Line BPH - Rice, O. sativa - (農業試驗所特刊第139號)International Symposium on Rice Research in the Era

Rice, O. sativa

Chromosome 4 Line BPH

RM3536 RM16554 RM16563 RM16655 RM3317 RM7113 RM3308

TNG71 7.7 A A A A A A A

852T034 3.7 B B B B B B B

R006 4.3 B B B B B B B

R008 9.0 A A A A A B B

R010 7.7 H A A A A B A

R011 3.7 A B B B B A A

R012 9.0 A A A A A B H

R019 8.3 H A A A A A A

R022 5.0 B B B B B A B

R029 7.7 H A A A A B B

R032 3.0 B B B B B B A

R033 6.3 H A A A A B B

R036 6.3 A A A A A B B

R047 7.0 A A A A A A A

R074 5.7 H H A H H B B

R099 5.0 B B B B B A B

R118 4.3 B B B B B A A

Fig 2. Phenotype (BPH resistance) and graphical genotype of randomly selected F10 individuals. A: Tainung 71 allele, B: 852T034 allele, H: heterozygous

MAS test for the BPH resistance gene linked to SSR markers

The SSR markers RM16655 and RM3317 were further validated as R- or S-associated DNA markers by genotyping the F2 progenies with different genetic background of Tainung 71. One hundred and forty-two F2 seedlings from a cross between 852T034 and a susceptible O. officinalis introgression line 871948 were genotyped and F2:3 families were used for BPH bioassay. 71% and 79%

homozygous allele of 852T034 at RM16655 and RM3317 loci in the F2

population showed resistance scores ≤5 , respectively. More than 90% F2

individuals detecting homozygous allele of 852T034 showed different degrees of increasing BPH resistance (Table 1). The results indicated linked SSR markers have great potential used in marker-assisted selection.

DISCUSSION

BPH is a major biotic stress in rice production in most Asian countries. In Taiwan, BPH resistance genes such as Bph1 and bph2 were introduced into japonica cultivars by conventional breeding methods. However, because of changes in BPH biotype and infestation patterns, varieties with the Bph1 gene for resistance have become susceptible. It is also reported that biotype changes occurred because of the immigration of new biotypes of BPH from China, Vietnam and Philippines by summer wind since BPH never overwinters in Taiwan (Cheng and Huang, 2004). Therefore, identification of a new source of BPH resistance genes followed by introducing into japonica cultivars is an important objective in breeding programs.

Table. Distribution of BPH resistance scores by genotypes of markers RM16655 and RM3317 in F3 of 852T034 x 871948

Number of lines in BPH resistance score class Marker Genotype^z

RS<=5 (%) 5<RS<7 (%) RS>=7 (%) Total Mean score

RM16655 1/1 14 26 14 26 26 48 54 6.74

1/2 15 50 7 23 8 27 30 5.57

2/2 41 71 13 22 4 7 58 4.72

RM3317 1/1 14 25 16 28 27 47 57 6.74

1/2 23 53 12 28 8 19 43 5.35

2/2 33 79 6 14 3 7 42 4.55

a 1/1 denotes the homozygous genotype of ‘871948’,1/2 denotes the genotype of the heterozygous, 2/2 denotes the homozygous genotype of ‘852T034’; RS, Resistance Score.

Wild rice species are important resources of disease and insect resistance in crop genetic improvement (Tanksley and McCouch, 1996). Several wild species, including O. latifolia, O. minuta, O. nivara, O. officinalis and O. punctata, possess resistance to various biotypes of BPH (Renganayaki et al. 2002; Yang et al, 2004). In this study, a BPH resistance gene explained 50% phenotypic variations in the F2 population was mapped on the short arm of chromosome 4 and the resistance was contributed by ‘852T034’ allele derived from O. nivara . This location is very close to BPH loci of Qbp2, Bph12(t), Bph15, Bph17 and Bph20(t) (Hunag et al. 2001; Yang et al. 2002, 2004; Huang et al. 2001; Sun et al.

2005; Rahman et al. 2009). It is very interesting to notice that BPH resistance genes derived from different wild rice species were mapped on the same region of chromosome 4. Disease resistance genes of plants were reported often clustering in the same chromosome region. It remains to be investigated whether the distribution of insect pests resistance genes is similar to that of disease resistance genes.

