Yong-Bao Pan

Highly polymorphic microsatellite dna markers for sugarcane germplasm evaluation and variety identity testing

The objective of this study was to evaluate the entire set of 221 sugarcane microsatellite (SSR) markers from the International Sugarcane Microsatellite Consortium for their utility on molecular characterization of elite U.S. germplasm. Five elite U.S. sugarcane clones were tested, including two cultivars LCP 85-384 and HoCP 96-540 from Louisiana, two cultivars CP 72-1210 and CP 85-1308 from Florida, and Green German, an active parental clone used in both states. The 5’ ends of all forward primers were labeled with the fluorescent phosphoramidite dye, FAM and PCR-amplified DNA fragments detected using a semi-automatic capillary electrophoresis system. The sizes of DNA fragments were computed accurately by running genotyping software calibrated against 16 fluorescence-labeled DNA size standard fragments. Evaluation criteria included PCR robustness, extent of the presence of irregular peaks, and the polymorphism information content (PIC). Sixty-seven SSR markers (30% of the total) were found to be highly robust, with PIC values ranging from 56% to 80%. Of these, 40 (60%) markers contained dinucleotide repeats, 11 (16%) markers contained trinucleotide repeats, and 16 (24%) markers contained composite repeats. A total of 467 alleles were scored, of which 350 were polymorphic, averaging five polymorphic alleles per marker, with their sizes ranging from 80 to 460 bp. Several of these highly polymorphic SSR markers have proven useful in sugarcane germplasm evaluation, variety identity tests, cross fidelity assessment, and polycross paternity analysis.

Genetic Analysis of Diversity within a Chinese Local Sugarcane Germplasm Based on Start Codon Targeted Polymorphism

In-depth information on sugarcane germplasm is the basis for its conservation and utilization. Data on sugarcane molecular markers are limited for the Chinese sugarcane germplasm collections. In the present study, 20 start codon targeted (SCoT) marker primers were designed to assess the genetic diversity among 107 sugarcane accessions within a local sugarcane germplasm collection. These primers amplified 176 DNA fragments, of which 163 were polymorphic (92.85%). Polymorphic information content (PIC) values ranged from 0.783 to 0.907 with a mean of 0.861. Unweighted pair group method of arithmetic averages (UPGMA) cluster analysis of the SCoT marker data divided the 107 sugarcane accessions into six clusters at 0.674 genetic similarity coefficient level. Relatively abundant genetic diversity was observed among ROC22, ROC16, and ROC10, which occupied about 80% of the total sugarcane acreage in China, indicating their potential breeding value on Mainland China. Principal component analysis (PCA) partitioned the 107 sugarcane accessions into two major groups, the Domestic Group and the Foreign Introduction Group. Each group was further divided based on institutions, where the sugarcane accessions were originally developed. The knowledge of genetic diversity among the local sugarcane germplasm provided foundation data for managing sugarcane germplasm, including construction of a core collection and regional variety distribution and subrogation.

Biplot evaluation of test environments and identification of mega-environment for sugarcane cultivars in China

Test environments and classification of regional ecological zones into mega environments are the two key components in regional testing of sugarcane cultivars. This study aims to provide the theoretical basis for test environment evaluation and ecological zone division for sugarcane cultivars. In the present study, sugarcane yield data from a three-year nationwide field trial involving 21 cultivars and 14 pilot test locations were analysed using both analysis of variance (ANOVA) and heritability adjusted-genotype main effect plus genotype-environment interaction (HA-GGE) biplot. The results showed that among the interactive factors, the GE interaction had the greatest impact, while the genotype and year interaction showed the lowest impact. Kaiyuan, Lincang and Baoshan of Yunnan, Zhangzhou and Fuzhou of Fujian, and Hechi, Liuzhou and Chongzuo of Guangxi, and Lingao of Hainan were ideal test environments with a demonstrated high efficiency in selecting new cultivars with a wide adaptability, whereas Baise of Guangxi was not. Based on HA-GGE biplot analysis, there are three ecological sugarcane production zones in China, the Southern China Inland Zone, the Southwestern Plateau Zone, and the Southern Coastal Zone. The HA-GGE biplot analysis here presents the ideal test environments and also identifies the mega-environment for sugarcane cultivars in China.

