Non–redundancy of Rice Mutant Library for Male Gametogenesis Confirmed by Bulked Segregants Analysis Using Illumina BeadsArray

概要

九州大学学術情報リポジトリ
Kyushu University Institutional Repository

Non–redundancy of Rice Mutant Library for Male
Gametogenesis Confirmed by Bulked Segregants
Analysis Using Illumina BeadsArray
Anh Tuan NGUYEN
Plant Breeding Laboratory, Division of Agrobiological Sciences, Department of Bioresource
Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

YOSHIMURA, Atsushi
Plant Breeding Laboratory, Division of Bioresource Sciences, Department of Bioresource
Sciences, Faculty of Agriculture, Kyushu University

YAMAGATA, Yoshiyuki
Plant Breeding Laboratory, Division of Bioresource Sciences, Department of Bioresource
Sciences, Faculty of Agriculture, Kyushu University

https://doi.org/10.5109/6796252
出版情報：九州大学大学院農学研究院紀要. 68 (2), pp.123-134, 2023-09. 九州大学大学院農学研究院
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J. Fac. Agr., Kyushu Univ., 68 (2), 123–134 (2023)

Non–redundancy of Rice Mutant Library for Male Gametogenesis
Confirmed by Bulked Segregants Analysis Using Illumina BeadsArray
Anh Tuan NGUYEN1, Atsushi YOSHIMURA and Yoshiyuki YAMAGATA*
Plant Breeding Laboratory, Division of Bioresource Sciences, Department of Bioresource Sciences,
Faculty of Agriculture, Kyushu University, Fukuoka 819–0395, Japan
(Received May 17, 2023 and accepted May 18, 2023)
Mutant resources play a crucial role in unraveling the genetic network controlling traits of interest. In
this study, we focused on pollen–sterile mutants as important genetic resources for understanding the
genetic regulation of male gametogenesis. We employed high–throughput genotyping technologies using
Illumina BeadArray to map the responsible genes for male gametogenesis in rice; four SPOROPHYTIC
POLLEN STERILITY (SPS2, SPS3, SPS4, SPS7) and four GAMETOPHYTIC POLLEN STERILITY (GPS1,
GPS2, GPS9, GPS10). Using 287 single nucleotide polymorphism (SNP) markers, we detected polymorphisms between the japonica cultivar Taichung 65 (T65) and Hinohikari. Bulked segregant analysis (BSA)
based on DNA marker analysis was performed to identify candidate markers tightly linked to the causal
genes. The analysis revealed candidate markers for each mutant line, such as Mk240 for SPS2 and Mk94
and Mk126 for GPS1. Linkage mapping using the PCR–based markers confirmed the map positions of these
candidate markers. Our study demonstrates the utility of high–throughput genotyping technologies combined with BSA for the genetic characterization of mutant stocks. The identified candidate markers provide
valuable resources for future studies aiming to understand the molecular mechanisms underlying pollen
development and male gametogenesis in rice. The systematic gathering and reduction of redundancy in pollen sterile mutants are essential for a comprehensive understanding of the molecular networks involved in
post–meiotic male gametogenesis.
Key words: Rice, Mutant, Pollen sterility, Bulked segregant analysis, Illumina Beadsarray

four haploid cells (microspores) through the meiosis,
and a microspore generates a tricellular pollen grain containing one vegetative cell and two sperm cells by two
rounds of asymmetric mitosis. During the development,
zygotic nursery tissues, tapetum, supply nutrition indispensable for developing male gametophytes and eventually disappears at bicellular to mature stages of male
gametogenesis.
Genetic defects for gametogenesis can be classified
into zygotic (sporophytic) and gametic (gametophytic)
types based on their genetic basis. Typical mutants for
sporophytic pollen sterility expressed aberrant zygotic
phenotypes in a recessive manner in anther wall layer,
tapetum formation and degradation, and lipid metabolism and transportation (Ariizumi and Toriyama, 2011;
Jiang et al., 2013). In contrast, the sterility of pollen
grains is controlled by the genotype of gametophytes in
gametophytic pollen sterility mutants.
MOR1/GEM
encodes a member of the MAP215 family of microtubuleassociated proteins for the establishment of interphase
arrays of cortical microtubules in plant cells, and the
MOR1/GEM deficient mutant in Arabidopsis, gemini
pollen1 (gem1), showed pollen semi–sterility due to
defective cytokinesis of pollen containing a T–DNA
insertion (Twell et al., 2002). The male gametophytic rice
mutant, rice immature pollen1 (rip1), carry defect protein with conserved five WD40 repeats sequences and
showed semi–sterility in pollen in heterozygous condition
(Han et al., 2006). An arabinokinase–like protein defective mutant, collapsed abnormal pollen1 (cap1),
showed pollen sterility in a gametophytic manner, possibly
due to the lack of UDP–L–arabinose (Ueda et al., 2013).
The RICE GLYCOSYLTRANSFERASE1 (OsGT1) gene is

