Loss of function of OsMADS3 via the insertion of a novel retrotransposon leads to recessive male sterility in rice (Oryza sativa)

Highlights

• A spontaneous mutant R127S exhibiting recessive male sterile.

• Loss of function of OsMADS3 resulted in the recessive sterility of R127S.

• A retrotransposon insertion destroyed the function of OsMADS3.

• OsMADS3 is very conserved across cultivars and wild rice.

• R127S is a good mediator for current selection due to its wide compatibility.

Abstract

Natural mutation is the source of natural variation, which is the fundamental basis for the genetic improvement of crops. During the process of developing a recombinant inbred line (RI), a spontaneous mutagenesis in RI127 led to the production of the recessive male-sterile line RI127S. Via a map-based cloning approach, the gene controlling the male sterility was identified as OsMADS3, which was previously reported to be associated with floral organ development and male sterility. Thermal asymmetric interlaced PCR isolated one 1633-bp insertion in OsMADS3 in RI127S, which damaged its function due to failed transcription. The 1633-bp insertion was derived from a fragment flanked by retrotransposon genes on chromosome 5. Seven haplotypes of OsMADS3 were observed among 529 cultivars and 107 wild rice accessions, and 98% of the investigated genotypes carried the same H2 haplotype, indicating that OsMADS3 is highly conserved. RI127S has the combined genome constitution of its parents, indica rice Teqing and japonica 02428, and carries the widely compatible S5 gene donated by 02428. RI127 exhibits good performance in regard to its agronomic traits and has a wide compatibility. Therefore, RI127S would be an elite mediator for recurrent breeding in cases requiring a tedious hand-crossing-based inter-crossing phase. RI127S can be crossed not only with indica rice but also with japonica rice, thus providing breeders with flexible arrangements in recurrent breeding programs.

Introduction

Variation is the prerequisite for genetic research and genetic improvement. Variation is produced not only via recombination during sexual crossing but also via natural and artificial mutations. Natural and artificial mutants are the most important resources for functional genomics. In addition to the variations caused by sexual recombination, mutants are valuable resources for generating novel germplasms. Generally, the frequency of natural mutations is very low, at only 10⁻⁵–10⁻⁸ in higher plants [1]. A number of rice mutants have been collected during the long process of domestication and genetic improvement [1], [2], and several mutant germplasms have made great contributions to the process of rice production. For example, semi-dwarfing gene 1 (sd-1) is one of the most important genes deployed in modern rice breeding. The recessive characteristic of sd-1 results in a shortened culm with improved lodging resistance and a greater harvest index, enabling the increased use of nitrogen fertilizers [3], [4]. Another example is the application of the cytoplasmic male sterile (CMS) and photoperiod-sensitive genic male sterile rice lines, which are widely used to develop hybrid rice seeds for commercial release [5].

