Linkage Mapping and Discovery of Candidate Genes for Drought Tolerance in Rice During the Vegetative Growth Period.

Drought is a major abiotic stress affecting crop yields. Mapping quantitative trait loci (QTLs) and mining genes for drought tolerance in rice are important for identifying gene functions and targets for molecular breeding. Here, we performed linkage analysis of drought tolerance using a recombinant inbred line population derived from Jileng 1 (drought sensitive) and Milyang 23 (drought tolerant). An ultra-high-density genetic map, previously constructed by our research team using genotype data from whole-genome sequencing, was used in combination with phenotypic data for rice grown under drought stress conditions in the field in 2017-2019. Thirty-nine QTLs related to leaf rolling index and leaf withering degree were identified, and QTLs were found on all chromosomes except chromosomes 6, 10, and 11. qLWD4-1 was detected after 32 days and 46 days of drought stress in 2017 and explained 7.07-8.19% of the phenotypic variation. Two loci, qLRI2-2 and qLWD4-2, were identified after 29, 42, and 57 days of drought stress in 2018. These loci explained 10.59-17.04% and 5.14-5.71% of the phenotypic variation, respectively. There were 281 genes within the QTL interval. Through gene functional annotation and expression analysis, two candidate genes, Os04g0574600 and OsCHR731, were found. Quantitative reverse transcription PCR analysis showed that the expression levels of these genes were significantly higher under drought stress than under normal conditions, indicating positive regulation. Notably, Os04g0574600 was a newly discovered drought tolerance gene. Haplotype analysis showed that the RIL population carried two haplotypes (Hap1 and Hap2) of both genes. Lines carrying Hap2 exhibited significantly or extremely stronger drought tolerance than those carrying Hap1, indicating that Hap2 is an excellent haplotype. Among rice germplasm resources, there were two and three haplotypes of Os04g0574600 and OsCHR731, respectively. A high proportion of local rice resources in Sichuan, Yunnan, Anhui, Guangdong and Fujian provinces had Hap of both genes. In wild rice, 50% of accessions contained Hap1 of Os04g0574600 and 50% carried Hap4; 13.51%, 59.46% and 27.03% of wild rice accessions contained Hap1, Hap2, and Hap3, respectively. Hap2 of Os04g0574600 was found in more indica rice resources than in japonica rice. Therefore, Hap2 has more potential for utilization in future drought tolerance breeding of japonica rice.

Genomic footprints of Kam Sweet Rice domestication indicate possible migration routes of the Dong people in China and provide resources for future rice breeding.

The Dong people are one of China's 55 recognized ethnic minorities, but there has been a long-standing debate about their origins. In this study, we performed whole-genome resequencing of Kam Sweet Rice (KSR), a valuable, rare, and ancient rice landrace unique to the Dong people. Through comparative genomic analyses of KSR and other rice landraces from south of the Yangtze River Basin in China, we provide evidence that the ancestors of the Dong people likely originated from the southeast coast of China at least 1000 years ago. Alien introgression and admixture in KSR demonstrated multiple migration events in the history of the Dong people. Genomic footprints of domestication demonstrated characteristics of KSR that arose from artificial selection and geographical adaptation by the Dong people. The key genes GS3, Hd1, and DPS1 (related to agronomic traits) and LTG1 and MYBS3 (related to cold tolerance) were identified as domestication targets, reflecting crop improvement and changes in the geographical environment of the Dong people during migration. A genome-wide association study revealed a candidate yield-associated gene, Os01g0923300, a specific haplotype in KSR that is important for regulating grain number per panicle. RNA-sequencing and quantitative reverse transcription-PCR results showed that this gene was more highly expressed in KSR than in ancestral populations, indicating that it may have great value in increasing yield potential in other rice accessions. In summary, our work develops a novel approach for studying human civilization and migration patterns and provides valuable genomic datasets and resources for future breeding of high-yield and climate-resilient rice varieties.

Kam Sweet Rice (Oryza sativa L.) Is a Special Ecotypic Rice in Southeast Guizhou, China as Revealed by Genetic Diversity Analysis.

Kam Sweet Rice (KSR) is a special kind of rice landrace that has been domesticated for thousands of years by the local Dong people in southeast Guizhou province, China. KSR has many distinguishing characteristics including strong fragrance; high resistance to diseases, pests, and adverse abiotic conditions; difficulty of threshing; and glutinous texture. There is a lack of systematic research on its genetic diversity. In this study, we analyzed the levels and patterns of genetic diversity and nucleotide variation in 1,481 rice germplasm using simple sequence repeat (SSR) markers and single nucleotide polymorphism (SNP) haplotype analysis of six unlinked nuclear loci. The accessions included 315 KSR resources from southeast Guizhou, 578 rice landraces from six rice-growing ecological zones in Guizhou, 546 rice landraces from nine provinces around Guizhou, and 42 wild rice sources. Genetic diversity and heterozygosity of KSR were both low, and thus KSR might be close to a pure rice line. Population structure analysis showed that KSR was isolated into a single type of rice, which had a large genetic distance and a unique genetic background compared to the local varieties in Guizhou province, indicating that KSR is a special rice ecotype. Haplotype analysis of the target genes showed that the population of KSR was rich in haplotypes for resistance to bacterial blight (Xa23) and rice blast (Pid3), and identified unique haplotypes that were different from those of the six rice ecotypes in Guizhou. This study shows that KSR is an excellent rice germplasm resource, provides important information for the improvement and utilization of rice landraces, and serves as a reference for formulating effective rice conservation measures.

