Sugar Tech (2009) 11(3) : 296-299

SHORT COMMUNICATION

Use of simple sequence repeats for authentication of sugarcane hybrids generated from Yunnan Erianthus rockii Yan-Fen Jing · Lian-an Tao · Xin-long Liu · Ru-dong An · Xue-kuan Chen

Received: 14 July 2009 / Accepted: 20 August 2009

Abstract An experiment was conducted by Yunnan Academy of Agricultural Sciences in which three clones of E. rockii, F1 2000-7, F12000-15, F12000-16 were crossed with ROC10, F172 and Yunnan E. arundinaceum. The use of 4 simple sequence repeat (SSR) primers, Msscir16, Msscir26, Msscir33 and Msscir36 authenticated that E. rockii F1 2000-15 [ROC10 (Female) x E.rockii 99-9 (Male)] and E. rockii F1 2000-16 [F172 (Female) x E. rockii 99-4(Male)] were the true hybrids of Yunnan E.rockii. Also, the progenies obtained by crossing between E. arundinaceum as a female parent and E. rockii F1 2000-7 and E. rockii F1 2000-15 as a male parent showed that F1 06-75 (Yunnan E. rockii with E. arundinaceum) was the true hybrids. The findings may be utilization in the breeding programs tailored to develop new high yielding sugarcane varieties using E. rockii genetic stocks. Keywords Sugarcane, Yunnan E. rockii clones, SSR makers Wild Saccharum species and its related genera are the main source for developing high yielding sugarcane cultivars which are also resistant to most of the biotic and abiotic stresses. In recent years, various high yielding and resistant sugarcane cultivars have been bred to cope with the world sugar demands. Recently, use of Saccharum species and its related genera, especially, the E. rockii has been investigated for imparting high sucrose yielding and resistance to different stresses. In China, various hybrids have been made using different Erianthus species (Zhu and Huang, 1996; Wang et Yan-fen Jing · Lian-an Tao · Ru-dong An ( ) Ruili Station of Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, Rui Li 678600, Yunnan, P.R. China email : [email protected] Xin-long Liu · Xue-kuan Chen Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, Kaiyuan 661600, Yunnan, P.R. China

al., 2003; Tao, 1996; Jing et al., 2001; Yang, 2004; Deng et al., 2004), particularly the E. fulvus (Li et al., 2003). The studies using E. rockii have been started in early 1990’s, the first progenies obtained in Guangxi and Yunnan (P. R, China) in 1996 (Chu and Huang, 1996; Wang et al.,2003). Until now only few isozyme markers and the testing of smut disease have been reported for clones generated from E. rockii by Jing (Jing et al., 2001) and Yang (Yang, 2004), but there is scarcity of the molecular markers to identify the authenticity of progenies of E. rockii. E. rockii has been identified for its high tillering abilities and has been showed to have tolerance to cold and resistant and also to certain diseases. However, it is difficult to use E. rockii to get progenies and to perform further crossing, because both of the genera, E. rockii and Saccharum are distantly related. In the present study, we have explored the combination method of F1 progenies (Deng et al., 2004) and identified the authenticity using SSR markers. Clone collection F172, ROC10, Ruili E. rockii 99-4,99-9; Yunnan E. rockii F12000-7, F12000-15, F12000-16; Yunnan E. arundinaceum 83177, 83-180, 83-182 [Ruili E. rockii is wild type and collected in Ruili, the clones generated in F1 called Yunnan E. rockii; Yunnan E.arundinaceum is wild type) Germplasm innovation Initially, the crosses have been made using F172 and Ruili E. rockii 99-4, ROC10 and Ruili E. rockii 99-9, and then by using F1 Yunnan E. rockii F12000-15, Yunnan E. rockii F120007 and Yunnan E. arundinaceum. The crosses and progenies generated were as follows: i. F172xRuili E. rockii 99-4→Yunnan E. rockii F12000-7, 2000-16

Sugar Tech (2009) 11(3) : 296-299

ii. iii. iv.

ROC10 x Ruili E. rockii 99-9 → Yunnan E. rockii F1 2000 -15 Yunnan E. rockii F12000-15 xYunnan E. arundinaceum 83-177→Yunnan E. rockii with E. arundinaceum F10678 Yunnan E. rockii F12000-7 x Yunnan E. arundinaceum (83-180+83-182)→Yunnan E. rockii with E. arundinaceum F106-73, F106-74, F106-75, F106-76 ,F10677.

297

in parents. This suggests that it was a true hybrid generated from ROC10xRuili E. rockii 99-9. Similarly, Yunnan E. rockii F12000-16 has the 17th typical band while Ruili E. rockii 99-4 do not have any other bands; therefore, Yunnan E.Rockii F12000-16 is likely to be true hybrid generated from F172xRuili E. rockii 99-4. In case of Yunnan E. rockii F12000-7, 7th band was not observed but it was present in Ruili E. rockii 99-4. Further, the presence of 24th and 25th bands in this indicating some doubt for being hybrid (Fig.1-B).