The efficient utilization of new mutants, including those formed via natural and artificial mutagenesis, requires the mutations to be mapped. Map-based cloning, which has been confirmed to be an efficient method to isolate target genes responsible for natural variation, is the first option for identifying random mutations. In fact, many yield-related genes have been isolated and functionally characterized, such as GRAIN SIZE 3 (GS3), GRAIN WIDTH and WEIGHT 2 (GW2), Grains number per panicle, plant height and heading date 7 (Ghd7), Ghd7.1 and Ghd8 [6], [7], [8], [9], [10]. Transposons are DNA fragments that can be integrated at different sites along a chromosome and generate duplicate copies during transposition. The first transposon, Ac/Ds, was discovered in maize by Barbara McClintock [11]. Transposons can be divided into two classes according to their transposable modes, as proposed by Finnegan [12]. Class I retrotransposons are produced during reverse transcription by RNA intermediates using a “copy and paste” transposable mechanism. Class II DNA transposons are directly transposed from one site to another in the genome using the “cut and paste” mechanism without any RNA intermediates. Another type of MITE (miniature inverted repeat transposable element) was also found to employ the “copy and paste” mechanism but without using RNA intermediates. Helitron, a new transposon, is transposed by cutting a single strand via a rolling-circle mechanism [13]. The maize transposon Ac and Ds elements have been successfully used as insertion mutagens for rice insertion mutagenesis [14], and a series of mutants defective in anther and pollen development have been identified. An Ac-type transposon Tok, a member of the hAT family in rice, is inactive in most vegetative organs but active in reproductive organs occasionally and alters the floral organ numbers [15]. The ANTHER INDEHISCENCE1 gene, which encodes a single MYB domain protein, is involved in anther development in rice [16]. ORYZA SATIVA MYOSIN XI B (OSMYOXIB) controls pollen development via photoperiod-sensitive protein localization [17]. The no pollen (Osnop) rice mutant, which has a pollen-less phenotype at the flowering stage, was identified during a phenotype screening of Ds insertional lines [18]. In addition to transposons, active retrotransposons have also been detected during rice tissue culture [19]. Retrotransposons are mobile genetic elements that transpose via the reverse transcription of an RNA intermediate. Tos17 is an endogenous rice retrotransposon that has been used as an important insertion mutagen [20]. Usually, Tos17 elements have no activity under normal growth conditions, but they are activated during tissue culture, and the copy number will increase to 5–30 [19].

Male sterility in plants is a widespread phenomenon in nature [21], and it has been well characterized due to its important roles in hybrid production in rice. Male sterility is classified into nucleic male sterility (NMS) or cytoplasmic male sterility (CMS) depending on the controlling gene source. In a previous study, CMS in plants was found to be a classical example of genomic conflict, either opposing maternally inherited cytoplasmic genes (mitochondrial genes in most cases), which induce male sterility, or nuclear genes, which restore male fertility [22]. In fact, the incompatibility caused by genome barriers between the nucleus and foreign mitochondria causes the pollen to be dead in cytoplasmic male sterile line, and studies of CMS have confirmed that pollen fertility is associated with anterograde/retrograde signaling [23], [24]. The recessive/dominant genes in the nucleus control NMS, and a recessive mutation trait can be inherited through heterozygotes and will follow Mendelian laws. Anther spatiotemporal expression profile in rice showed that 28,141 anther-expressed genes were classified into 20 clusters, which contained 3468 anther-enriched genes. These genes are related to important biological events in anther development, such as pollen maturation, pollen germination, pollen tube elongation and pollen wall formation [25]. Mutations of these genes may cause male gamete sterility. Therefore, many male-sterile mutants have been identified, and many crucial genes involved in anther development have been characterized [26], [27], [28]. Photoperiod/thermo-sensitive male sterile lines were identified in many species and have been used in breeding [29], [30]. In rice, UDP-glucose pyrophosphorylase 1 is essential for pollen callose deposition, and its co-suppression results in a new type of thermo-sensitive genic male sterility. The OSMYOXIB gene controls pollen development via photoperiod-sensitive protein localization, and the osmyoxib mutant shows male sterility under short-day-length (SD) and fertility under long-day-length (LD) conditions [17]. A long noncoding RNA regulates male sterility in photoperiod-sensitive male-sterile lines, and it is an essential component of two-line hybrid rice [31]. The cloning of these genes will greatly promote research on rice breeding.

In this study, a male-sterile mutant was identified in RI127 when we developed a set of recombinant inbred lines (RIs) using Teqing (TQ) and 02428. The sterile mutant exhibited an abnormal stamen and complete male sterility. To identify the mutation, the mutant was first crossed with the indica rice Minghui 63 (MH63), and fertile F₁ plants were obtained. Then, an F₂ population from the cross between RI127/MH63 was used to identify the mutation via a map-based cloning approach. Finally, we identified the gene as OsMADS3, whose function was destroyed by a 1633-bp insertion in the sterile mutant line. The value of the sterile line for recurrent selection was tested and discussed.