Y. B. Pan

An assessment of the phylogenetic relationship among sugarcane and related taxa based on the nucleotide sequence of 5S rRNA intergenic spacers.

Abstract5S rRNA intergenic spacers were amplified from two elite sugarcane (Saccharumhybrids) cultivars and their related taxa by polymerase chain reaction (PCR) with 5S rDNA consensus primers. Resulting PCR products were uniform in length from each accession but exhibited some degree of length variation among the sugarcane accessions and related taxa. These PCR products did not always cross hybridize in Southern blot hybridization experiments. These PCR products were cloned into a commercial plasmid vector PCR™ 2.1 and sequenced. Direct sequencing of cloned PCR products revealed spacer length of 231–237u2009bp for S. officinarum, 233–237 for sugarcane cultivars, 228–238u2009bp for S. spontaneum, 239–252u2009bp for S. giganteum, 385–410u2009bp for Erianthusspp., 226–230u2009bp for Miscanthus sinensisZebra, 206–207u2009bp for M. sinensisIMP 3057, 207–209u2009bp for Sorghum bicolor, and 247–249u2009bp for Zea mays. Nucleotide sequence polymorphism were found at both the segment and single nucleotide level. A consensus sequence for each taxon was obtained by Align X. Multiple sequences were aligned and phylogenetic trees constructed using Align X, CLUSTAL and DNAMAN programs. In general, accessions of the following taxa tended to group together to form distinct clusters: S. giganteum, Erianthusspp., M. sinensis, S. bicolor, and Z. mays. However, the two S. officinarumclones and two sugarcane cultivars did not form distinct clusters but interrelated within the S. spontaneumcluster. The disclosure of these 5S rRNA intergenic spacer sequences will facilitate marker-assisted breeding in sugarcane.

Genetic analysis of the sugarcane (Saccharum spp.) cultivar ‘LCP 85-384’. I. Linkage mapping using AFLP, SSR, and TRAP markers

Sugarcane hybrids are complex aneu-polyploids (2nxa0=xa0100–130) derived from inter-specific hybridization between ancestral polyploid species, namely S. officinarum L. and S. spontaneum L. Efforts to understand the sugarcane genome have recently been enhanced through the use of new molecular marker technologies. A framework genetic linkage map of Louisiana’s popular cultivar LCP 85-384 was constructed using the selfed progeny and based on polymorphism derived from 64 AFLP, 19 SSR and 12 TRAP primer pairs. Of 1,111 polymorphic markers detected, 773 simplex (segregated in 3:1 ratio) and 182 duplex (segregate in 77:4 ratio) markers were used to construct the map using a LOD value of ≥4.0 and recombination threshold of 0.44. The genetic distances between pairs of markers linked in the coupling phase was computed using the Kosambi mapping function. Of the 955 markers, 718 simplex and 66 duplex markers were assigned to 108 co-segregation groups (CGs) with a cumulative map length of 5,617xa0cM and a density of 7.16xa0cM per marker. Fifty-five simplex and 116 duplex markers remained unlinked. With an estimated genome size of 12,313xa0cM for LCP 85-384, the map covered approximately 45.6% of the genome. Forty-four of the 108 CGs were assigned into 9 homo(eo)logous groups (HGs) based on information from locus-specific SSR and duplex markers, and repulsion phase linkages detected between CGs. Meiotic behavior of chromosomes in cytogenetic studies and repulsion phase linkage analysis between CGs in this study inferred the existence of strong preferential chromosome pairing behavior in LCP 85-384. This framework map marks an important beginning for future mapping of QTLs associated with important agronomic traits in the Louisiana sugarcane breeding programs.

An assessment of the genetic diversity within a collection ofSaccharum spontaneum L. with RAPD-PCR

A local collection of 33Saccharum spontaneum L. clones and two sugarcane cultivars (LCP 82-89 and LCP 85-384) were assessed for genetic variability using random amplified polymorphic DNA (RAPD)-PCR. A total of 157 polymorphic RAPD-PCR bands were scored with 17 primers. The number of RAPD-PCR products per primer ranged from four to 16. The data were analyzed with two multivariate analysis software programs, NTSYSpc and DNAMAN®. Although these two programs yielded similar results, a bootstrapped phylogenetic tree could only be generated with the DNAMAN® software. A substantial degree of genetic diversity was found within the localS. spontaneum collection. Pairwise genetic homology coefficients ranged from 65% (SES, 196/Tainan 2n = 96) to 88.5% (IND 81-80/IND 81-144). LCP 82-89 and LCP 85-384 shared a greater similarity (82%) than either was to any clone ofS. spontaneum (ranging from 60.5 to 75.2%). The 33S. spontaneum clones were assigned to eight groups independent of their geographic origin or morphology, while the two sugarcane cultivars were assigned to the ninth group. All but two pairs ofS. spontaneum clones could be distinguished by a single RAPD primer OPBB-02. The use of a second primer, either OPBE-04 or Primer 262, separated allS. spontaneum clones. One amplification product from the RAPD primer OPA-11, OPA-11-336, proved to be cultivar-specific and has been adopted for use in our breeding program. Information from this study would help conserve the genetic diversity ofS. spontaneum.

