The biotron breeding system: a rapid and reliable procedure for genetic studies and breeding in rice

Oryza sativa is widely used as a model organism for many aspects of research in monocots and cereals. However, it has certain disadvantages as a model species compared with Arabidopsis thaliana, the eudicot species most widely used in plant sciences: first, it has a long cultivation time; and second, it requires considerably more space for growth. Here, we introduce a biotron breeding system, which allows rapid and reliable rice cultivation using a well-equipped artificial environmental chamber. This system involves use of regulation of CO? levels, removal of tillers and embryo rescue to overcome the disadvantages of rice cultivation. The rice cultivars Nipponbare, Koshihikari, Taichung 65 and Kasalath all showed vigorous growth and sufficient seed production in the biotron breeding system with accelerated flowering time. Nipponbare, which was the earliest among these cultivars, flowered at about 50 d after sowing. The life cycle of these plants could be further shortened using an embryo rescue technique on immature seeds at 7 d after pollination, thereby avoiding the lengthy process of seed maturation. Overall, it was possible to shorten the life cycle of Nipponbare to about 2 months under the controlled conditions. Furthermore, controlled crosses, which can be difficult with conventional cultivation methods, were easy to perform as we could control the exact timing of anther dehiscence. Thus, our biotron breeding system offers a valuable new approach to genetic and breeding studies in rice.     

利用染色体片段置换系定位水稻落粒性主效QTL

水稻落粒性是与其生产密切相关的重要性状之一。以7个染色体片段置换系为材料, 采用重叠群代换作图法对控制落粒性的2个主效QTL进行定位。结果表明, 104个SSR标记在亲本间具有多态性, 多态率为68.0%; 4个置换系的落粒性与亲本日本晴的落粒性相似, 表现难落粒。3个置换系与亲本93-11的落粒性相似, 表现易落粒; 7个染色体片段置换系在第1和第6染色体上检出7个置换片段, 其长度分别为23.6、16.5、 6.6、 9.9、 10.4、 20.2和7.1 cM; qSH-1-1被定位在第1染色体RM472－RM1387之间, 遗传距离约为6.6 cM。qSH-6-1为新发现的落粒性主效QTL, 被定位在第6染色体RM6782－RM3430之间,遗传距离约为4.2 cM。利用染色体片段置换系能准确地定位水稻落粒性QTL, qSH-1-1与qSH-6-1的鉴定和初步定位为其进一步的精细定位、图位克隆及分子标记辅助选择奠定了基础。     

利用染色体片段置换系定位水稻落粒性主效QTL

水稻落粒性是与其生产密切相关的重要性状之一。以7个染色体片段置换系为材料,采用重叠群代换作图法对控制落粒性的2个主效QTL进行定位。结果表明,104个SSR标记在亲本间具有多态性,多态率为68.0%;4个置换系的落粒性与亲本日本晴的落粒性相似,表现难落粒。3个置换系与亲本93-11的落粒性相似,表现易落粒;7个染色体片段置换系在第1和第6染色体上检出7个置换片段,其长度分别为23.6、16.5、6.6、9.9、10.4、20.2和7.1 cM;qSH-1-1被定位在第1染色体RM472-RM1387之间,遗传距离约为6.6 cM。qSH-6-1为新发现的落粒性主效QTL,被定位在第6染色体RM6782-RM3430之间,遗传距离约为4.2 cM。利用染色体片段置换系能准确地定位水稻落粒性QTL,qSH-1-1与qSH-6-1的鉴定和初步定位为其进一步的精细定位、图位克隆及分子标记辅助选择奠定了基础。     

Development and high-throughput genotyping of substitution lines carring the chromosome segments of indica 9311 in the background of japonica Nipponbare

