Building Near-Isogenic Introgression Lines of Elite Maintainer Gangxiang B and Identifying Some Specific Traits  

Yeqing Xiao , Xiaoyan Wu , Lanxiang Hu , Wenchang Wu , Shiyou Luo , Wei Deng , Dazhou Chen
Rice Research Institute, Jiangxi Academy of Agricultural Sciences, Nanchang, 330200, P.R. China
Author    Correspondence author
Rice Genomics and Genetics, 2012, Vol. 3, No. 2   doi: 10.5376/rgg.2012.03.0002
Received: 16 Dec., 2011    Accepted: 17 Jan., 2012    Published: 31 Jan., 2012
© 2012 BioPublisher Publishing Platform
This article was first published in Molecular Plant Breeding in Chinese, and here was authorized to translate and publish the paper in English under the terms of Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Xiao et al., 2012, Building Near-Isogenic Introgression Lines of Elite Maintainer Gangxiang B and Identifying Some Specific Traits, Rice Genomics and Genetics, Vol.3, No.2, 8-12 (doi: 10.5376/rgg.2012.03.0002)


Wild rice is abundant with types of genetic variation formed in the long evolution of natural selection, which contains a great number of favorable genes as well as develops a strong adaptability to the environment. Efficiently exploring the favorable genes of wild rice would be a prerequisite to achieve a breakthrough in rice breeding program. In this research we developed a population consisting of 892 near-isogenic introgression lines (NILs) by crossing and subsequent backcrossing by using eleven wild rice accessions as donors came from different origins and using Gangxiang B as recipient, an elite maintainer to rice cytoplasm male sterility of three-line hybrid system developed by Rice Research Institute of Jiangxi Academy of Agricultural Sciences. There are significant differences of phenotypic traits existing in the populations of the NILs, mainly including Growth period, plant height, tillering ability, panicle shape, grain shape, awn etc. We preliminary identified a batch of materials using the near-isogenic introgression lines we developed in this research, such as materials with drought resistance or high temperature resistance screened under the conditions of drought or high temperature above 35℃, materials with low nitrogen tolerance or blast resistance identified under the conditions of low nitrogen levels or in the prone areas of rice blast, as well as materials with seedling flooding tolerance or herbicide-resistance assessed under the conditions of flooding or spraying of the herbicides such as phosphine glycosides. Using of these excellent materials, we will attempt to pyramid the favorable traits to further improve the features of stress resistance, low nitrogen tolerant capacity and disease resistance in the maintainer of Gangxiang B in order to lay a foundation to achieve the goal of green super rice breeding. In the scanning outcome of large-scale NILs, most of the targeted traits could be identified in the progenies derived from the donors of wild rice, indicating that there should be many favorable genes existing in wild rice.

Rice (Oryza sativa L.); Wild rice (Oryza rufipogon L.); Near-isogenic introgression line; Evaluation of target trait; Maintainer of three-line hybrid rice

Agronomic wild plants are not only very important wealth of human beings, but also important genetic resources for human survival and development. Since wild rice has been found in 1917, China made remarkable achievements across the world for utilizing the wild rice germplasms in the research. As far back as 1929, Prof. Ding Ying bred Zhongshan No.1 and its series of derivatives by crossing between cultivated rice and wild rice. In 1970s, Chinese rice breeders were the first to realize the matching sets of indica three-line hybrid rice through transferring the cytoplasmic male sterility gene of common wild rice into cultivated rice. Increase of rice yield and resistance is benefits from the discovery and utilization of germplasm resources. In the long term process of natural selection, wild rice forms a rich variation of types of which led to stronger resistance to pests and diseases as well as strong environmental adaptability, such as excellent gens conferring resistant to leaf blight, rice blast, sheath blight, and plant hoppers as well as tolerant to drought, cold, high temperature, salinity and flooding, and so on. Therefore, wild rice germplasms have important values for enriching genetic basis of cultivated rice varieties to provide excellent agronomic genes conferring resistance to pests and other abiotic stresses, as well as to improved varieties of cultivated rice (Li, 2005; Luo, 2005; Xu et al., 2005). In this study, we attempted to build a set of near-isogenic introgression lines by crossing and subsequent backcrossing by using eleven wild rice accessions as donors came from different origins and using Gangxiang B as recipient, an elite maintainer to rice cytoplasm male sterility of three-line hybrid system. The primarily phenotypic screening were performed for disease resistance, drought resistance, nutrient efficiency, high temperature tolerance, flooding and other target traits in order to lay a the foundation to develop "green super rice".