BPH populations can quickly overcome single resistance gene under natural conditions. New resistance genes are always needed for rice improvement and breeding against BPH. Therefore, the resistance genes of ‘852T034’ can be a useful BPH resistance donor for a new resource of BPH resistance. The transfer of BPH resistance genes from wild species to different variety backgrounds can be greatly facilitated by using MAS (Tanksley and Nelson. 1996; Moncada et al.

2001). There has been great progress in the development of MAS for BPH in recent years. SSR and CAPS markers linked to Bph1, bph2 and Bph3 were used widely in Japan, Korean and Thailand MAS programs (Sharma et al. 2004; Cha et al. 2008; Jairin et al. 2009). But relatively few varieties or lines have been reported to be successfully developed by this method. One of the problems lies in the crossovers between markers and genes caused the segregation of the resistance in the population and deviated selection.

In this study, we conducted SSR mapping of wild species O. nivara and identified the SSR markers closely linked to BPH resistance locus, and verified by the

efficiency of MAS. Both RM16655 and RM3317 showed over 90% accuracy predicting BPH resistance in the F2 generation derived from 852T034 x 871948.

Thus RM16655 and RM3317 could be applied to the MAS of the trait of BPH resistance in rice breeding programs.

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International Symposium (2009)

Rice Research in the Era of Global Warming 66~78

Integration of genomics into breeding in rice

Masahiro Yano^*, Shuichi Fukuoka, Kaworu Ebana, Toshio Yamamoto

QTL Genomics Research Center, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

*Corresponding author: myano@nias. affrc.go.jp

ABSTRACT

Elucidation of the association between nucleotide and phenotypic changes has been a big challenge in plant molecular genetics. Toward this goal, we have been involved in the genetic dissection of natural phenotypic variations in rice and have identified several genes involved in complex traits, including heading date, shattering habit, pre-harvest sprouting, root morphology, disease resistance and eating quality. To enhance the power of genetic dissection of complex phenotypes, we are developing several mapping populations, such as recombinant inbred lines and chromosome segment substitution lines, which will be required to extract the useful alleles from natural variants. Marker assisted selection has been applied for the development of heading date and durable resistance. To facilitate allele mining using novel plant materials, we have also embarked on the genome-wide discovery of single nucleotide polymorphisms (SNPs). In particular, to overcome a shortage of SNPs among temperate japonica cultivars, we have attempted whole-genome sequencing of several cultivars using next-generation sequencing approaches. Although Japanese cultivars are closely related genetically, about 67,000 SNPs have been discovered between Nipponbare (reference) and Koshihikari by this approach. This SNP discovery has led to the development of an array-based SNP genotyping system in Japanese rice cultivars. Large-scale

genotyping of these SNPs has made it possible to visualize pedigree haplotypes for the Japanese landraces and modern cultivars. These new efforts in genomics have opened up new opportunities to accelerate not only the genetic dissection of complex traits, but also the improvement of rice cultivars.

Key words: Quantitative trait locus, Genetic mapping, Marker assisted selection, Single nucleotide polymorphism

INTRODUCTION

It is about 4 years since the whole rice genome was decoded (IRGSP 2005). The sequence information has provided new tools for genetics and has created a new paradigm of selection strategy in plant breeding. Many phenotypic traits of economic interest are controlled by multiple genes and often show complex and quantitative inheritance. Recent progress in rice genomics has had a great impact on the genetic dissection of such traits into single genetic factors or quantitative trait loci (QTLs) (Yamamoto et al., 2009). These resources have already contributed to both our understanding of biological phenomena in plants, and to the application of genomics tools to the development of new crop cultivars.

Genes with agronomic value have been tagged by DNA markers and have been introduced into elite cultivars by marker-assisted selection (MAS). Technological innovations in large-scale sequencing and genotyping have opened new possibilities in rice genetics and breeding. This paper introduces our recent activity on the genetic dissection of complex traits, marker-assisted introgression, and pyramiding of agronomic traits, and future prospects in genomics-assisted breeding of rice.