Chromosome segment substitution lines (CSSLs) are useful for the precise mapping of quartitative trait loci (QTLs) and dissection of the genetic basis of complex traits.In this study,two whole-genome sequenced rice cultivars,the japonica Nipponbare and indica 9311 were used as recipient and dtonor,respectively.A population with 57 CSSLs was developed after crossing and back-crossing assisted by mo lecular rnarkers,and genotypes were identified using a high-throughput resequencing strategy,Detailed graphical genotypes of 38 lines were constructed based on resequencing data.These CSSLs had a total of 95 substituted segments derived from indica 9311,with an average of about 2.5 segments pet CSSL and eight segments per chromosome,and covered about 87.4％ of the rice whole genome.A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight.The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice.These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome discovery and functional study of essential genes/QTLs in rice,and offer ideal materials and foundations for japonica breeding.     

野败型水稻细胞质雄性不育恢复基因Rf-4的分子标记定位

为了用分子标记准确定位野败型水稻细胞质雄性不育恢复基因Rf-4,将日本水稻基因组项目（Rice Genome Program,RGP)构建的水稻遗传连锁图谱第10染色体分子遗传图上的分子标记R1877和G2155之间对应区域YAC物理图上的6个YAC克隆进行了亚克隆,获得119个片段,对这些探针进行多态性探查,获得了2个多态分子标记,用珍汕97A和恢复基因近等基因系的杂种F2分离群体中的117完全不育株进行连锁分析表明,从YAC4892获得的亚克隆Y3－8与Rf－4座位的连锁距离为0．9cM,从YAC4630获得的亚克隆Y1－10与Rf－4座位的连锁距离为3．2cM,根据以上结果把Rf－4座位定位于第10染色体的特定位置,为该基因的分子标记辅助选择和定位克隆打下了基础。     

Detection of quantitative trait loci controlling pre-harvest sprouting resistance by using backcrossed populations of japonica rice cultivars

Backcrossed inbred lines (BILs) and a set of reciprocal chromosome segment substitution lines (CSSLs) derived from crosses between japonica rice cultivars Nipponbare and Koshihikari were used to detect quantitative trait loci (QTLs) for pre-harvest sprouting resistance. In the BILs, we detected one QTL on chromosome 3 and one QTL on chromosome 12. The QTL on the short arm of chromosome 3 accounted for 45.0% of the phenotypic variance and the Nipponbare allele of the QTL increased germination percentage by 21.3%. In the CSSLs, we detected seven QTLs, which were located on chromosomes 2, 3 (two), 5, 8 and 11 (two). All Nipponbare alleles of the QTLs were associated with an increased rate of germination. The major QTL for pre-harvest sprouting resistance on the short arm of chromosome 3 was localized to a 474-kbp region in the Nipponbare genome by the SSR markers RM14240 and RM14275 by using 11 substitution lines to replace the different short chromosome segments on chromosome 3. This QTL co-localized with the low-temperature germinability gene qLTG3-1. The level of germinability under low temperature strongly correlated with the level of pre-harvest sprouting resistance in the substitution lines. Sequence analyses revealed a novel functional allele of qLTG3-1 in Nipponbare and a loss-of-function allele in Koshihikari. The allelic difference in qLTG3-1 between Nipponbare and Koshihikari is likely to be associated with differences in both pre-harvest sprouting resistance and low-temperature germinability.     

Development of Chromosome Segment Substitution Lines Derived from Backcross between Two Sequenced Rice Cultivars, <Emphasis Type="Italic">Indica</Emphasis> Recipient 93-11 and <Emphasis Type="Italic">Japonica</Emphasis> Donor Nipponbare

Chromosome segment substitution lines (CSSLs) are powerful tools for detecting and precisely mapping quantitative trait loci (QTLs) and evaluating gene action as a single factor. In this study, 103 CSSLs were produced using two sequenced rice cultivars: 93-11, an elite restorer indica cultivar as recipient, and Nipponbare, a japonica cultivar, as donor. Each CSSL carried a single chromosome substituted segment. The total length of the substituted segments in the CSSLs was 2,590.6 cM, which was 1.7 times of the rice genome. To evaluate the potential application of these CSSLs for QTL detection, phenotypic variations of seed shattering, grain length and grain width in 10 CSSLs were observed. Two QTLs for seed shattering and three for grain length and grain width were identified and mapped on rice chromosomes. The results demonstrate that CSSLs are excellent genetic materials for dissecting complex traits into a set of monogenic loci. These CSSLs are of great potential value for QTL mapping and plant marker-assisted breeding (MAB).     

