Marker-assisted introgression of QTLs for yield under moisture stress into elite varieties of rice (Oryza sativa)

Rice production is severely constrained by the moisture stress. The present study was undertaken to transfer two important QTLs viz., (qDTY2.2 and qDTY4.1), which controls yield under moisture stress (DTY) into two elite varieties, that is, MTU1010 and NLR34449 through marker-assisted breeding. Foreground and background selections of backcross generations, that is, BC₁F₁, BC₂F₁ and BC₂F₂ identified several promising introgression lines (ILs) with two QTLs and single QTLs with an appreciable level of recovery of recipient parent genome. In ILs, the recovery of MTU1010 genome content was estimated to be 83%–93% while the recovery of NLR34449 was 84%–92%. The two-QTL ILs of MTU1010 and NLR34449 backgrounds (qDTY2.2 and qDTY4.1) have shown substantial yield advantage (32 to 84%) over the single-QTL ILs (either qDTY2.2 or qDTY4.1) under moisture stress conditions. Among all, two ILs, MBC-124 and NBC-127 are the best high yielding lines under moisture stress conditions. These outyielded ILs have the potential to be released as varieties in rainfed ecosystem and also can be used as donors in the existing breeding programme in rice.     

Detection of quantitative trait loci (QTL) associated with resistance to whitebacked planthopper (Sogatella furcifera) in rice (Oryza sativa)

Whitebacked planthopper (WBPH) is an important insect pest of rice. In this study, we report quantitative trait loci (QTL) associated with resistance to WBPH using a doubled‐haploid (DH) mapping population derived from the cross IR64/Azucena. We evaluated a set of 91 DH lines using various screening tests which measure seedling resistance, antibiosis and tolerance to WBPH. QTL analysis involving a RFLP map of 175 markers detected a significant QTL on chromosome 7 (RG511‐RG477) associated with seedling resistance to WBPH. In addition, QTL analysis involving available defence related candidate genes as markers on a sub set of 60 DH lines showed significant association of genomic regions on chromosome 1 (W1‐pMRF1), 2 (XLRfrI7‐RG157) and 7 (RG711‐CDO418) with resistance to WBPH. Several suggestive QTL were detected on chromosomes 2, 3, 6, 7, 8 and 11 showing the possibility of their association with resistance to WBPH. The phenotypic contribution of the QTL ranged from 8.4% to 32.1%. Some of the WBPH resistance QTL detected in this study showed similar map positions with the QTL reported for resistance to brown planthopper (BPH) in the same mapping population. These results would be useful for attempts to trace the genes associated with resistance to planthoppers in rice.     

Marker‐assisted breeding for improvement of anaerobic germination in japonica rice (Oryza sativa)

Direct seeding is increasingly being practiced in rice cultivation areas because it saves labour and reduces cost of production and management. In this study, four japonica‐type breeding lines were developed from a cross of a japonica cultivar and an AG‐tolerant donor, KHO, using marker‐assisted selection. Validation of the target genes AG1 and AG2 in the selected lines was examined by PCR analysis and the background recovery rate by the recurrent parent was confirmed by genotyping using a 6K SNP chip. Compared to the recurrent, the survival rates of the selected lines were improved in a range from 33% to 115% in three conditions; in particular, the survival rate was increased in more than twice that of the recurrent parent under the hypoxic condition. The main agronomic trait, yield and quality‐related traits were similar to those of the recurrent parent. The use of these lines will contribute to the expansion of the cultivation area due to the proven economic benefits of direct seeding in Korea.     

STS and microsatellite marker-assisted selection for bacterial blight resistance and waxy genes in rice, Oryza sativa L.

DNA marker-assisted selection was employed to select Xa-21 bacterial blight resistance and waxy (Wx) genes. Genotypes with both genes were selected from four F₂ populations involving indica × indica, indica × intermediate and japonica × indica crosses. With the assistance of PCR marker, 13 true breeding lines carrying Xa-21 were identified from F₂ generation of IRBB 21 × G 11353 cross. Similarly ten, eleven and fifty two plants having Xa-21 gene were isolated from G 3005-4-1 × IRBB 21, IRBB 21 × HJX 74 and IRBB 21 × SY 2crosses respectively. The lines with Wx gene in both homozygous and heterozygous state were also scored from the above crosses. Twenty plants having both Xa-21 and Wx loci in homozygous state were identified. DNA-based progeny testing was conducted to ensure the selection of homozygous lines for Xa-21 and Wx genes. Finally,twenty true breeding lines with high amylose content and Xa-21 gene were isolated from four crosses. These homozygous lines are phenotypically superior and resistant to Chinese race 5 of the bacterial blight pathogen. Fifty-six germplasm sources were surveyed for PCR polymorphism in order to facilitate future PCR-based marker assisted transfer of bacterial blight resistance genes xa-5, xa-13 and Xa-21 to any desired varietal background which will be useful for selection of parents in breeding programmes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular mapping of a new genic male-sterility gene causing chalky endosperm in rice ( Oryza sativa L.)