1 Results and Analysis
1.1 Building Near-isogenic introgression lines
In 2007, we made a series of crosses using Ganxiang B as maternal parent and employing wild rice as donors including Dongxiang wild rice, Guangxi wild rice, Jiangyong wild rice, Chaling wild rice, and Yuanjiang common wild rice, Hehuatang No.2, No.4, and No.10 as well as a series of wild rice germplsms of 06YD2, 06YD5, 06YD6 and 06YD12 provided by Huazhong Agricultural University. The backcrossed were carried out 2 or 3 times by using Ganxiang B as paternal parent (Individual with more traits of wild rice might be ackcrossed three times), prior to self-crossing. Until in 2010, 892 lines including lines of BC2F5 and BC3F4 were achieved in this research (Table 1).


Table 1 Library of near isogenic introgression lines (ILS) by using Ganxiang B as the recipient

1.2 Screening for tolerance to drought
The weather with consecutive high temperature and less humidity occurred in Nanchang city from 1st to 18th, August, 2010, the highest temperature reached 35℃ or more, which would be facilitate to identify the drought-resistant materials. Where soil in field appeared paling, cracking, grass wilting, materials that had poor drought tolerance appeared the symptoms of leaf curling and wilting of the whole plant that could not recovered by rehydration. The 6 996 individuals with high drought tolerance from 11 combinations were preliminary screened, while the control varieties exhibited a strong drought tolerance (Table 2).


Table 2 The screening results of near isogenic introgression lines for tolerance to drought

1.3 Screening for tolerance to low nitrogen
In the low nitrogen conditions, 892 lines derived from the different combinations showed significantly differences in the use of nitrogen, of which some lines had the superiority of vegetative growth stronger than that of parental receptors Ganxiang B, mainly exhibiting excellent characters such as strong tillering, large panicle, stem thickening, etc., indicating that some of the NILs should have a strong capacity of tolerance to low nitrogen. 123 lines with excellent tolerance to low nitrogen were preliminary screened out (Table 3).


Table 3 The screening results of near isogenic introgression lines for tolerance to low nitrogen

1.4 Screening for resistance to rice blast
892 lines were planted in Fengtian village of Kenzhi Station in Jingguangshang where was heavy blast-prone area for nature induced screening, there were 585 lines with resistance to leaf blast, 502 lines with resistance to panicle-neck blast, The line of R752 had the highest susceptible to rice blast comparing to the control of Ganxiang B, the results shown in Table 4.


Table 4 The screening results of near isogenic introgression lines for resistance to rice blast

1.5 Screening for tolerance to high temperature
The weather with continuous hot days happened from 1st to 18th, August in 2010, in region of Nanchang City, the highest temperature reached 35℃ and or 39℃, during this period, there were 723 lines occurring in the stages of heading and flowering, which was facilitate to screen the resistant lines to high-temperature. Most of lines heading under the conditions of the high temperature exhibited low seed setting rate, generally about 40%, some lower about 10%. Whereas 18 NIL lines heading from 3rd to 12th August, 2010, had higher seed setting rate of 70% to 76.3%, of which exhibited high tolerance to high temperature.

1.6 Screening for flooding-resistance
Seedlings submerged 7 days later, then drained water off in 5 days, field survey for counting living seedlings was carried out. The results showed that most of the seedlings in flooded condition were almost no growing and dead in the beginning of the 5th day. Whereas the flood-resistant seedlings were still growing in flooded condition, after draining water off, the seedlings become thin and green seedlings. The 157 lines with flooding tolerance from 11 combinations had been initially screened out.