QTL analysis of fertility-restoration against cytoplasmic male sterility in wheat

The cytoplasm of Triticum timopheevi causes cytoplasmic male sterility (CMS) in common wheat (T. aestivum) cv. 'Chinese Spring' (CS), and that of Aegilops kotschyi causes CMS in spelt wheat (T. spelta) var. duhamelianum (Sp). CS has fertility-restoring (Rf) genes against the latter cytoplasm and Sp has the ones against the former. To know the genetic system concerning to CMS, we crossed 66 F8 recombinant inbred lines (RILs) derived from a cross between CS and Sp as males to the alloplasmic lines of CS and Sp having the cytoplasms of T. timopheevi and Ae. kotschyi, respectively. The fertilities of respective F1 plants derived from the crosses were examined for QTL analysis. The major QTLs detected in both systems were located on the short arm of chromosome 1B. One minor QTL on chromosome 2B was also commonly detected in both of the systems, while other minor QTLs against T timopheevi cytoplasm were distributed on the chromosomes 2A, 4B, and 6A.     

Novel QTLs for photoperiodic flowering revealed by using reciprocal backcross inbred lines from crosses between japonica rice cultivars

The rice japonica cultivars Nipponbare and Koshihikari differ in heading date and response of heading to photoperiod (photoperiod sensitivity). Using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers, we conducted quantitative trait locus (QTL) analyses for heading date in a set of reciprocal backcross inbred lines (BILs) from crosses between Nipponbare and Koshihikari. Under natural-day conditions, transgressive segregation in days to heading (DTH) toward both early and late heading was observed in both BIL populations. QTL analyses revealed that two QTLs-on chromosomes 3 and 6-were involved in the difference in heading date between the parental cultivars. The Nipponbare allele at the QTLs on chromosomes 3 and 6 showed, respectively, increasing and decreasing effects on DTH in both BIL populations. The transgressive segregation observed in the BILs could be accounted for mainly by the complementary action of a set of alleles with opposing effects. Both QTLs were finely mapped as single Mendelian factors in secondary mapping populations (BC(2)F(2) plants/BC(2)F(3) lines). The QTL on chromosome 3 was mapped in the 1,140-kb interval between 94O03-4 (SSR) and OJ21G19-4 (SNP) and was designated Hd16. The QTL on chromosome 6 was mapped in the 328-kb interval between P548D347 (SSR) and 0007O20 (SSR) and was designated Hd17. Both Hd16 and Hd17 were involved in photoperiod sensitivity, as revealed by observation of the DTH of nearly isogenic lines of Nipponbare under short- and long-day conditions, suggesting that allelic differences in both Hd16 and Hd17 account for most of the difference in photoperiod sensitivity between the parental cultivars.     

Improvement of lodging resistance with QTLs for stem diameter in rice (Oryza sativa L.)

Varietal differences among ten rice cultivars showed that stem diameter is a key factor in lodging resistance (measured in terms of pushing resistance). Two near-isogenic lines (NILs) were selected from a series of chromosome segment substitution lines developed between cultivars Nipponbar and Kasalath, one containing a single stem diameter QTL (sdm8; NIL114), and another with four stem diameter QTLs (sdm1, sdm7, sdm8, sdm12; NIL28). Compared with the Nipponbare control, stem diameters were larger in NIL114 and NIL28 by about 7 and 39%, respectively. Pushing resistance in NIL28 was significantly greater than in Nipponbare, but NIL114 was similar to Nipponbare. The two NILs had greater weight of lower stem and culm wall thickness than Nipponbare. NIL28 had higher plant height, which is a negative effect on lodging resistance, than Nipponbare. The non-structural carbohydrate contents of NIL stems were higher than that of Nipponbare, whereas the silicon contents were lower in the NILs, and cellulose contents were lower only in NIL28. The basal internodes of the two NILs were significantly stiffer than those of Nipponbare. These results suggest that increasing stem diameter in rice breeding programs would improve lodging resistance, although the combination of multiple QTLs would be necessary to produce thicker stems with higher pushing resistance, whereas the higher plant height could also result from the combination of multiple QTLs.     