A genic male-sterility gene newly induced by chemical mutagenesis, tentatively designated as ms-h(t), was located on the molecular map of rice and tested for its effect on chalky endosperm. Bulked segregant analysis was used to determine the chromosomal location of the ms-h(t) locus by screening four to five RFLP markers per chromosome. After confirming that the gene was located on chromosome 9, twenty-four RFLP markers from chromosome 9 were surveyed for polymorphism in the parents of the mapping population. Of these, eleven markers were mapped around the ms- h(t) locus. RG451 and RZ404 flanked the ms-h(t) gene, at 2.5cM and 3.3cM, respectively. Heterozygous F2 to F4 progenies were tested for co- segregation of male-sterility and chalky endosperm and it was revealed that ms-h(t) might have a pleiotropic effect on chalky endosperm. This mutant would be a good biological material to characterize the biochemical mechanism of male sterility and related pleiotropic effects. Further studies should be needed to know the usefulness of this mutant for hybrid seed production. This revised version was published online in July 2006 with corrections to the Cover Date.     

利用东乡普通野生稻染色体片段置换系定位产量相关性状QTL

以东乡普通野生稻和日本晴为亲本构建的染色体片段置换系为研究材料, 2019年分别在北京、山东临沂和江西南昌对分蘖数、穗粒数和粒形等11个产量相关性状进行多环境鉴定,结合染色体片段置换系基因型数据定位水稻产量相关性状QTL。3个环境共检测到68个QTL,包括株高4个、穗长5个、分蘖数2个、一次枝梗数7个、一次枝梗粒数8个、二次枝梗数8个、二次枝梗粒数10个、每穗粒数6个、千粒重7个、粒长8个和粒宽3个; LOD值介于2.50～12.66之间,贡献率变幅为4.67%～27.79%,15个QTL的贡献率大于15%;24个QTL与已报道位点/基因位置重叠,44个QTL为新发现位点; 6个QTL在2个环境能被检测到, 1个QTL qTGW2能在3个环境检测到,且是还未报道的新位点。最后,利用BSA法验证了qPH7、qPBPP8-2和qGW10三个QTL的可靠性。本研究将为后续产量相关性状基因克隆以及进一步解析其遗传基础和分子调控机制奠定基础。     

Genetic analysis and fine mapping of a dominant dwarfness gene from wild rice (Oryza barthii)

Plant architecture has been proposed as a means to enhance the potential yield of rice, especially by reducing height to provide lodging resistance. In this study, we developed a near‐isogenic line (NIL) using cultivar ‘Dianjingyou 1’ (DJY1) as a recipient parent and wild rice (Oryza barthii) as the donor parent. The NIL had semi‐dwarf stature and more tillers than DJY1. Cytological examination showed decreased numbers of cells in the stems of the NIL compared to DJY1. Genetic analysis indicated that this phenotype was controlled by a newly identified dominant dwarf gene, tentatively named as Dd7. A large population derived from the hybrid NIL‐Dd7/DJY1 was developed to fine‐map Dd7. Based on the physical location of molecular markers, the Dd7 locus was finally delimited to an 82‐kb region in chromosome 7. Gene prediction identified 14 open reading frames (ORFs) within this region. NIL‐Dd7 seems to confer no undesirable pleiotropic effects and therefore has potential value for rice breeding.     

Simultaneous improvement in cold tolerance and yield of temperate japonica rice (Oryza sativa L.) by introgression breeding

To combine high yield and improved cold tolerance (CT) in a japonica rice variety, ‘Chaoyou1’ (CY1), 324 BC₂F₅ introgression lines (ILs) selected for CT from 11 CY1 BC₂F₄ populations were evaluated in replicated experiments for their CT at the reproductive and seedling stages. A mean realized heritability of 0.747 was achieved in this study for CT. Evaluation of 116 ILs from five BC populations in replicated experiments under stress and normal conditions identified 18 promising ILs that had greatly improved CT and yield compared with CY1. Detailed comparisons between the ILs and CY1 for CT and yield‐related traits under stress and non‐stress conditions provided useful information and better understanding of important issues such as donor selection, selection efficiency and associated changes in non‐target traits in the BC breeding process. The large numbers of CT ILs developed provide useful materials for genetic, physiological and molecular dissection of CT and yield traits using DNA markers and ‐omic tools, and as parents for further improving these traits by designed QTL pyramiding.     

