The rice local population was clearly differentiated into six groups over the 100-year history of rice breeding programs in the northern limit of rice cultivation over the world. Genetic improvements in plant breeding programs in local regions have led to the development of new cultivars with specific agronomic traits under environmental conditions and generated the unique genetic structures of local populations. Understanding historical changes in genome structures and phenotypic characteristics within local populations may be useful for identifying profitable genes and/or genetic resources and the creation of new gene combinations in plant breeding programs. In the present study, historical changes were elucidated in genome structures and phenotypic characteristics during 100-year rice breeding programs in Hokkaido, the northern limit of rice cultivation in the world. We selected 63 rice cultivars to represent the historical diversity of this local population from landraces to the current breeding lines. The results of the phylogenetic analysis demonstrated that these cultivars clearly differentiated into six groups over the history of rice breeding programs. Significant differences among these groups were detected in five of the seven traits, indicating that the differentiation of the Hokkaido rice population into these groups was correlated with these phenotypic changes. These results demonstrated that breeding practices in Hokkaido have created new genetic structures for adaptability to specific environmental conditions and breeding objectives. They also provide a new strategy for rice breeding programs in which such unique genes in local populations in the world can explore the genetic potentials of the local populations.

SummaryIn higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMSencoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

Cold temperature during the reproductive phase leads to seed sterility, which reduces yield and decreases the grain quality of rice. The fertilization stage, ranging from pollen maturation to the completion of fertilization, is sensitive to unsuitable temperature. Improving cold tolerance at the fertilization stage (CTF) is an important objective of rice breeding program in cold temperature areas. In this study, we characterized fertilization behavior under cold temperature to define the phenotype of CTF and identified quantitative trait loci (QTLs) for CTF. A wide variation in CTF levels has been identified among local cultivars in Hokkaido, which is one of the most northern regions for rice cultivation in the world. Clear varietal differences in pollen germination, and pollen tube elongation due to cold temperature have been observed. These differences may confer a degree of CTF among this population. We conducted QTL analysis for CTF using 120 backcrossed inbred lines derived from a cross between Eikei88223 (vigorous CTF) and Suisei (very weak CTF). Three QTLs for CTF were identified. A clear effect by QTL, qCTF7, for increasing the level of CTF was validated using advanced progeny. These results will facilitate marker-assist selection for desirable QTLs for CTF in rice breeding program.

Low-temperature is one of the most common environmental stresses that affect plant growth and development and places a major limit on plant productivity. Tolerance to low-temperature is an important objective of rice breeding programs in temperate areas and at high altitudes in tropical and sub-tropical areas. Vigorous rice growth during the seedling stage at low-temperature is an important characteristic for stable seedling establishment (SES) in direct seeding methods, in which rice is sown directly into a flooded field. The aim of this study was to identify QTLs controlling SES using 3 mapping populations of backcrossed inbred lines (BILs) with Arroz Da Terra and Italica Livorno as the donor parents. SES was evaluated by a field evaluation system in this study, which is used in current rice breeding programs because of the complexity of environmental conditions in SES in rice cultivation in a paddy field. A total of 9 chromosomal regions for SES were identified, which explained 10.9% to 25.6% of total phenotypic variation. For all of QTLs except for qSES5-2, alleles from the donor parents increased phenotypic values. These QTLs should be useful for the improvement of SES in rice breeding programs in low-temperature regions.