SSR assays DNA extraction from fresh leaf and PCR test were carried out as described in Cai et al. (2005) with the following modifications. Four microsalellite primers which were polymorphic were selected from a number of microsatellites developed by CIRAD as part of the International Consortium of Sugarcane Biotechnology Sugarcane Microsatellite Collection (Rossi et al., 2003), they were msscir 21(EMBL accession number: AJ293493), msscir 26 (EMBL accession number: AJ293497), msscir16 (EMBL accession number: AJ293488), msscir33 (EMBL accession number: AJ293504), msscir36 (EMBL accession number: AJ401313). 20 µL PCR reactions contain 25 ng templates DNA, 6 µM of forward and reverse primers, 0.2 mM of each dNTP, 3.0 mM MgCl2 and 0.4UTaq polymerase (MBI) with accompanying buffer. PCR procedure was performed on Eppendorf Master Cycle Gradient. Cycling conditions were 1 min at 94oC followed by 35 cycles of 30 sec at 94oC, 1 min at the appropriate annealing temperature (54oC), 30sec at 72oC and a final extension step of 5 min at 72oC. The PCR products were run on a denaturing 5% polyacrylamide, the gels were dyed with rapid silver staining. Marker identification for the F1 generated between Saccharum and Yunnan E. rockii Analysis of the DNA with primer Msscir16 showed the presence of 2 common bands (12 thand13th) in Ruili E. rockii 99-4 and 99-9, revealing its relationship with E. rockii, these 2 bands were absent in F172 and ROC10. F172 has a typical band 8th, similarly 6th and 7th bands were present in ROC10, Yunnan E. rockii F12000-7 and F12000-16 have the 8th, 12th, 13th bands while the parents have no any other bands. This shows that they are the hybrids generated from F172xYunnan E. rockii. Yunnan E. rockii F12000-15 has the 6th, 7th ,12th ,13th bands similar to ROC10 showing that this is a true hybrids of ROC10xRuili E. rockii 99-9 (Fig.1-A). Analysis of the DNA with primer Msscir36 showed that Ruili E. rockii 99-4 has the 17th band, Ruili E. rockii 99-9 has the 13th, 14th bands, ROC10 has the 4th,15th, 22nd, 23rd typical bands, F172 has the 2nd, 3rd, 20th,21sttypical bands. Yunnan E. rockii F12000-15 has the 4th, 13th, 14th bands which are absent

Fig. 1. The molecular identification of the hybrid progenies from the cross between Saccharum and E. rockii using primer Msscir16 (A), Msscir36 (B) and Msscir33 (C) Note: In these figures used YD00-16 which full name is Yunnan E. rockii F1 2000-16, Er99-4 means Ruili E.rockii99-4, Ea is same with E. arundinaceum, the others all follow this named way. The number used on the figures named follow this way: one figure is a whole, band is in the topic of figure called 1st, and next band called 2nd, and so on. We did not write down all number, just wrote the typical bands. Same numbers on different figures mean different bands.

13

298

Sugar Tech (2009) 11(3) : 296-299

Fig. 2. The molecular identification of the hybrid and backcross progenies from the cross between Saccharum and E. rockii, E. rockii and E. arundinaceum respectively using primer Msscir33 (A) and Msscir26 (B)

Band profile with the primer Msscir33 showed that 10th band was typical for F172, the 3rd, 4th and 14th bands were typical for ROC10, the 5th ,6th were typical bands for Ruili E. rockii 99-9, the 11th, 12th were common bands for Ruili E. rockii 99-4 and 99-9. All bands appeared from Yunnan E. rockii F1 2000-7 were shared with F172 which had no typical bands (except 11th and the12th), making difficulties in identification. Yunnan E. rockii F1 2000-15 had 11th, 12thand 14th bands which were also present in parents, it’s likely to be the hybrid generated from ROC10 x Ruili E. rockii 99-9. Yunnan E. rockii F1 2000-16 had the 10th, 11th and 12th bands indicating that its hybrid generated from F172 x Ruili E. rockii 99-4 (Fig.1 C). Msscir26 showed the presence of 1st, 2nd, 3rd, 17th, 18th and 19 bands in Ruili E. rockii 99-9 and the 4th and 5th in Ruili E. rockii 99-4 and 99-9, the 15th and 16th bands were typical for Ruili E. rockii 99-4, the 7th ,8th and 23rd for F172 and 9th for ROC10. Yunnan E. rockii F12000-7 besides possessing the parents bands (4th, 5th, 7th, 8th), also had the 17th ,18th and 19th bands which were common with Ruili E. rockii 99-9. Yunnan E. rockii F1 2000-15 had the 2nd ,3rd ,17th ,18th bands which were common with Ruili E. rockii 99-9 showing that its true hybrids of ROC10 x Ruili E. rockii 99-9; Yunnan E. rockii F1 2000-16 had the 4th ,5th ,15th and 16th common with male parent so it is the true hybrids of F172 x Ruili E. rockii 99-4 (Fig.2-B). It can be concluded from the above studies that Yunnan E. rockii F12000-15 and Yunnan E. rockii F12000-16 are the true hybrid generated from ROC10 x Ruili E. rockii 99-9 and F172 x Ruili E. rockii 99-9, respectively. It was also observed that the Yunnan E. rockii F12000-7 was not hybrid of E. rockii and Sacccharam. th