I N T RODUCTION
Mutants are important genetic resources to reveal
genetic networks controlling traits of interest by cloning
causative genes and their physiological and biological
characterizations. To understand the functions of the
genes that regulate biological processes, molecular biological and biochemical analyses using mutant resources
are extensively used in plant model species. At the
beginning of a post–genome era, the forward genetics
approach by positional cloning and reverse genetics
approaches by T–DNA or transposon tagging and targeting induced local lesions in genomes (TILLING) have
dramatically promoted cloning and characterization of
genes functionally unknown (Osborne et al., 1991;
Hirochika et al., 2001; Tzfira et al., 2004; Comai et al.,
2006). Moreover, high–throughput sequencing technologies by next–generation sequencers (NGS) would allow
rapid identification of causal mutation (for review,
Schneeberger, 2014). Therefore, developing a series of
mutant libraries associated with specific biological phenomena of interest becomes more important to clarify
molecular players on a pathway.
Pollen–sterile mutants have been considered critical
genetic resources for understanding male gametogenesis’s genetic regulation (Twell, 2011; McCormick, 2004).
On male gametogenesis, one male meiocyte produces
Plant Breeding Laboratory, Division of Agrobiological Sciences,
Department of Bioresource Sciences, Graduate School of
Bioresource and Bioenvironmental Sciences, Kyushu
University
* Corresponding author (E–mail address: yoshiyuk@agr.
kyushu–u.ac.jp)
1

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T. A. NGUYEN et al.

suggested to localize in a Golgi apparatus of pollen
essential for intine wall formation and pollen maturation
(Moon et al., 2013). Although gene cloning and characterization studies for pollen formation have been extensively conducted in the mutant approach, the isolated
genes involved in diverged biological processes to each
other and their spatially and temporally different expression and function let our knowledge be partially limited.
Therefore, systematic gathering and reduction of redundancy in the pollen sterile mutants are necessary for a
more efficient comprehensive understanding of complicated molecular networks during post–meiotic male
gametogenesis.
For the genetic characterization of mutant stocks,
an allelic test is a simple and powerful approach to classify mutant stocks into allelic groups. However, an allelic
test among pollen–sterile mutants is generally tricky
because mutant alleles do not transmit to progeny via
male gamete due to sterility, especially, for the gametophytic pollen sterility. In this case, gene tagging using a
DNA marker is available to investigate the allelism of
mutants. Bulked segregant analysis (BSA) has been frequently used for rapid gene identification (Michelmore
et al., 1991). The BSA uses two types of pooled
(bulked) DNA samples from normal and mutant segregants for DNA marker analysis, and the DNA markers
with a high frequency of alleles derived from the mutant
stock have a high possibility to tightly link to the causal
gene in the mutant bulked DNA. Today, single nucleotide polymorphisms (SNP) markers are rapidly replacing
simple sequence repeats (SSR) markers because they
are more abundant, stable, conveniently automated, efficient, and increasingly cost–effective (McCouch et al.,
2010; Thomson et al., 2012). Furthermore, modern SNP
genotyping techniques provide a high–throughput and
cheaper allele calling process. It also produces accurate
and stable data that can be flexible, merged across
groups, and stored in databases no matter which genotyping platform is used. (Thomson et al., 2012).
So far, we have identified twelve mutants of
SPOROPHYTIC POLLEN STERILITY (SPS1–SPS12)
and twelve GAMETOPHYTIC POLLEN STERILITY
(GPS1–GPS12) by gamma–ray mutagenesis to japonica
cultivar Taichung 65 (T65) (Yamagata et al., 2007). To
understand the genetic basis of pollen development,
linkage mapping of these mutants has been conducted.
Although SPS6, SPS9, SPS12, GPS4, GPS5, GSP6, and
GPS12 have been mapped on the rice linkage map.
However, the map position of the causal gene for the
other mutant lines has not been revealed. Here, we conducted bulked segregants analysis assisted by Illumina
Beadarray–based genotyping system in the F2 population
for four SPS mutant lines and four GPS mutant lines.
Using 287 SNP markers designed for Japanese rice
accessions, we detected signal peaks on candidate SNP
markers involved in phenotypes. Linkage analysis was
conducted by PCR–based markers which are linked to
the candidate SNP markers for identification of the
genes for sporophytic or gametophytic pollen sterility.

M AT ER I A LS A N D M ET HODS
Plant materials
Eight gamma–ray–induced mutants derived from
T65 were used in this study (Table 1). For bulked segregant analysis and linkage analysis of the mutants, the
F2 population derived from a cross between the mutants
and the Japonica rice cultivar Hinohikari was grown. For
the recessive SPS2, SPS3, SPS4, and SPS5 mutants
showing complete male and female sterility, pollen–fertile plant segregated in mutant lines were crossed by
Hinohikari pollen, and the F2 populations in which segregation of pollen sterile plants was observed were used
for the gene mapping. ...

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