Vigorous cold tolerance at the fertilization stage (CTF) is a very important characteristic for stable rice production in cold temperature conditions. Because CTF is a quantitatively inherited trait, pyramiding quantitative trait loci (QTLs) using marker-assisted selection (MAS) is effective for improving CTF levels in rice breeding programs. We previously identified three QTLs controlling CTF, qCTF7, qCTF8 and qCTF12, using backcrossed inbred lines derived from a cross between rice cultivar Eikei88223 (vigorous CTF) and Suisei (very weak CTF). However, pyramiding of these QTLs for the application of MAS in practical rice breeding programs have not yet been elucidated. In this study, we examined the effect of pyramiding QTLs for improvement of CTF level using eight possible genotype classes from the 152 F3 population derived from a cross between Eikei88223 and Suisei. Increasing of CTF levels in combinations between qCTF7 and qCTF12 and between qCTF8 and qCTF12 were detected. Furthermore, we compared the haplotype pattern around the QTLs for CTF among the rice cultivars from Hokkaido. These results are useful for improvement of new cultivars with high CTF levels using MAS and identification of genetic resources with the novel QTL(s) for CTF.

Improving the eating quality of cooked rice has been one of the most important objectives in rice breeding programs. Eating quality of cooked rice is a complex trait including several components, such as external appearance, taste, aroma, and texture. Therefore, dissection of these components followed by marker-assisted selection of detected QTL(s) may be a useful approach for achieving desirable eating quality in rice breeding. Whiteness of cooked rice (WCR) is an important factor related to the external appearance of cooked rice. WCR is known to be associated with the amylose and protein contents of the endosperm. However, the genetic basis of WCR remains unclear. In this study, we evaluated phenotypic variation in WCR among recently developed rice cultivars from Hokkaido, Japan. Then, we developed doubled haploid lines (DHLs) derived from a cross between two cultivars from Hokkaido, Joiku No. 462 (high WCR) and Jokei06214 (low WCR). Using the DHLs, we detected two QTLs for WCR, qWCR3 and qWCR11, on chromosomes 3 and 11, respectively. We also examined the dosage effect of the two QTLs based on both the categorized segregants in the DHLs and the relationship between the WCR phenotype and inheritance around the QTL regions in cultivars from Hokkaido.

Differential screening of a sugar beet (normal cytoplasm line TK81‐O) cDNA library made with anther tissues of various stages resulted in the isolation of a clone (#74‐29) that hybridized to flower bud RNA but did not hybridize to RNA of vegetative organs. The clone contained an open reading frame (ORF) (designated bvLTP‐1) that encoded a putative lipid transfer protein. We also identified a second copy (bvLTP‐2) of the gene. In situ hybridization analysis demonstrated that expression of bvLTP‐1 was confined to the tapetal cells of the anthers at the young microspore stage. Flower bud RNA was prepared from male‐sterile sugar beet with Owen cytoplasm and fertility‐restored plants and used for northern hybridization with the bvLTP‐1 probe. Interestingly, bvLTP‐1 was found to be expressed in the flower buds from the restored plants producing 30% or more stainable pollen, but not in the flower buds from completely sterile or poorly fertility‐restored plants. These results lead us to suppose that the expression of bvLTP‐1 is strongly reduced in the tapetum in response to mitochondrial dysfunction and subsequent physiological changes caused by the Owen cytoplasm.

Cold stress at the booting stage in rice induces spikelet sterility because of aberrant microspore development, which often seriously damages seed production. Some breeding lines with high cold tolerance were developed by using tropical japonica variety Silewah as a donor of cold tolerance; however, the genetic factors that confer cold tolerance of this variety have not been comprehensively analyzed. In this study, phenotypic and molecular characterization of novel cold-tolerant strains derived from crosses with Silewah was performed to identify quantitative trait loci (QTLs) responsible for cold tolerance. Molecular marker analysis revealed that 2 cold-tolerant strains carried chromosomal segments of Silewah at the same genomic regions on chromosomes 3, 4 and 11. Single marker analysis in segregating population confirmed that the allele of Silewah on chromosome 3 (qCTB3-Silewah) conferred cold tolerance. The effect of qCTB3-Silewah was supported by the fact that this allele had been a target of selection during developing a breeding line by phenotypic selection from backcrossed progenies with an elite variety as a recurrent parent. qCTB3 is a different QTL from those reported for Silewah previously, suggesting that different QTLs might be exploited in different breeding programs depending on the genetic backgrounds and environmental conditions.