Fine mapping of the qLOP2 and qPSR2-1 loci associated with chilling stress tolerance of wild rice seedlings.

KEY MESSAGE: Using leaf osmotic potential and plant survival rate as chilling-tolerant trait indices, we identified two major quantitative trait loci qLOP2 and qPSR2 - 1 (39.3-kb region) and Os02g0677300 as the cold-inducible gene for these loci. Chilling stress tolerance (CST) at the seedling stage is an important trait affecting rice production in temperate climate and high-altitude areas. To identify quantitative trait loci (QTLs) associated with CST, a mapping population consisting of 151 BC(2)F(1) plants was constructed by using chilling-tolerant Dongxiang wild rice (Oryza rufipogon Griff.) as a donor parent and chilling-sensitive indica as a recurrent parent. With leaf osmotic potential (LOP) and plant survival rate (PSR) as chilling-tolerant trait indexes, two major QTLs, qLOP2 (LOD = 3.8) and qPSR2-1 (LOD = 3.3), were detected on the long arm of chromosome 2 by composite interval mapping method in QTL Cartographer software, which explained 10.1 and 12.3% of the phenotypic variation, respectively. In R/QTL analyzed result, their major effects were also confirmed. Using molecular marker RM318 and RM106, qLOP2 and qPSR2-1 have been introgressed into chilling-sensitive varieties (93-11 and Yuefeng) by marker-assisted selection procedure (MAS), which resulted in 16 BC(5)F(3) BILs that chilling tolerance have significantly enhanced compare with wild-type parents (P < 0.01). Therefore, two large segregating populations of 11,326 BC(4)F(2) and 8,642 BC(4)F(3) were developed to fine mapping of qLOP2 and qPSR2-1. Lastly, they were dissected to a 39.3-kb candidate region between marker RM221 and RS8. Expression and sequence analysis results indicated that Os02g0677300 was a cold-inducible gene for these loci. Our study provides novel alleles for improving rice CST by MAS and contributes to the understanding of its molecular mechanisms.

Fine mapping of qRC10-2, a quantitative trait locus for cold tolerance of rice roots at seedling and mature stages.

UNLABELLED: Cold stress causes various injuries to rice seedlings in low-temperature and high-altitude areas and is therefore an important factor affecting rice production in such areas. In this study, root conductivity (RC) was used as an indicator to map quantitative trait loci (QTLs) of cold tolerance in Oryza rufipogon Griff., Dongxiang wild rice (DX), at its two-leaf stage. The correlation coefficients between RC and the plant survival rate (PSR) at the seedling and maturity stages were -0.85 and -0.9 (P = 0.01), respectively, indicating that RC is a reliable index for evaluating cold tolerance of rice. A preliminary mapping group was constructed from 151 BC2F1 plants using DX as a cold-tolerant donor and the indica variety Nanjing 11 (NJ) as a recurrent parent. A total of 113 codominant simple-sequence repeat (SSR) markers were developed, with a parental polymorphism of 17.3%. Two cold-tolerant QTLs, named qRC10-1 and qRC10-2 were detected on chromosome 10 by composite interval mapping. qRC10-1 (LOD = 3.1, RM171-RM1108) was mapped at 148.3 cM, and qRC10-2 (LOD = 6.1, RM25570-RM304) was mapped at 163.3 cM, which accounted for 9.4% and 32.1% of phenotypic variances, respectively. To fine map the major locus qRC10-2, NJ was crossed with a BC4F2 plant (L188-3), which only carried the QTL qRC10-2, to construct a large BC5F2 fine-mapping population with 13,324 progenies. Forty-five molecular markers were designed to evenly cover qRC10-2, and 10 markers showed polymorphisms between DX and NJ. As a result, qRC10-2 was delimited to a 48.5-kb region between markers qc45 and qc48. In this region, Os10g0489500 and Os10g0490100 exhibited different expression patterns between DX and NJ. Our results provide a basis for identifying the gene(s) underlying qRC10-2, and the markers developed here may be used to improve low-temperature tolerance of rice seedling and maturity stages via marker-assisted selection (MAS). KEY MESSAGE: With root electrical conductivity used as a cold-tolerance index, the quantitative trait locus qRC10-2 was fine mapped to a 48.5-kb candidate region, and Os10g0489500 and Os10g0490100 were identified as differently expressed genes for qRC10-2.

Screening for and genetic analysis on T-DNA-inserted mutant pool in rice.

T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants, and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype. In this study, we screened 4,416 rice T1 tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents, ZH11 and ZH15. We found many lines showed obvious morphological mutations, including two types--fake-homozygous mutation and separating mutation. The mutation phenotype was related to 14 kinds of trait such as plant height, heading date, leaf shape, leaf color, tiller number, panicle shape, spikelet number, grain shape, disease-like mutation, male sterility, awn, and so on. Among them, plant height, heading date, leaf color and male sterility had a comparatively high mutation frequency (over 1%). The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents, but the frequency of heading date and male sterility varied greatly, suggesting that environment had a great effect on the expression of latter two traits. By conducting continuously co-segregating analyses in T1 and T2 generation, we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets, which would provide a base for cloning correlative functional genes. At the same time, we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR, of which, 26 were vector backbone sequence, 14 had good identity to rice genome sequence. The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.