Cultivar Evaluation and Essential Test Locations Identification for Sugarcane Breeding in China

The discrepancies across test sites and years, along with the interaction between cultivar and environment, make it difficult to accurately evaluate the differences of the sugarcane cultivars. Using a genotype main effect plus genotype-environment interaction (GGE) Biplot software, the yield performance data of seven sugarcane cultivars in the 8th Chinese National Sugarcane Regional Tests were analyzed to identify cultivars recommended for commercial release. Fn38 produced a high and stable sugar yield. Gn02-70 had the lowest cane yield with high stability. Yz06-407 was a high cane yield cultivar with poor stability in sugar yield. Yz05-51 and Lc03-1137 had an unstable cane yield but relatively high sugar yield. Fn39 produced stable high sugar yield with low and unstable cane production. Significantly different sugar and cane yields were observed across seasons due to strong cultivar-environment interactions. Three areas, Guangxi Chongzuo, Guangxi Baise, and Guangxi Hechi, showed better representativeness of cane yield and sugar content than the other four areas. On the other hand, the areas Guangxi Chongzuo, Yunnan Lincang, and Yunnan Baoshan showed strong discrimination ability, while the areas Guangxi Hechi and Guangxi Liuzhou showed poor discrimination ability. This study provides a reference for cultivar evaluation and essential test locations identification for sugarcane breeding in China.

Rational regional distribution of sugarcane cultivars in China

Knowing yield potential and yield stability of sugarcane cultivars is of significance in guiding sugarcane breeding and rationalising regional distribution of sugarcane cultivars. In the present study, a heritability-adjusted genotype main effect plus genotype × environment (HA-GGE) biplot program was used to analyze the cane and sucrose yields of 44 newly released sugarcane cultivars at eight pilot test sites. The cane and sucrose yields of nine cultivars were higher than those of the control cultivar ROC22. From the perspective of cane yield, cultivars FN 40 and YZ 06–407 were well adapted to a wider range of conditions and produced relatively high cane yields in several pilot sites. From the perspective of sucrose yield, cultivars LC 03–1137, FN 38, FN 41, MT 01–77 and LC 05–136 were well adapted to a wide range of conditions and produced relatively high sucrose yields. Based on these results, three high yielding and widely adapted cultivars, namely, FN 39, LC 05–136, and YZ 05–51 were recommended for production in three major Chinese sugarcane planting areas. The results will provide a theoretical basis for recommending the effective use and rational regional distribution of sugarcane cultivars in China.

Biogeographical Variation and Population Genetic Structure of Sporisorium scitamineum in Mainland China: Insights from ISSR and SP-SRAP Markers

A total of 100 Sporisorium scitamineum isolates were investigated by inter simple sequence repeat (ISSR) and single primer-sequence related amplified polymorphism (SP-SRAP) markers. These isolates were clearly assorted into three distinct clusters regardless of method used: either cluster analysis or by principal component analysis (PCA) of the ISSR, SP-SRAP, or ISSR + SP-SRAP data set. The total gene diversity (H t) and gene diversity between subpopulations (H s) were estimated to be 0.34 to 0.38 and 0.22 to 0.29, respectively, by analyzing separately the ISSR and SP-SRAP data sets, and to be 0.26–0.36 by analyzing ISSR + SP-SRAP data set. The gene diversity attributable to differentiation among populations (G st) was estimated to be 0.35 and 0.22, and the gene flow (Nm) was 0.94 and 1.78, respectively, when analyzing separately ISSR and SP-SRAP data set, and was 0.27 and 1.33, respectively, when analyzing ISSR + SP-SRAP data set. Our study showed that there is considerable genetic variation in the analyzed 100 isolates, and the environmental heterogeneity has played an important role for this observed high degree of variation. The genetic differentiation of sugarcane smut fungus depends to a large extent on the heterogeneity of their habitats and is the result of long-term adaptations of pathogens to their ecological environments.

Photosynthetic and Canopy Characteristics of Different Varieties at the Early Elongation Stage and Their Relationships with the Cane Yield in Sugarcane

During sugarcane growth, the Early Elongation stage is critical to cane yield formation. In this study, parameters of 17 sugarcane varieties were determined at the Early Elongation stage using CI-301 photosynthesis measuring system and CI-100 digital plant canopy imager. The data analysis showed highly significant differences in leaf area index (LAI), mean foliage inclination angle (MFIA), transmission coefficient for diffused light penetration (TD), transmission coefficient for solar beam radiation penetration (TR), leaf distribution (LD), net photosynthetic rate (PN), transpiration rate (E), and stomatal conductance (GS) among sugarcane varieties. Based on the photosynthetic or canopy parameters, the 17 sugarcane varieties were classified into four categories. Through the factor analysis, nine parameters were represented by three principal factors, of which the cumulative rate of variance contributions reached 85.77%. A regression for sugarcane yield, with relative error of yield fitting less than 0.05, was successfully established: sugarcane yield = −27.19 − 1.69 × PN + 0.17 ×  E + 90.43 × LAI − 408.81 × LD + 0.0015 × NSH + 101.38 ×  D (R 2 = 0.928**). This study helps provide a theoretical basis and technical guidance for the screening of new sugarcane varieties with high net photosynthetic rate and ideal canopy structure.