Novel quantitative trait loci for yield and yield related traits identified in Basmati rice (Oryza sativa)

Increasing crop productivity is one of the prime goals of crop breeding research. Rice grain yield is a complex quantitative trait governed by polygenes. Although several QTLs governing grain yield traits have been reported and limited attempts have been made to map QTLs for grain yield parameters in Basmati rice. A population from the cross Sonasal and Pusa Basmati 1121 comprising 352 RILs was generated through the single seed descent method. A total of 12 QTLs governing yield and yield-related traits were mapped on six chromosomes, namely, 1, 2, 3, 7, 8 and 9, of which five QTLs were novel. We identified a novel and robust epistatic QTL (qPH1.1 and qPL1.1) governing plant height and panicle length, flanked by the markers RM5336-RM1 on chromosome 1. The gene encoding brassinosteroid insensitive 1-associated receptor kinase 1 precursor is the putative candidate gene underlying this epistatic QTL. Another novel QTL, qNT3.1, governing tiller number was bracketed to a region of .77 Mb between the markers RM15247 and RM15281 on chromosome 3. Of the 57 annotated gene models, Os03g0437600 encoding alpha/beta-fold hydrolase, a homologous to AtKai2 is a putative candidate gene underlying the novel QTL qNT3.1. The other QTLs such as qDFF1.1 governing days to 50% flowering co-localizes with the gene Ghd7, QTL for plant height qPH1.2 co-localizes with the gene sd1, the QTLs for panicle length co-localizes with FUWA and DEP2, the QTL for tiller number co-localizes with OsRLCK57 and QTLs for thousand-grain weight co-localize with the major gene GS3. The QTLs identified in the current study can be effectively used in marker-assisted selection for developing Basmati rice varieties with a higher yield.     

Effectiveness of different classes of molecular marker for classifying and revealing variation in rice (Oryza sativa) germplasm

We have examined the effectiveness of similar numbers of markers from four molecular marker systems (AFLP, isozymes, ISSR and RAPD) for revealing genetic diversity and discriminating between infraspecific groups of Oryza sativa germplasm. Each marker system classifies the germplasm into three major groups (most effectively with isozymes and AFLPs), but with differences (primarily with ISSR) between the precise classifications generated. However, at the highest levels of genetic similarity there was only partial agreement as to relationships between individual accessions when different markers were used. When variance was partitioned among and within the three subspecific groups, although the differences were not significant, greater variation was found among than within groups using AFLP and isozymes, with the reverse for RAPD and ISSR. Measurement of polymorphism using average heterozygosity and effective number of alleles gave similar results for each marker system. These results are discussed in relation to various genetic resources conservation activities, and the advisability of extrapolating to other sets of germplasm particularly of other crop species. This revised version was published online in July 2006 with corrections to the Cover Date.     

RFLP facilitated analysis of tiller and leaf angles in rice (Oryza sativa L.)

Plant type is an important composite trait associated with the yield potential in rice and other cereal crops. Several characters associated with the plant type of modern rice cultivars including tiller angle, leaf and flag leaf angle, were investigated using a complete linkage map with 115 well distributed RFLP markers and progeny testing of 2418 F₂ derived F₄ lines from a cross between O. sativa ssp. japonica cv. ‘Lemont’ and spp. indica cv. ‘Teqing’. One major gene (Ta) and 11 QTLs were largely responsible for the tremendous variation of the three plant type characters in the Lemont/Teqing F₂ population. The major gene, Ta, located between RZ228 and RG667 on chromosome 9, accounted for 47.5% of the phenotypic variation in tiller angle and had large pleiotropic effects on both leaf and flag leaf angles. This gene plus four QTLs accounted for 69.1% of the genotypic variation in tiller angle. Eight additional QTLs for leaf and flag leaf angles were also identified, which collectively explained 52.0 and 66.4% of the genotypic variation of these traits. Ta and three QTLs ( QFla2, QFla5 and QFla7) apparently affected the related plant type characters differently, suggesting their possible differential expression in different developmental stages of rice plants or possibly clustering of different genes affecting these traits. Plant type, and consequently grain yield of rice, may be improved by deliberately manipulating these QTLs in a marker-assisted selection program. This revised version was published online in July 2006 with corrections to the Cover Date.     