Uncovering the Naturally Occurring Variations in Complex Traits

In the last decade, much effort has been paid to the genetic and molecular dissection of complex traits. An excellent example is the analysis of heading date in rice. Heading date is a key determinant for the adaptation of rice to different

cultivation areas and cropping seasons. Therefore, control of heading date is a leading objective in rice breeding. Many genetic studies have been conducted for QTL mapping of heading date using advanced backcross progeny (Yano et al., 2001). Fifteen QTLs, called Heading date (Hd)1–Hd3a and Hd3b–Hd14, have been detected by using several kinds of progeny from a cross between japonica cultivar 'Nipponbare' and indica cultivar Kasalath (Yano et al., 2001). Among them, nine QTLs—Hd1, Hd2, Hd3a, Hd3b, Hd4, Hd5, Hd6, Hd8, and Hd9—were mapped as single Mendelian factors (Yano et al., 2001; Lin et al., 2000; 2003).

Detection of QTLs for heading date has allowed further genetic analyses, such as the development of nearly isogenic lines (NILs), analysis of epistatic interactions among QTLs, and map-based cloning. Hd1 has been found to encode a protein with zinc finger and CCT motifs and to be an ortholog of Arabidopsis CONSTANS (Yano et al., 2000). Hd6 and Hd3a were found to encode a casein kinase 2 alpha and an Arabidopsis FT-like protein (Takahashi et al., 2001; Kojima et al., 2002). A major QTL, Early heading date 1 (Ehd1), for heading date was detected on chromosome 10 by using a BC1F1 population derived from a cross between cultivar T65 and an accession of another cultivated species, Oryza glaberrima (Doi et al., 1998). Further analysis revealed that Ehd1 encodes a B-type response regulator (Doi et al., 2004). In all cases of QTL cloning for heading date, large-scale linkage mapping was required to narrow the candidate genomic region for the QTLs. These efforts led us to identify functional nucleotide polymorphisms in Hd1, Hd6, and Ehd1. To comprehensively dissect the genetic factors controlling naturally occurring variations in rice flowering, we have performed a QTL analysis using 12 populations derived from crosses of the japonica cultivar Koshihikari with cultivars and lines that originate from various regions in Asia. By QTL mapping, several QTLs were detected on the 12 rice chromosomes, some of which were shared among the different cross combinations. The chromosomal locations of these QTLs corresponded to those detected in Nipponbare and Kasalath (Yano et al., 2008). However, the allelic

effects of each QTL varied among the parental combinations used, suggesting that a large proportion of the wide range of phenotypic variations in flowering time could be generated by the combination of different alleles of the corresponding QTLs. These genetic and molecular studies have definitely contributed to our understanding of heading date in rice (Izawa, 2007; Tsuji et al., 2008).

Much effort has also been paid to the analysis of other complex traits, such as grain size, shattering habit, disease resistance, and environmental stress tolerance.

These activities are summarized in the “Gramene-QTL” database (Jaiswal et al.

2002). Around 8000 QTLs have been detected so far. Among them, several with major effects have already been cloned by a map-based strategy (Yamamoto et al., 2009).

Current Status of Marker-assisted Selection in Rice Breeding

Once genes controlling traits with economic and agricultural interest are cloned or mapped precisely on the respective chromosomes, it becomes possible to use information such as gene sequences and chromosomal locations in breeding programs. Since the paradigm of MAS emerged nearly 20 years ago, much effort has been invested in the practice of MAS. Several examples of the development of NILs with particular traits in elite cultivars have already been reported.

Submergence by deep water causes severe stress to rice in Southeast Asia, where flooding occurs during the monsoon season. A major QTL, Submergence 1 (Sub1), was detected near the centromere of chromosome 9 (Xu et al., 2000). The underlying gene was cloned (Xu et al., 2006), and the Sub1A allele was introgressed by MAS into an elite cultivar grown widely in Asia. The resultant lines showed promising performance in yield and other agronomic traits, as well as tolerance to submergence (Neeraja et al., 2007; Septiningsih et al., 2009). Four QTLs for rice heading date—Hd6, Hd1, Hd4, and Hd5—were introgressed from Kasalath into Koshihikari by MAS to enhance the cropping potential of Koshihikari, one of the leading cultivars in Japan (Takeuchi et al., 2006). As a

result, NILs of Koshihikari with early and late heading dates have been successfully developed. The size of the introgressed chromosomal segments in those lines was very small: 300 to 600 kb in NILs for Hd1, Hd6, and Hd5. Precise information on the chromosomal locations of the genes allowed breeders to minimize the length of the substituted chromosome segments containing the target QTLs. That study clearly demonstrated the potential power of MAS in rice breeding. Recently, cloning of a durable blast resistance gene, pi21, has been cloned and this achievement led us to solve a linkage drag of pi21, associating with high level of blast resistance and inferior eating quality. Clarification of precise location of pi21 allowed us to develop several markers, which could be used for the dissection between blast resistance and eating quality (Fukuoka et al.