Application of Molecular Markers in Sugarcane Germplasm Innovation and Breeding: New Germplasm with Cytoplasm from Saccharum spontaneum

All current sugarcane cultivars (Saccharum hybrids spp.) are interspecific hybrids of S. officinarum, S. robustum, and S. spontaneum that bear the same cytoplasm of S. officinarum. Until the end of twentieth century, S. spontaneum was exclusively used as male parents to confer such traits as vigor, ratoon ability, and disease and insect resistance. There was no report on S. spontaneum being used as female parents, due to S. spontaneum being regulated noxious weeds with substantial self-fertilization and vigorous rhizomes. This situation changed when two series of innovative crosses (S. spontaneum 9 elite cultivars) were made in 1997 and 2001 at the USDA-ARS, SRU. Flowers of S. spontaneum were pretreated by trimming off both dehisced and immature florets, immersing in 45 C (or 50 C) circulating water bath for 10 (or 5) min, and being placed underneath the flowers of elite varieties. The 1997 cross was made between S. spontaneum clone Djatiroto and cultivar LCP 85-384 (Pan et al. 2004). The 2001 crosses were made between ten S. spontaneum clones and six elite varieties (Pan et al. 2006). One F1 progeny from the cross (SES 234A 9 LCP 85-384) survived a hard frost in March 2003 and was commercially released as an energy cane cultivar Ho 02-113. From the 1997 cross, ten F1 progenies were selected based on DNA marker and phenotypic evaluations. Their authenticity was further confirmed by SSR fingerprinting. One progeny, US 99-43, produced the largest stalks with the highest Brix values and was chosen for further improvement. Three cycles of backcrossing, field evaluation, and selection were completed. Five BC2 progenies were selected in 2008. One BC2 progeny, Ho 08-9504, produced eight large stalks and a Brix of 23.8. It never flowered in Louisiana, but flowers readily in Florida. A BC3 backcross (Ho 08-9504 9 HoCP 04-852) was made at Canal Point, FL; 216 BC3 progenies were planted in the field in 2015, of which 36 were advanced to first-line trials in 2016. Seven of the 36 BC3 progenies were advanced to second-line trials in 2017 that were free of diseases and borers, produced 11–20 stalks with 25–32 mm diameter and 19.6–22.4 Brix. Pedigree indicated that these seven BC3 progenies may inherit nuclear genes from S. spontaneum, S. robustum, and Erianthus. Availability of these S. spontaneum cytoplasm-containing BC3 progenies may enhance genetic diversity analysis of Saccharum germplasm and enable sugarcane breeders to explore the possible contribution of S. spontaneum cytoplasm in the development of new sugarcane cultivars.

A Comparative Study of Three Detection Techniques for Leifsonia xyli Subsp. xyli, the Causal Pathogen of Sugarcane Ratoon Stunting Disease

The ratoon stunting disease (RSD), caused by the bacterium Leifsonia xyli subsp. xyli (Lxx), is one of the most economically devastating diseases impacting sugarcane. RSD causes significant yield losses and variety degradation. Diagnosis of RSD is challenging because it does not exhibit any discernible internal and external symptoms. Moreover, the Lxx bacteria are very small and difficult to isolate, cultivate, and detect. In this study, conventional polymerase chain reaction (PCR), real-time quantitative PCR (RT-qPCR), and Lxx-loop-mediated isothermal amplification (Lxx-LAMP) were utilized to specifically detect the presence of Lxx pathogens in the juice from Lxx-infected sugarcane stalks and an Lxx-pMD18-T recombinant plasmid. The results showed that Lxx was a highly specific causal pathogen for RSD. All three techniques provided great reproducibility, while Lxx-LAMP had the highest sensitivity. When the DNA extract from Lxx-infected sugarcane juice was used as a template, Lxx-LAMP was 10 and 100 times more sensitive than RT-qPCR and conventional PCR, respectively. When the Lxx-pMD18-T recombinant plasmid was used as a template, Lxx-LAMP was as sensitive as RT-qPCR but was 10 times more sensitive than conventional PCR. Based on the Lxx-LAMP detection system established, adding 0.4 μM loop primers (LF/LP) can accelerate the reaction and reduce the total time required. In addition, the optimal amount of Bst DNA polymerase for Lxx-LAMP reactions was determined to be 6.0 U. The results provide technical support for the detection of RSD Lxx pathogen that will help manage sugarcane RSD.