Mutation of DEP1 facilitates the improvement of plant architecture in Xian rice (Oryza sativa)

Plant architecture is a complex trait and has a profound impact on crop performance and productivity. We applied the CRISPR/Cas9 system to mutate the DEP1 gene, which has been reported to function as regulator of plant architecture, in elite Xian cultivar ‘Huhan1509’. Sequencing analysis of T₀ transformed plants showed that the CRISPR/Cas9 system was highly efficient in mutagenesis of targeted DEP1 gene, with 30% of the homozygous mutations and 70% of the heterozygous mutations. T₂ homozygous mutants without T-DNA were further examined for the agronomic traits. The DEP1 mutants exhibited an altered plant architecture along with a shorter plant height and grain size and increased spikelets and grain density. Furthermore, phenotypes of raising primary branches and stem diameters were observed in the DEP1 mutants. Our results demonstrate that favourable alleles of the DEP1 gene, developed by CRISPR/Cas9 system, could be used to improve plant architecture in Xian rice.     

Potential of marker-assisted selection in hemp genetic improvement

Summary The development and applications of molecular markers to hemp breeding are recent, dating back only to the mid-1990s. The main achievements in this field are reviewed. The analysis of Cannabis germplasm by RAPD, AFLP and microsatellites is discussed, with its consequence for the still debated species concept in Cannabis. DNA-based markers have also been exploited in the field of forensic science, in an attempt to discriminate licit from illicit crop. The main applications of the molecular markers to the breeding, however, have been achieved with the development of markers closely linked to the male sex and to some of the most relevant chemotypes. Active research is carried out by several groups in the field of identification and characterization of the genes involved in fiber quality and quantity, and in the determination of monoecy, another very important target of hemp breeding. Besides, markers associated to new, potentially useful chemotypes are being developed, for the marker-assisted breeding of pharmaceutical Cannabis.     

Crosses between cultivars and tissue culture-selected plants for salt resistance improvement in rice, Oryza sativa

Several R₂ somaclonal families were derived from plants regenerated from a salt‐resistant callus of the salt‐sensitive rice cultivar ‘I Kong Pao’ (IKP). The family R₂‐1‐23, in the presence of NaCl exhibited higher yield performances than the initial cultivar. This improvement in salinity resistance, however, was not transmitted to following generations; despite a higher number of spikelets per plant, family R₃‐1‐23 did not perform better than the initial cultivar because of a very low seed set. This somaclonal family, its initial being the cultivar IKP, the breeding line IR₃1785 (extremely salt‐sensitive) and the cultivar ‘Aiwu’ (moderately salt‐resistant), were used as parents for production ofhybrids. Four crosses, IKP×R3‐1‐23, IR31785 ×R3‐1‐23, IR31785× IKP and IKP בAiwu’, were performed. Most of the F₁ hybrids cultivated in the absence of salt exhibited increased performances compared with the mid‐parent, suggesting an heterosis effect for yield‐related parameters. F₂ populations were screened for salinity resistance and a clear improvement for yield in stress conditions was recorded for populations derived from IK×R3‐1‐23, IR31785×R3‐1‐23 and IR31785×IKP, although the mean level of increase over the mid‐parent (RIMP) varied depending on the population, the presence or absence of stress, and the quantified parameters. The results are discussed in relation to the usefulness of in vitro selection for obtaining interesting somaclonal variants useful to be integrated in classical breeding programmes for salinity resistance in rice.     

水稻抽穗期的QTL剖析

利用沈农265/丽江新团黑谷的F2群体对抽穗期进行定位分析表明,该群体共定位到7个控制抽穗期的 QTLs,分别位于第2,3,6和7染色体上.单个QTL对性状表型贡献率在9.7%～23%之间.与其他研究结果比较表明,多数控制抽穗期的QTLs在不同的材料、不同遗传背景下重演性较好.同时,发现一个新的QTL位点,这些为北方粳稻育种的生育期选择提供基础性数据.     

Role of the rice blast resistance gene Pi-cd in rice (Oryza sativa) breeding programmes

A series of DNA markers for various agronomic traits may accelerate the success of marker-assisted selection in practical plant breeding programmes. Here, we developed DNA markers for the blast resistance gene Pi-cd. In this study, we examined the effects of the Pi-cd locus on not only blast resistance but also agronomic traits in agriculture. We developed three pyramiding lines (PLs) coupling Pi-cd with three blast resistance genes, pi21, Pi35 and Pi39. The effect of Pi-cd on blast resistance was dependent on the coupled resistance genes. Then, we evaluated the effects of Pi-cd on 13 agronomic traits. Amylose content and 1,000-grain weight showed significant differences between the PLs and current commercial varieties, which had no negative effects on agronomic trait values. Furthermore, we investigated the distribution of genotype for the Pi-cd locus among varieties of upland rice. The KT genotype specific to rice blast resistance may be predominant in the varieties. The results suggested that Pi-cd has the potential to be useful for improving blast resistance in rice breeding programmes.     