2009). MAS also offers a new concept in breeding: Once NILs with particular economic value are developed, gene pyramiding can be performed by simple crossing between them (Ashikari and Matsuoka, 2006). To develop a new line with lodging resistance and high yield, the combination of two genes controlling semi-dwarfing and grain number were successfully introduced into Koshihikari (Ashikari et al., 2005). This concept can also be applied to multiple genes controlling specific traits. Four QTLs controlling partial resistance to rice blast in upland rice have been successfully pyramided into lowland rice cultivars by MAS (Fukuoka and Saka, 2006; Saka et al., 2007).

Allele Mining for Rice Breeding

Many successes have been achieved in cloning and MAS of particular QTLs in rice. However, these successes have largely depended on allelic differences. In most cases, the allelic difference was relatively large, allowing reliable determination of the chromosomal location. One major QTL, Grain number 1a, was successfully isolated by a map-based strategy (Ashikari et al., 2005). This finding contributed both to our understanding of the genetic control of spikelet development in rice, and to MAS to improve grain number per panicle. However,

in general, one major QTL alone is not enough to acquire the level of phenotypic performance needed. To this end, it would be necessary to combine genes with major and minor effects. Owing to the statistical power of detection, it is usually very difficult to detect QTL with minor effects in QTL analysis using F2 and recombinant inbred lines (RILs). To solve this problem, we developed chromosome segment substitution lines (CSSLs) (Ebitani et al., 2005; Takai et al., 2007; Ando et al., 2008). In these novel plant materials, a particular chromosomal segment from a donor line is substituted into the genetic background of the recurrent line. The substituted segments cover all chromosomes in a whole set of lines. The potential of CSSLs in QTL detection has been demonstrated in many ways. We have established a systematic research flow for exploration and cloning of useful genes (Fig. 1). For example, CSSLs can be used in genetic analysis to associate QTLs with particular chromosomal regions and to quickly develop NILs of target regions containing QTLs of interest. In general, when an association is detected between a chromosomal region and a trait, it is often difficult to validate the QTLs, especially those with very small genetic effects. In such a case, NILs are required in order to analyze genetic effects in detail (Miura et al., 2001; Sato et al., 2003; Ueda et al., 2004). Because CSSLs normally have one chromosomal region substituted, they can be used as NILs themselves or as starting material to develop NILs. Such NILs enable us to combine two or three QTLs in one genetic background in order to clarify epistatic interactions among them (Lin et al., 2000;

2003; Yamamoto et al., 2000). Furthermore, once we detect significant differences between the CSSLs and the recurrent parental line, comparison of the size of the substituted segments enables us to delineate candidate chromosomal regions of QTLs (substitution mapping). If a significant difference is found between a particular CSSL and the recurrent parent, a large mapping population can be easily produced by a simple crossing of the CSSL with the recurrent parent.

Map-based cloning of the QTLs detected can be started quickly by using such plant materials. A simple survey of target traits on the CSSLs allows us to detect

Fig.1 Systematic research flow for the expｌoration and utilization of natural variations in rice

minor phenotypic differences with reference to the recurrent parent, because there is almost no effect of background genetic noise (i.e., allelic effect of other QTLs).

Although the resolution of QTLs in terms of linkage mapping and the power to detect epistatic interactions are not comparable with those given by primary mapping populations such F2s and RILs, the use of CSSLs can facilitate the discovery of valuable alleles from donor chromosome segments. To enhance the potential of CSSLs, we are now developing CSSLs from a wide range of cross combinations, with Koshihikari as the recurrent parent. Donor parental lines are indica and japonica cultivars, including some of our core collections (Kojima et al., 2005).

Genome-wide Discovery of SNPs and Development of Genotyping Arrays The genetic dissection of natural variations requires novel and effective genotyping and phenotyping. New technology recently enabled us to perform massive analysis of sequences (Blow, 2007; Hutchison, 2007). This method,

在文檔中 (農業試驗所特刊第139號)International Symposium on Rice Research in the Era of Global Warming (頁 77-95)