1.7 Screening for resistance to herbicide
We had the NIL lines sown in the nursery field to be growing at 2 leaf stage, and then uniform spraying herbicide glyphosate isopropylamine, using Ganxiang B as control, the number of alive seedlings were investigated in 10 days after spraying. The results showed that the seedlings began to be suffering in the next day, and after 10 days later, the control's seedlings of Ganxiang B were dead at all. Three NIL lines derived from 11 combinations had some surviving seedlings, of which total of nine individuals were identified to have some resistance to herbicide.

2 Discussion
In this study, a batch of wild rices come from different sources of as the donors, elite maintainer Ganxiang B as the receptor, a large number of crossing and backcrossing were carried out to build near-isogenic introgression lines with the genetic background of, elite maintainer, having achieved a large number of materials with different phenotypic characteristics of wild rice. Regarding the large-scale screening process for rice favorable traits, we considered the effects of selections were dependent on whether the selection criteria could extreme match the favorable goals. For example, Conditions for screening drought tolerance in rice blooming growth stage must be ensured under the continuing high temperature and drought, was well as the control of drought-susceptive material has to appear leaf curling, wilting, basically not being restored. Conditions for screening the low-nitrogen tolerant materials must to be guaranteed to applying no nitrogen fertilizer that will facilitate to screen low tolerant materials, otherwise if nitrogen fertilizer were applied too much there would be very small differences exhibiting among the lines. Screening high-temperature tolerant materials must be required under the condition of a high temperature at 35℃ or more during the rice heading stage. It is necessary for screening the flooding tolerant materials to completely submerged rice seedlings as well as to ensure that the flooding condition was maintained even in the process of water changing, the majority of seedlings died at all. The screening results also need to be further validated under the controllable conditions. In this study, we acquired a batch of potential breeding values and prospective use of new germplasms though a large scale screening the favorable traits, such as drought, low nitrogen, high temperature, blast, flooding, herbicides, etc. Using these favorable germplasms could enhance the characters of Ganxiang B including abilities of anti-abiotic stresses, capacity of low nitrogen tolerance and disease resistance, it would be the foundations for breeding the cultivar of green super rice. Also, in the view of the screening results for building a large-scale NILs, we have screened most of the improved target traits from the NILs derived from the wild rice as donors, which further indicated that there should be many favorable target genes in wild rice.

3 Materials and Methods
3.1 Recurrent parent of Ganxiang B as genetic background
Gangxiang B, an elite maintainer of three-line hybrid rice developed by Rice Institute of Jiangxi Academy of Agricultural Sciences, bred by multiple crossing among five different sources of rice germplsams of 03B, IR58025B, Jing 23B, Xinlu B and Jiangnong B. The ganxiang B has excellent figures with thickened stem, strong individual strengths and with 12~13 leaves of main stem. In the mature stage, functional leaves keep vigorous, no pre-aging, good leaf color transiting. The plant architecture is moderate tight with pendulous panicle under the flag leaf, strong tillering ability, well developed root system, the flag leaf erecting, large spike with more grains, dense grain setting and 22 cm of panicle in length, the average number of 150 grains per panicle, Kilo-seed weight 28~29 g, not easy shattering, 3.3 of ratio of length to width, aromatic flavor, stigma extruding, high content of straight chain starch and susceptible to temperature. F1 hybrid mating with the genetic background of Ganxiang B has a strong early maturity but normal ability to high temperature tolerance.

3.2 Donor parents
Eleven different sources of wild rice germplasms, named Dongxiang wild rice, Guangxi wild rice, Jiangyong wild rice, Chaling wild rice, Yuanjiang common wild rice Hehuatang No 2, 4 and 10. Four wild rice germplasms namely 06YD2, 06YD5, 06YD6, and 06YD12 provided by Huazhong Agricultural University.