Genetic diversity of North American and Old World Saccharum assessed by RAPD analysis

Saccharum (= Erianthus) native to North America is an untapped germplasm for genetic improvement of sugarcane (Saccharum spp. hybrids). There are five species and two varieties native to North America: S. alopecuroideum, S. baldwinii, S. brevibarbe vars. brevibarbe and contortum, S. coarctatum, and S. giganteum. There are three cytotypes of S. giganteum (2n = 30, 60, 90), and they overlap in gross morphology. Our objectives were to compare genetic diversity of North American and Old World members of Saccharum. Bulked DNA for five North American species, three Old World Erianthus spp. sect. Ripidium clones, and five sugarcane cultivars was tested by PCR with 13 RAPD primers. A total of 283 repeatable RAPD bands was scored for the nine taxa. Genetic distance coefficients ranged from 0.365 to 0.767 indicating substantial diversity among taxa. Taxa were assigned to one of three cluster groups: 1) S. baldwinii, S. brevibarbe var. contortum, S. coarctatum, and S. giganteum 2n = 90; 2) S. gig anteum 2n = 30 and 2n = 60, S. alopecuroideum, and sugarcane cultivars; and 3) Old World Erianthus spp. The RAPD analysis indicated that sugarcane was genetically more similar to North American Saccharum than it was to Old World Erianthus. This was unexpected given that North American Saccharum is geographically, cytologically, morphologically, and possibly reproductively isolated from Old World Erianthus and sugarcane. The data support the taxonomic separation of cytotypes of S. giganteum.

SSR marker-based analysis of genetic relatedness among sugarcane cultivars (Saccharum spp. hybrids) from breeding programs in China and other countries

AbstractCapillary electrophoresis-based molecular genotyping was conducted on 35 sugarcane cultivars (Saccharum spp. hybrids) and 5 clones of related wild species with 20 polymorphic SSR DNA markers. A total of 251 alleles were identified with 248 alleles displaying varying degrees of polymorphism and the remaining three alleles being monomorphic. The total number of alleles by any SSR marker varied from as few as 7 to as many as 18, with an average of 12.5 alleles per marker. Diversity index (DI = 1 −

Quick detection of Leifsonia xyli subsp. xyli by PCR and nucleotide sequence analysis of PCR amplicons from Chinese Leifsonia xyli subsp. xyli isolates

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Registration of ‘Ho 95-988’ Sugarcane

) for these SSR markers ranged from 0.71 to 0.91, with a mean of 0.83. A composite parameter NDI, representing a product of the number of alleles (N) and DI, is an indicator on the general usefulness of a DNA marker. Ten SSR markers, namely, mSSCIR43, mSSCIR66, SMC119CG, SMC24DUQ, SMC278CS, SMC31CUQ, SMC336BS, SMC597CS, SMC703BS, and SMC851MS, have NDI values of greater than 12 in comparison to less than 10 from the rest markers, indicating that these 10 SSR markers provide much information for genotyping the 40 clones. A finding to minimize stutters and minus-Adenine peaks gave a guideline for the selection of best SSR markers for other SSR research. The 35 cultivars were clustered into five groups based on pairwise similarity coefficient values and their relationships to the wild species were demonstrated. Inclusion of CP67-412, CP72-1210, N21, N27, and S. officinarum clone Badila into the cultivar groups is due to the fact that these clones have been extensively used as parental material in sugarcane breeding programs. The results are in general agreement with the evolutionary course of the sugarcane cultivars that the order of contributing species in modern sugarcane cultivars is S. officinarum, S. spontaneum, S. robustum Brandes et. Jesw.ex., S. sinense Roxb., and S. barberi Jesweit. The accordance of molecular results with recorded evolution of sugarcane verified the fidelity and usefulness of these 20 SSR markers in progeny selection and allele transmission study in this aneu-polyploidy crop.

Analysis of Primer-Derived, Nonspecific Amplification Products in RAPD-PCR

New lines of Saccharum hybrids with an array of S. spontaneum cytoplasm backgrounds are reported. To expand the genetic base of sugarcane, we made eleven bi-parental crosses between ten S. spontaneum (S) and six commercial-type sugarcane (C) clones during the 2001 crossing season. Prior to crossing, all the maternal S. spontaneum inflorescences were emasculated by immersion in a 50°C circulating water bath for 5 minutes. Analysis of microsatellite fingerprints between parents and progeny allowed us to classify 1,952 progeny grown out from these crosses into four genotypic classes. Class H progeny inherited microsatellite alleles from both the S. spontaneum and the commercial-type parents and were, therefore, considered being F1 hybrids. Class S and Class C progeny inherited microsatellite alleles only from one parent and were considered to be either selfs of either parent or F1 hybrids that only inherited allele(s) from one parent. Class X progeny inherited non-parental microsatellite allele(s) in addition to the allele(s) from the maternal S. spontaneum parent and were considered to be contaminants. With the exception of one cross, eight to ten Class H progeny were pre-selected from each cross while still in seedling greenhouse and were backcrossed with commercial-type sugarcane clones. The remaining progeny were transplanted into a breeding nursery for phenotypic evaluation that concurred with the molecular classification. Pearson Correlation Coefficients between molecular and phenotypic classifications were inconsistent that justified the need of molecular markers in the selection process. This study demonstrated that the molecular approach of fingerprinting progeny to confirm parentage prior to field planting even with only one microsatellite marker might substantially increase selection efficiency.