Classification of rice germplasm: III. High-resolution fingerprinting of cytoplasmic genetic male-sterile (CMS) lines with AFLP

 The cytoplasmic genetic male-sterile (CMS) lines developed at the International Rice Research Institute are valuable in producing tropical rice hybrids. Efficient use of CMS lines in hybrid rice production will depend on their level of genetic diversity. Aside from morphological characterization, molecular analysis based on DNA markers can provide information on the genetic diversity of the germplasm. The Amplified Fragment Length Polymorphism (AFLP) technique was used to fingerprint 71 CMS lines and four rice cultivars, ‘IR64’, ‘Azucena’, ‘IR74’, and ‘FR13A’. Eleven primer pair combinations specific to the enzymes PstI and MseI were used to generate 530 AFLP markers, 176 of which were polymorphic. Each CMS line revealed a distinct fingerprint. The AFLP marker-based dendrogram depicted genetic variation among the CMS lines. The CMS lines developed in japonica background grouped with ‘Azucena’, a japonica cultivar. None of the CMS lines clustered with ‘FR13A’, a flood-tolerant traditional indica variety. ‘IR64’ was found to be distinct from the other indica CMS lines and clustered with lines developed in its background. The grouping of CMS lines into a few groups is useful for breeders in selecting genetically diverse CMS lines for hybrid rice production and in avoiding test crossing every CMS line empirically. This study demonstrated that AFLP is a powerful and reliable tool in determining the genetic relationships and in producing distinct fingerprints of rice cultivars. Received: 20 December 1996 / Accepted: 9 October 1997     

籼稻二元不育系9730A及三交种的初步研究

通过对所选育的籼稻二元不育系9730A的花粉育性、自交结实率、雌性育性、对"920"的敏感性、柱头外露情况、花时、雄性不育的可恢复性及三交种的抽穗整齐度、有效穗、株高、单株产量、结实率、主茎穗长、主茎叶长、穗粒等变异度的分析,对杂交水稻三交法育种的应用价值进行初步探讨.结果认为:通过引进具有不同优良品种特性的第二保持系,可以在保持所选育杂种品种主要农艺性状整齐一致的前提下,较大幅度地拓宽品种的遗传基础,改进雄性不育系的异交性能,从而为提高杂种种子生产水平、及时利用优良常规稻育种成果材料和综合实现杂交稻品种选育过程中的多抗、广适、高产、优质育种目标提供新的途径.     

Mapping 49 quantitative trait loci at high resolution through sequencing-based genotyping of rice recombinant inbred lines

Mapping chromosome regions responsible for quantitative phenotypic variation in recombinant populations provides an effective means to characterize the genetic basis of complex traits. We conducted a quantitative trait loci (QTL) analysis of 150 rice recombinant inbred lines (RILs) derived from a cross between two cultivars, Oryza sativa ssp. indica cv. 93-11 and Oryza sativa ssp. japonica cv. Nipponbare. The RILs were genotyped through next-generation sequencing, which accurately determined the recombination breakpoints and provided a new type of genetic markers, recombination bins, for QTL analysis. We detected 49 QTL with phenotypic effect ranging from 3.2 to 46.0% for 14 agronomics traits. Five QTL of relatively large effect (14.6–46.0%) were located on small genomic regions, where strong candidate genes were found. The analysis using sequencing-based genotyping thus offers a powerful solution to map QTL with high resolution. Moreover, the RILs developed in this study serve as an excellent system for mapping and studying genetic basis of agricultural and biological traits of rice.     

