG domain (Figure 4).In the ORF and the upstream from −1 to −824 base of G1, there were 8 and 10 base substitutions between Indica and Japonica rice, respectively, and 2 of the 8 mutants located at the ORF caused the changes of the amino acid sequence.The aminoacid No. 133, V, becomes G, and the No. 190, M, becomes R (Figure 4).These 2 amino acids are located at the ALOG domain, but do not impact on the gene function.It is known that ALOG domain is far different from the MADS domain tightly linked with the development of the floral organ in eudicots and monocots, which suggested that the molecular mechanism of rice sterile lemma identity is different from that of other floral organs, particularly in eudicots.However, in the transgenic rice plants with Oryza sativa MADS box gene 1 (OsMADS1) and the rice actin1 promoter, the 2 sterile lemmas elongated and developed palea-like and lemma-like glumes (Jeon et al. 2000), which seemed that the sterile lemma identity should also be related to the MADS gene family.
How is the rice sterile lemma regulated?Is there a real link between ALOG domain and MADS domain?How do the 2 domains work together?Now all of these questions are little known to us.The research on gene regulation and signal path should be further conducted in the future.The present study might provide some good mutant materials and theoretical basis for further studying the key domain, gene regulation, and signal path related to rice sterile lemma.

cOnclUsiOn
Rice sterile lemma is a unique outer floral organ in gramineous plants.Long sterile lemma is a recessive trait and regulated by a key gene, G1.The mutant genes, sl-1(t), in JF11, JF12, JF13 and JNY-7 are alleles of G1.JF11, JF12 and JF13 have an 11-base deletion, which gives rise to a frame-shifting mutation.JNY-7 has a base substitution which triggers a missense mutation.
G1 codes an ALOG domain, which is a distinct N-terminal DNA-binding domain with an additional zinc ribbon.In this study, we found 2 mutations.One locates the downstream of the ALOG domain, and the other, in the third helix of the ALOG domain.These 2 mutations both affect the gene function.Also, 8 and 10 base substitutions happen in the ORF and the upstream from −1 to −824 base of G1 between Indica and Japonica rice, respectively, and only 2 of them in the ORF lead to the change of amino acid sequence.Although these 2 polymorphisms are located at the ALOG domain, there is no impact on the gene function, which shows these polymorphism loci are not the key conservative ones for supporting the structure and function of ALOG domain.
In a word, this study makes the researcher to further understand the structure and function of the G1 gene and provides 2 available mutants for exploring the development mechanism of rice floral organ.

Figure 2 .Figure 3 .
Figure 2. The location of sl-1(t) in the molecular linkage map on the short arm of chromosome 7.The bar is sl-1(t), and the numbers on the left of the linkage map represent genetic distance (cM).

Figure 4 .
Figure 4.The CDS, protein, and ALOG domain of G1.Pink words represent the mutant loci of 4 mutants in the present study.Blue words mean the different loci between Indica and Japonica rice.Black lines and orange cylinders show the secondary structure of ALOG domain (Iyer and Aravind 2012).