Status and factors influencing on-farm conservation of Kam Sweet Rice (Oryza sativa L.) genetic resources in southeast Guizhou Province, China.

BACKGROUND: Kam Sweet Rice (KSR) is a special kind of rice landrace that has been cultivated for thousands of years in the borders of Guizhou, Hunan, and Guangxi Provinces of China, and is mainly distributed in southeast Guizhou Province of China currently. KSR has many unique qualities, including strong resistance to diseases, pests, and adverse abiotic conditions, difficulty of threshing, and well glutinous features. KSR germplasm resources are an indispensable material and cultural symbol in the production and daily life and customs of the Dong people. Related studies showed that numerous traditional KSR varieties and cultivation area of KSR decreased sharply from the Qing dynasty to 2015, but many KSR varieties are still conserved in Dong villages of southeast Guizhou Province compared to other areas. However, the number of KSR varieties that are conserved on farms in southeast Guizhou Province and factors influencing the erosion and conservation of KSR genetic resources is unclear. Therefore, this study was an on-farm conservation investigation of KSR genetic resource in China's major KSR producing areas-Liping, Congjiang, and Rongjiang counties in Guizhou Province and influencing factors analysis of KSR abandonment and conservation. METHODS: The information of KSR conservation status and variety characteristics, typical villages, Dong's cultural customs, and factors influencing KSR abandonment and conservation was obtained using ethno-biology methods, mainly through field research interviews, including participatory observation, semi-structured interviews, key informant interviews, focus group discussions, and cultural anthropology. The altitude, plant height, awn color and length, hull color, and rice color of 156 KSR accessions in 28 villages were recorded. The variety quantity and cultivation area of KSR were investigated in 33 ethnic villages. Questionnaire surveys were conducted in typical Dong villages to obtain local farmers' attitudes toward cultivation and protection of KSR. We randomly selected 26 farmers from Sizhai village and 30 farmers from Huanggang village and chose 3 social characteristics including age, gender, and education levels of farmers, and adopted the method of face-to-face interviewing to complete the questionnaires. Then, we analyzed the correlation and determined the significance between farmers with different social characteristics and farmers' attitudes to KSR development and protection using SPSS 17.0 software. RESULTS: (1) On-farm conservation status of KSR: a total of 156 KSR varieties were collected from 28 ethnic minority villages from 13 townships (accounting for 21% of three counties) in Liping, Congjiang, and Rongjiang counties. KSR accessions accounted for more than 90% of local rice varieties in each village. According to local farmers, although the quantity of KSR varieties decreased more than 50% in the investigated villages compared to the past 10-20 years, some Dong villages have still cultivated KSR, accounting for more than 50% of the rice field area in 10 villages. This result showed that many KSR varieties are still conserved by in Dong villages, and these KSR varieties have a high genetic diversity of phenotypes. (2) Typical villages investigation: the cultivation area of KSR in Congjiang was the highest, 6.7 times larger than Liping and eight times larger than Rongjiang. In addition, the cultivation area of KSR in Dong villages was larger than that in other ethnic villages, and villages that had a higher planting area of KSR had more KSR accessions. (3) Farmers' attitude toward the development and conservation of KSR: Dong farmers hold the negative attitudes concerning the development of KSR resources, but they thought it is necessary to protect KSR landraces. Especially, a high level of education and female, young, and old farmers played more important roles in the cultivation and protection of KSR. CONCLUSIONS: Until now, some Dong ethnic villages have still cultivated KSR for thousands of years in Qiandongnan area, although the number of varieties and the planting area of KSR have been greatly reduced. In addition, ethnic traditional culture and social customs were the main influencing factors of KSR conservation; economic, management, and policy factors were the main influencing factors of KSR abandonment. Through the analysis of the correlation between farmers with different social characteristics and their attitudes toward the cultivation, reasons for conservation and abandonment, development tendency, and protection of KSR, we found that a high level of education and female, young, and old farmers play more important role in the cultivation and protection of KSR. Therefore, in order to promote the protection and sustainable utilization of KSR, it is necessary to build on-farm conservation of KSR and improve the position of female farmers and the education level of young people, and encourage the old people to educate the middle-aged to conserve and protect KSR as well as Dong's traditional culture and social customs. This study is of great significance to promote better protection and optimal utilization of KSR and enable the public, government, and related researchers pay more attention to conserving ethnic traditional cultures.