Introgression and molecular tagging of <Emphasis Type="Italic">Rf</Emphasis><Subscript>4</Subscript>, a new male fertility restoration gene from wild sunflower <Emphasis Type="Italic">Helianthus maximiliani</Emphasis> L.

Cytoplasmic male sterility (CMS) and its fertility restoration (Rf) genes are critical tools for hybrid seed production to utilize heterosis. In sunflower, CMS PET1 and the associated Rf gene Rf (1) is the only source extensively used in commercial hybrid production. The objective of this research was to develop new sources of CMS and fertility restorers to broaden the genetic diversity of hybrid seed production. We identified a new type of CMS, named as CMS GIG2, from an interspecific cross between Helianthus giganteus accession1934 and H. annuus cv. HA 89. Based on reactions to a set of standard Rf testers, CMS GIG2 is different from all previously reported CMS types, including the CMS GIG1 from another H. giganteus accession. We also identified an Rf gene for CMS GIG2 from wild species H. maximiliani accession 1631. The CMS GIG2 and its restoration gene were introduced into HA 89 background through recurrent backcross and single plant selection techniques. Genetic analysis revealed that the CMS GIG2-Rf system is controlled by a completely dominant gene, named as Rf (4), and the gene additive and dominance effects were estimated as 39.9 and 42.2%, respectively, in the HA 89 background. The gene Rf (4) was mapped onto linkage group 3 with simple sequence repeat (SSR) markers and RFLP-derived STS-marker, and is about 0.9 cM away from the SSR marker ORS1114 based on a segregation population of 933 individuals. The CMS GIG2-Rf (4) system tagged by molecular markers provides an alternative genetic source for hybrid breeding in the sunflower crop.     

主要农作物细胞质雄性不育系育性恢复基因研究进展

提升作物产量、抗逆性和品质的主要手段之一是利用杂种优势,其中细胞质雄性不育/恢复(CMS/Rf)系统是应用最广的雄性不育系统。研究细胞质雄性不育系育性恢复机理是“三系”法选育的重要分子遗传学基础。目前,各个作物已经创制了不同类型的不育系。随着分子技术、基因组学和测序技术的深入,大量育性恢复基因已被定位,且部分已被克隆和功能鉴定。针对主要作物中恢复基因的遗传模式,分子标记定位、克隆及CMS/Rf系统在杂交育种中的应用进行了系统总结。希望本文能为今后恢复系分子标记辅助选育,或利用转基因、基因编辑手段创制新恢复系提供思路。     

Two non-allelic nuclear genes restore fertility in a gametophytic pattern and enhance abiotic stress tolerance in the hybrid rice plant

In indica rice, the HongLian (HL)-type combination of cytoplasmic male sterility (CMS) and fertility restoration (Rf) is widely used for the production of commercial hybrid seeds in China, Laos, Vietnam and other Southeast Asian countries. Generally, any member of the gametophytic fertility restoration system, 50% of the pollen in hybrid F(1) plants displays recovered sterility. In this study, however, a HL-type hybrid variety named HongLian You6 had approximately 75% normal (viable) pollen rather than the expected 50%. To resolve this discrepancy, several fertility segregation populations, including F(2) and BC(1)F(1) derived from the HL-CMS line Yuetai A crossed with the restorer line 9311, were constructed and subjected to genetic analysis. A gametophytic restoration model was discovered to involve two non-allelic nuclear restorer genes, Rf5 and Rf6. The Rf5 had been previously identified using a positional clone strategy. The Rf6 gene represents a new restorer gene locus, which was mapped to the short arm of chromosome 8. The hybrid F(1) plants containing one restorer gene, either Rf5 or Rf6, displayed 50% normal pollen grains with I(2)-KI solution; however, those with both Rf5 and Rf6 displayed 75% normal pollens. We also established that the hybrid F(1) plants including both non-allelic restorer genes exhibited an increased stable seed setting when subjected to stress versus the F(1) plants with only one restorer gene. Finally, we discuss the breeding scheme for the plant gametophytic CMS/Rf system.     

