Complete Genome Resource of a Hypervirulent Xanthomonas oryzae pv. oryzae Strain YNCX Isolated from Yunnan Plateau Japonica Rice.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight (BLB), is one of the most destructive bacterial pathogens in rice production worldwide. Although several complete genome sequences of Xoo strains have been released in public databases, they are mainly isolated from low-altitude indica rice cultivating areas. Here, a hypervirulent strain, YNCX, isolated from the high-altitude japonica rice-growing region in Yunnan Plateau, was used to extract genomic DNA for PacBio sequencing and Illumina sequencing. After assembly, a high-quality complete genome consisting of a circular chromosome and six plasmids was generated. The genome sequence of YNCX provides a valuable resource for high-altitude races and enables the identification of new virulence TALE effectors, contributing to a better understanding of rice-Xoo interactions.

Genetic differentiation and restricted gene flow in rice landraces from Yunnan, China: effects of isolation-by-distance and isolation-by-environment.

BACKGROUND: Understanding and identifying the factors responsible for genetic differentiation is of fundamental importance for efficient utilization and conservation of traditional rice landraces. In this study, we examined the spatial genetic differentiation of 594 individuals sampled from 28 locations in Yunnan Province, China, covering a wide geographic distribution and diverse growing conditions. All 594 accessions were studied using ten unlinked target genes and 48 microsatellite loci, and the representative 108 accessions from the whole collection were sampled for resequencing. RESULTS: The genetic diversity of rice landraces was quite different geographically and exhibited a geographical decline from south to north in Yunnan, China. Population structure revealed that the rice landraces could be clearly differentiated into japonica and indica groups, respectively. In each group, the rice accessions could be further differentiated corresponded to their geographic locations, including three subgroups from northern, southern and middle locations. We found more obvious internal geographic structure in the japonica group than in the indica group. In the japonica group, we found that genetic and phenotypic differentiation were strongly related to geographical distance, suggesting a pattern of isolation by distance (IBD); this relationship remained highly significant when we controlled for environmental effects, where the likelihood of gene flow is inversely proportional to the distance between locations. Moreover, the gene flow also followed patterns of isolation by environment (IBE) whereby gene flow rates are higher in similar environments. We detected 314 and 216 regions had been differentially selected between Jap-N and Jap-S, Ind-N and Ind-S, respectively, and thus referred to as selection signatures for different geographic subgroups. We also observed a number of significant and interesting associations between loci and environmental factors, which implies adaptation to local environment. CONCLUSIONS: Our findings highlight the influence of geographical isolation and environmental heterogeneity on the pattern of the gene flow, and demonstrate that both geographical isolation and environment drives adaptive divergence play dominant roles in the genetic differentiation of the rice landraces in Yunnan, China as a result of limited dispersal.

Genomic analyses reveal selection footprints in rice landraces grown under on-farm conservation conditions during a short-term period of domestication.

Traditional rice landraces grown under on-farm conservation conditions by indigenous farmers are extremely important for future crop improvement. However, little is known about how the natural selection and agriculture practices of indigenous farmers interact to shape and change the population genetics of rice landraces grown under on-farm conservation conditions during the domestication. In this study, we sequenced DNA from 108 core on-farm conserved rice landraces collected from the ethnic minority regions of Yunnan, China, including 56 accessions collected in 1980 and 52 accessions collected in 2007 and obtained 2,771,245 of credible SNPs. Our findings show that most genetic diversity was retained during the 27 years of domestication by on-farm conservation. However, SNPs with marked allele frequency differences were found in some genome regions, particularly enriched in genic regions, indicating changes in genic regions may have played a much more prominent role in the short-term domestication of 27 years. We identified 186 and 183 potential selective-sweep regions in the indica and japonica genomes, respectively. We propose that on-farm conserved rice landraces during the short-term domestication had a highly polygenic basis with many loci responding to selection rather than a few loci with critical changes in response to selection. Moreover, loci affecting important agronomic traits and biotic or abiotic stress responses have been particularly targeted in selection. A genome-wide association study identified 90 significant signals for six traits, 13 of which were in regions of selective sweeps. Moreover, we observed a number of significant and interesting associations between loci and environmental factors, which implies adaptation to local environment. Our results provide insights into short-term evolutionary processes and shed light on the underlying mechanisms of on-farm conservation.