Construction and characterization of 3-S Lines, an alternative population for mapping studies in rice (Oryza sativa L.)

Most traits of agronomic importance in rice are quantitative in nature and are controlled by polygenes, called quantitative trait loci (QTL). Understanding the nature and effect of QTLs are important for rice breeding to achieve higher yield and stability. Single segment substitution lines (SSSLs or 3-S Lines) were developed through simple sequence repeats (SSR) marker-facilitated backcrossing methods for Hua-Jing-Xian 74 (HJX74) with the donor segment from six elite germplasm and was characterized. Complete genome survey was carried out with 258 polymorphic SSR markers. Polymorphism of the donors with the recurrent parent varied between 32.98 and 60.73% with an average of 47.81%. Japonica donors were more polymorphic than indica donors. Number of substitution segments per plant decreased with the advancement of backcross generations. In BC₂F_1, BC₃F₁, BC₃F₂ and BC₃F₃ the average number of substitution segment per plant were 12.5, 5.98, 1.69 and 1.46, respectively. Average size of substitution segments also decreased with the number of times plants were backcrossed and selfed. In BC₂F₁, BC₃F₁, BC₃F₂ and BC₃F_3, average size of the segments was 25.43, 22.38, 20.78 and 18.15 cM, respectively. The rate of reduction of segment size was more in backcross (11.99%) than selfing (7.15%) generations. Percent recovery of recurrent parent genome in BC₂F₁, BC₃F₁, BC₃F₂ and BC₃F₃ was 82.24, 92.55, 98.04 and 98.52%, respectively. A total of 111 SSSLs comprising of 43 unique types were developed in BC₃F₂ and BC₃F₃. The estimated length of the segments in SSSLs ranged from 2.00 to 64.80 cM with an average of 21.75 cM, and 6.05 to 48.90 cM with an average of 20.95 cM in BC₃F₂ and BC₃F₃, respectively. Total length of all substitution segments was 2367.5 cM that covered 704.50 cM (39.25%) of the entire rice genome. Effective development and successful utilization of 3-S Lines for analysis of QTLs and mapping of genes established the suitability of the SSR marker facilitated backcross breeding approach for 3-S Lines development and its utilization.     

Trisomic genetic analysis of aroma in three Japanese native rice varieties (Oryza sativa L.)

Information on the genetics of aroma in rice facilitates breeding and selection of new aromatic varieties with high yield and good quality. Objective of the present study was to make clear the number of genes controlling aroma, and the allelism of aroma genes and the location of aroma gene(s) on the chromosome in three Japanese native aromatic rice varieties (Kabashiko, Shiroikichi and Henroyori). Lack of leaf aroma in all F₁ plants of non-aromatic/aromatic crosses indicated the recessive nature of aroma, and the segregation ratios (3:1) of non-aromatic to aromatic plants in its F₂ populations from Nipponbare/aromatic varieties crosses revealed that each of the three aromatic varieties contains a single recessive gene for aroma. Through trisomic analysis, the segregation of non-aromatic and aromatic plants in all F₂ populations from the crosses between trisomics lines NT8, with an extra chromosome 8, and aromatic varieties deviated significantly from disomic segregation of 3:1 ratios, and fitted to trisomic segregation, however, in other F₂ populations derived from other 7 types of trisomic F₁ plants, the segregation ratios of non-aromatic to aromatic were 3:1, indicating that the single recessive aroma gene was located on chromosome 8 in three aromatic varieties. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of heterotic loci detected in a cross between indica and japonica rice (Oryza sativa L.)

The study on the genetic basis of heterosis has received significant attention in recent years. In this study, using a set of introgression lines (ILs) and corresponding testcross F₁ populations, we investigated heterotic loci (HL) associated with six yield-related traits in both Oryza sativa L. subsp. indica and japonica. A total of 41 HL were detected on the basis of mid-parent heterosis values with single-point analysis. The F₁ test-cross population showed superiority in most yield-related traits and was characterized by a high frequency of overdominant HL. Thirty-eight of the 41 HL were overdominant, and in the absence of epistasis, three HL were dominant, suggesting that heterotic effects at the single-locus level mainly appeared to be overdominant in rice. Twenty-four HL had a real positive effect, suggesting that they are viable candidates for the improvement of rice yield potential. Compared with the quantitative trait loci (QTLs) detected in the ILs, only six out of the 41 (14.6%) HL were detected in QTL analysis under the same statistical threshold, indicating that heterosis and trait performance may be conditioned by different sets of loci.