Identification of <Emphasis Type="Italic">qSOR1</Emphasis>, a major rice QTL involved in soil-surface rooting in paddy fields

Specific Indonesian lowland rice (Oryza sativa L.) cultivars elongate thick primary roots on the soil surface of paddy fields. To clarify the genetic factors controlling soil-surface rooting, we performed quantitative trait locus (QTL) analyses using 124 recombinant inbred lines (RILs) derived from a cross between Gemdjah Beton, an Indonesian lowland rice cultivar with soil-surface roots, and Sasanishiki, a Japanese lowland rice cultivar without soil-surface roots. These cultivars and the RILs were tested for soil-surface rooting in a paddy field. We identified four regions of chromosomes 3, 4, 6, and 7 that were associated with soil-surface rooting in the field. Among them, one major QTL was located on the long arm of chromosome 7. This QTL explained 32.5–53.6% of the total phenotypic variance across three field evaluations. To perform fine mapping of this QTL, we measured the basal root growth angle of crown roots at the seedling stage in seven BC₂F₃ recombinant lines grown in small cups in a greenhouse. The QTL was mapped between markers RM21941 and RM21976, which delimit an 812-kb interval in the reference cultivar Nipponbare. We have designated this QTL qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1).     

胞质雄性不育系冀2658A细胞质对陆地棉主要性状的影响

利用棉花(Gossypium hirsutum)雄性不育不仅可以培育优质的杂交种,还能提高棉花制种效率并降低制种成本。该研究以冀2658系及其同核异质不育系冀2658A为母本,以6个恢复系为父本配制12个杂交组合。利用F1代研究棉花细胞质对棉花农艺性状、抗病性、种子中粗脂肪和粗蛋白含量、纤维品质及产量性状的影响。结果表明,冀2658A的细胞质主要影响棉花杂交种F1代的产量相关性状、黄萎病抗性及棉籽粗脂肪含量等,表现为衣分显著降低(比对照组低1.61%),黄萎病抗性增强(黄萎病指数比对照组低18.29%),棉籽中的粗脂肪含量降低(比对照组低2.88%)。该研究初步探讨了胞质不育型细胞质对陆地棉主要性状的影响,为陆地棉胞质雄性不育系的利用提供了理论参考。     

Mitotype-specific sequences related to cytoplasmic male sterility in Oryza species

Plant mitochondrial genomes contain a large number of mitotype-specific sequences (MSS) which establish a mitochondrial genome structure distinct from other mitotypes. In rice, nine mitochondrial genomes have been sequenced, which provides us with the possibility of characterizing the MSS of rice and probing their relationship to cytoplasmic male sterility (CMS) in rice. We therefore analyzed the mitochondrial genomes of CW-CMS, LD-CMS, WA-CMS, N and Nipponbare lines, and found 57 MSS with sizes ranging from 102 to 5,745 bp, and with an aggregate length of 92.4 kb. The MSS account for more than 14.5 % of the rice mitochondrial genome and are a significant contributing factor in the variation of mitochondrial genome sizes. Of the MSS tested, 34 MSS exhibited polymorphism among rice lines, and 14 MSS were further confirmed as being specific to CMS. This includes nine MSS specific to sporophytic CMS, three specific to gametophytic CMS, and two shared by all types of CMS. Interestingly, except for CMS genes orf(H)79 and orf352 which are partly or fully overlapping with some MSS fragments, there are ten more open reading frames of unknown function that were detected in CMS-specific MSS, hinting at their possible roles in plant CMS. These novel findings provide us with potential new molecular tools to direct the breeding of CMS lines in hybrid rice breeding programs.