Genetic structure and isolation by altitude in rice landraces of Yunnan, China revealed by nucleotide and microsatellite marker polymorphisms.

Rice landraces, a genetic reservoir for varietal improvement, are developed by farmers through artificial selection during the long-term domestication process. To efficiently conserve, manage, and use such germplasm resources, an understanding of the genetic structure and differentiation of local rice landraces is required. In this study, we analyzed 188 accessions of rice landraces collected from localities across an altitudinal gradient from 425 to 2, 274 m above sea level in Yunnan Province, China using ten target genes and 48 SSR markers. We detected clear differentiation of the rice landraces into indica and japonica groups and further separation of the accessions in each group into two subgroups according to altitude, including a lower altitude subgroup and higher altitude subgroup. The AMOVA results showed significant genetic differentiation among altitude zones at SSRs and most genes, except Os1977 and STS22. We further determined that differentiation among landrace populations followed a model of isolation by altitude, in which gene flow was higher among populations at similar altitude levels than across different altitude levels. Our findings demonstrated that both adaptation to altitude and altitude-dependent gene flow played key roles in the genetic differentiation of rice landraces in Yunnan, China.

Natural variation in CTB4a enhances rice adaptation to cold habitats.

Low temperature is a major factor limiting rice productivity and geographical distribution. Improved cold tolerance and expanded cultivation to high-altitude or high-latitude regions would help meet growing rice demand. Here we explored a QTL for cold tolerance and cloned the gene, CTB4a (cold tolerance at booting stage), encoding a conserved leucine-rich repeat receptor-like kinase. We show that different CTB4a alleles confer distinct levels of cold tolerance and selection for variation in the CTB4a promoter region has occurred on the basis of environmental temperature. The newly generated cold-tolerant haplotype Tej-Hap-KMXBG was retained by artificial selection during temperate japonica evolution in cold habitats for low-temperature acclimation. Moreover, CTB4a interacts with AtpB, a beta subunit of ATP synthase. Upregulation of CTB4a correlates with increased ATP synthase activity, ATP content, enhanced seed setting and improved yield under cold stress conditions. These findings suggest strategies to improve cold tolerance in crop plants.

Diachronic analysis of genetic diversity in rice landraces under on-farm conservation in Yunnan, China.

KEY MESSAGE: Diachronic analysis showed no significant changes in the level of genetic diversity occurred over the past 27 years' domestication, which indicated genetic diversity was successfully maintained under on-farm conservation. Rice (Oryza sativa L.) is one of the earliest domesticated crop species. Its genetic diversity has been declining as a result of natural and artificial selection. In this study, we performed the first analysis of the levels and patterns of nucleotide variation in rice genomes under on-farm conservation in Yunnan during a 27-year period of domestication. We performed large-scale sequencing of 600 rice accessions with high diversity, which were collected in 1980 and 2007, using ten unlinked nuclear loci. Diachronic analysis showed no significant changes in the level of genetic diversity occurring over the past 27 years' domestication, which indicated genetic diversity was successfully maintained under on-farm conservation. Population structure revealed that the rice landraces could be grouped into two subpopulations, namely the indica and japonica groups. Interestingly, the alternate distribution of indica and japonica rice landraces could be found in each ecological zone. The results of AMOVA showed that on-farm conservation provides opportunities for continued differentiation and variation of landraces. Therefore, dynamic conservation measures such as on-farm conservation (which is a backup, complementary strategy to ex situ conservation) should be encouraged and enhanced, especially in crop genetic diversity centers. The results of this study offered accurate insights into short-term evolutionary processes and provided a scientific basis for on-farm management practices.

[On-farm conservation and utilization of paddy rice, wheat and maize landrace varieties in 15 unique ethnic groups in Yunnan, China].