3.3 Screening methods for target characters
Screening for drought tolerance: Lines was sown by backcross combinations in upland of Nanchang. Seedlings were nursing in wet field, the growing season was arranged in hot dry season (7-8 months) prior to transplanting. It began to drain water off prior to weeding and fertilizing after transplanted seedlings reviving until the stages of heading and maturity. Both of drought-resistant varieties of “Brazilian upland rice and Jinggang upland rice No.1” and the recipient parent "Ganxiang B" were employed as the controls, of which began to be curling as criteria for selection, seed rate and other traits.

Screening for tolerance to low nitrogen: Transplanting 24 individuals per lines followed line by line, fertilizer of urea 75 kg per hectare was topdressed in the stage of seedling reviving after transplanting, on any additional fertilizer applied. Growing traits were recorded including plant biomass, tiller numbers, panicle length, grain number etc. The receptor of Ganxiang B used as control, the line that most of selection criteria was better than that of the control lines was screening out as low nitrogen-tolerant line.

Screening for rice blast resistance: All lines for blast identifying were planted in the Fengtian village of Kenzhi Station in Jingguangshang where was heavy blast-prone area, where is a national station of southern regions of rice and Jiangxi provincial station for rice blast identification. Incidence of leaf blast and panicle blast occurs severe every year. In Seeds sown in late May, transplanting followed by lines, 15 individuals per lines transplanted with in density of 16.5 cm×13.2 cm. Using LijiangXintuanHeigu and recovering line 752 were employed as references, the appropriate urea was applied. leaf blast was investigated during the vegetative growing stage and panicle blast were investigated in the early ripening stage based on the national standard GB/T 15970, grade 1~3 was classified as leaf blast resistance, grade 4~9 was classified as a susceptible to rice blast, incidence rate less than or equal to 5% stands for resistance to panicle blast while greater than 5% stands for susceptible to panicle blast.

Screening for high-temperature tolerance: Transplanting 24 individuals per lines followed line by line, The growing stages of booting, heading and filling were matched in the hot season, labeling spike blooming in hot temperature, after 20 days labeling the seed setting rate was investigated, if the seed setting rate was more than 70% that the line was considered as high temperature tolerant line.

Screening for flooding resistance: Seeds mixed by lines were sown in the deep bucket with soil placed outdoors, growing to 1 leaves with new emerging leaflet, seedlings were submerged, refreshing the water (filling water instead of pouring water) in bucket every three days to prevent possible toxic to seedling due to fermentation happening, after 7 days later draining water out, the alive seedlings were investigated in additional 5 days later in the outdoor.

Screening for herbicide resistance: Seeds mixed by lines were sown in the nursing field prior to soaking and germinating. The seedling with 2 leaves was uniform spraying herbicide glyphosate isopropylamine salt (41%), and re-spraying three days later after first spraying. Ganxiang B used as a control, the number of alive seedlings were investigated after 10 days of re-spraying.

Authors' contributions
Yeqing Xiao is the person who conducted experimental design and performances and wrote the manuscript. Xiaoyan Wu, Lanxiang Hu, Wenchang Wu, Shiyou Luo and Wei Deng involved in doing research work. Dazhou Chen is principal investigator who conceived the project and supervised the experimental designing, data analyzing, and paper writing and revising. All authors have read and agreed the final context of manuscript.

This research was jointly funded by 948 project (2006-G1) of the Ministry of Agriculture, 863 project (Innovations of drought-resistant germplasm of rice) and Project (51587-13) of the Bill & Melinda Gates Foundation. Authors thank the two anonymous peer reviewers for their critical comments and helpful revising. Thanks Dr. Xianhua Shen of our Institute for his revising this English version.

Li Z.K., 2005, Strategies for molecular rice breeding in China, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 3(5): 603-608

Luo L.J., 2005, Development of near isogenic introgression lines and molecular breeding on rice, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 3(5): 609-612

Xu J.L., Gao Y.M., Fu B.Y., and Li ZK., 2005, Identification and screening of favorable genes from rice germplasm in backcross introgression populations, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 3(5): 619-628

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