The E. rockii and Saccharum are distantly related generas, it is difficult to cross and obtain the viable seeding from these

13

two. The first F1 seeding had been obtained in 1996 (Chu et al., 1996; Deng et al., 2004), since then, no progenies were obtained by crossing or backcross. In this study, Yunnan E. rockii F12000-7 and F 12000-15 were pollened by Yunnan E. arundinaceum (83-180+83-182,83-177) at Ruili Breeding Station in 2006 and 6 progenies were obtained (F1: 06-73, 06-74, 06-75, 06-76, 06-77, 06-78). They have been identified using the primers Misscir 33 and Misscir 26. Analysis of the DNA with primer Msscir33 showed that 6 progenies insignificantly had 10th, 11th and 12th bands which maybe the typical bands for Yunnan E. arundinaceum. But F106-74 and F106-77 all had 2 nd, 9th bands, F106-73 and F106-76 all have 7 th band, F106-78 has 14th band while the parents did not. So, F106-73, F106-74, F106-76 and F106-77 are not the true hybrids of Yunnan E. rockii F 1 2000-7xYunnan E. arundinaceum (83-180+83-182). F106-78 is not the true hybrids of Yunnan E. rockii F12000-15 x Yunnan E. arundinaceum 83177. F1 06-75 is possible of the true hybrid progeny of Yunnan E.rockii F12000-7 x Yunnan E. arundinaceum (Fig.2A). Msscir 26 showed that F106-75 had 12th, 13th bands which are typical for Yunnan E. rockii F12000-7, and it had 14th band which is typical for Yunnan E. arundinaceum 83-180. So F10675 is the ture hybrids of Yunnan E. rockii F12000-7xYunnan E. arundinaceum 83-180. F106-78 is possible of the self-crossing progeny because it had no typical for Yunnan E. arundinaceum 83-177. F106-73, F106-76, F106-77 have 32nd,33td band which the parents no them and 06-74 has 9th,10th bands which the parents did not. So the 4 F1-type were not the true hybrid generated from Yunnan E. rockii F12000-7xYunnan E. arundinaceum(83180+83-182) (Fig. 2B).

Sugar Tech (2009) 11(3) : 296-299

The results concluded from the above results is that the progenies F106-75 is the true hybrid generated from Yunnan E. rockii F12000-7 x Yunnan E. arundinaceum 83-180. F1 06-78 has many bands common to their parents, and more similar to Yunnan E. rockii, which may be due to the self pollination between the parents. It seems difficult to obtain the progenies by crossing distant genera using traditional methods. In the present study, the true hybrids progenies (Yunnan E. rockii F12000-15, F 12000-16) have been obtained by crossing between Saccharum xYunnan E. rockii. Simultaniously, the true hybrid (F106-75) was also identified which was obtained from a cross between Yunnan E. rockii F12000-7xYunnan E. arundinaceum. These studies will help in understanding of the crosses using Yunnan E. rockii, Yunnan E. arundinaceum, and the stocks of related genus. The findings of our studies may be utilized in the breeding program in developing new and high yielding sugarcane varieties using E. rockii stocks.

299

References Cai Q, Aitken K, Deng HH, Chen XW, Fu C, Jackson PA, Mcintype CL (2005). Verification of the introgression of Erianthus arundinaceus germplasm into sugarcane using molecular markers. Plant Breeding 124:322-328 Deng HH, Li QW, Chen ZY (2004). Breeding and utilization of new sugarcane parents. Sugarcane 11(3):7-12. Jing YF, Tao LA, Yang LH (2001). Primary authenticity identification study of Saccharum progenies using isozyme. Sugarcane and Canesugar (6): 1-4. Li FS, He LL, Yang QH (2003). Evaluation of some Special Characters of Erianthus fulvus and Identification of Sugarcane Hybrids based on Chromosome Number and RAPD. Molecular Plant Breeding 1:775-781. Rossi M, Araujo PG, Paulet F, Garsmeur O, Dias VM, Cheu H, Van Sluys MA, D’Hont A (2003). Genome distribution and characterization of EST-derived resistance gene analogs (RGAs) in sugarcane. Mol. Genet. Genomics 269, 406-419. Tao LA (1996). Primary study and use of heterosis of wild sugarcane resources in Yunnan. Yunnan Agricultural Sciences and Technology (5):13-17. Wang LP, Fan YH, Cai Q (2003). Progress report on crossing and utilization of sugarcane resources. Sugarcane 10(3):17-23. Yang LH (2004) Smut resistance study of progenies of S.spontaneum, E.arundinaceum, E.Rockii of Yunnan. Sugarcane (1): 10-14. Zhu GY, Huang, J. M. (1996). Primary crossing study of Saccharum and its wild related plant. Guangxi Agricultural Sciences (1):5-6.

13