On-farm conservation and utilization of crop landraces have been proposed as means of conserving plant germplasm, in contrast to ex situ germplasm conservation, but little is known about the effectiveness of this approach. This paper reports the findings from a survey conducted in 15 unique ethnic groups of the Yunnan Province on the conservation and utilization of paddy rice, wheat (including wheat, barley, oats, and rye) and maize landrace varieties through participatory rural appraisal (PRA) and questionnaires. The surveyed regions covered 306 villages (the village group) from 237 administrative villages in 124 towns (township) distributed in 36 counties of 11 prefectures (city) in Yunnan Province. The survey showed that 44.8%, 77.5%, and 37.3% of the visited villages had lost their paddy rice, wheat, and maize landraces, respectively. A total of 901 landraces were collected, including 371 rice, 119 wheat, and 411 maize varieties, respectively. There were 2.9 on-farm varieties on average per village, 3.3, 8.0 and 5.2 varieties on average per 100 households, 1 000 peasants and 100 Ha farmland areas, respectively. Among the 306 villages, two villages (Laomian and Qingkou) maintained the highest crop diversity with 18 varieties (including 10 rice and 8 maize varieties) and 14 paddy rice varieties. Also, on-farm varieties in different ethnic groups varied significantly from each other, ranging from 16 to 120 varieties per group. The diversity of paddy rice varieties was the highest, ranging from 1 to 72, and the diversity was mainly distributed in southern, southwest Yunnan of tropical, subtropical ethnic regions. The wheat varieties ranged from 0 to 47 and distributed in northern, northwest Yunnan of high altitude, temperature, and cold ethnic regions. The maize varieties ranged from 4 to 40. These patterns of variation in on-farm varieties are directly associated with traditional culture and custom for the ethnic minorities and reflect the lack of improved varieties for the local special environments. These findings confirm the role of on-farm conservation in plant genetic resources in China and provide baseline information for better on-farm conservation and utilization of plant germplasm in China.

[Effects of different ecological conditions on grain quality and RVA profile of japonica rice from Yunnan of China and Korea].

Taking sixteen japonica rice varieties from Yunnan of China and Korea growing at three locations with different altitudes in Yunnan Plateau as test materials, this paper studied the effects of different ecological conditions on their grain quality, including brown rice length (BRL), brown rice width (BRW), length-width ratio (L/W), chalky rate (CR), whiteness (WH), amylose content (AC), protein content (PC), and alkali digestion value (ADV), and their starch RVA profile, including peak viscosity (PKV), hot viscosity (HTV), final viscosity (FLV), breakdown viscosity (BDV), setback viscosity (SBV), consistence viscosity( CTV), peak time (PeT), and pasting temperature (PaT). Of all the test parameters, SBV had the largest coefficient of variation. The coefficients of variation of BRL, CR, AC, PC, ADV, FLV, SBV, and PeT of Yunnan varieties were higher than those of Korean varieties, while the BRW, L/W, WH, PKV, HTV, BDV, CTV, and PaT of Korean varieties had higher coefficients of variation than those of Yunnan varieties. With increasing altitude, the BRL, BRW, L/W, WH, AC, ADV, FLV, and CTV of Yunnan varieties, and the BRL, BRW, WH, PC, PKV, HTV and BDV of Korean varieties decreased significantly, while the CR, PC, HTV, and PeT of Yunnan varieties, and the L/W, AC, ADV, SBV and CTV of Korean varieties increased significantly. The CR of Korean varieties had no obvious change. The PKV, BDV, and PaT of Yunnan varieties and the PaT of Korean varieties increased after an initial decrease, whereas the SBV of Yunnan varieties and the FLV and PeT of Korean varieties were in reverse.

Cloning and characterization of a novel avirulence gene (arp3) from Xanthomonas oryzae pv. oryzae.

A novel avirulence gene was cloned from Xanthomonas oryzae pv. oryzae strain PX0339, which is the standard representative of the Philippines race 9a. The full-length gene spans 2118 bp and encodes a protein of 705 amino acids. BLAST search in NCBI indicated that the gene belongs to avrBs3 gene family, and designated arp3 (AvrBs3-related protein 3, arp3). The central region of the arp3 contains only 5.5 copies of 102bp repeats, the smallest copy number of repeats found in avrBs3 gene family by now. Together with the repeats is heptad repeats, resembling leucine zippers. Three functional nuclear localization signals and an acidic activation domain are also found in the C-terminal region. However, the arp3 lacks of two segments in its N-terminal region, which is unique in avrBs3 gene family. Southern blotting data showed that the arp3 is present as a single-copy in genomic DNA of PX0339 and locus in plasmid clone. The arp3 could be expressed in vitro in Escherichia coli BL21 and a 128kDa fusion protein was detected by Western analysis.