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Research on genetic diversity and phylogeny of Saccharum spontaneum L. in China

Hui Chen1, Yuanhong Fan2, Qing Cai3 and Ya-ping Zhang4

1 Sugarcane Research Institute, Yunnan Academy of Agricultural Sciences, Kaiyuan, Yunnan , China, 661600
Email: chenhuiysri@ yahoo.com
2
Sugarcane Research Institute ,Yunnan Academy of Agricultural Sciences, Kaiyuan, Yunnan ,China, 661600
Email fyhysri@sohu.com.
3
Sugarcane Research Institute ,Yunnan Academy of Agricultural Sciences, Kaiyuan, Yunnan ,China, 661600
Email caiqingysri@ sohu.com.
4
Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology ,the Chinese Academy of Sciences,
Kunming ,Yunnan, China 650223 Email yapingzhang@mail.kiz.ac.cn

Abstract

In this study, 195 accessions of Saccharum Spontaneum L. from different geographical populations in China were studied using random amplified polymorphic DNA (RAPD) analysis to estimate their genetic diversity. Phylogenetic analysis was done to assess relationships among haplotypes. A total of 266 bands were scored of which 145 were reproducibly polymorphic. The extent of genetic variability was between 62.95% and 37.05% within populations. The topology of the phylogenetic tree of S. spontaneum corresponds to the population geographical distribution. The genetic variation and diversity among different populations was consistent with existing knowledge on the geographical distribution of S. spontaneum in China. We tentatively propose that Yunnan province was the center of origin for S. spontaneum in China, based on previous collections and archeological information when combined with these results.

Media summary

Based on our research on genetic diversity and phylogeny combined with previous collections and archeological information, Yunnan province is the center of origin for S. spontaneum in China. and

Key Words

Saccharum Spontaneum L., RAPD, genetic diversity, phylogeny

Introduction

China's vast territory, complicated geological formation and various climate types create favorable conditions for the evolution of wild sugarcane. Diverse sugarcane genetic resources evolved in these variable environments. Germplasm collections are an important component of sugarcane improvement programs as they provide breeders with sources of useful traits. Basic germplasm has often been used in sugarcane breeding. Most modern commercial sugarcane clones represent a complex interspecific combination between the genomes of S. officinarum and S. spontaneum (Besse et al. 1997). At present, sugarcane breeding in China is progressing towards the introduction of valuable genes, with positive effects on agronomically important traits, from new basic germplasm into modern sugarcane clones. This is especially true for genes from the species S. spontaneum, which has played a very important role in sugarcane breeding in the development of interspecific hybrids. S. spontaneum has the widest geographical distribution of the six species of the genus Saccharum (S. officinarum L., S. spontaneum L., S. barberi Jeswiet., S. sinense Roxb., S. robustum Brandes & Jeswiet ex Grassl. and S. edule Hassk.). S. spontaneum has many desirable characters including disease resistance, vigour, tillering ability, drought tolerance, water tolerance, frost tolerance, and general adaptability. Because of these traits, sugarcane breeders all over the world have considerable interest in the collection, maintenance, evaluation, and exploitation of its genetic potential (Tai et al. 1999). S. spontaneum clones are distributed throughout 8 provinces in China. Clones have been collected on a number of occasions, and maintained in the National Nursery for Sugarcane Germplasm Resources, Yunnan Sugarcane Research Institute, Kaiyuan, Yunnan province, China. In this paper, we examine the use of RAPDs for estimating the level of genetic diversity among 195 S. spontaneum accessions from different geographical populations in China, and discuss the implications of our results for the origin and diffusion of the wild sugarcane.

Materials and Methods

Materials

195 accessions from eight geographic populations were considered in this study (Table 1). All samples were obtained from the National Nursery for Sugarcane Germplasm Resources in Yunnan province, China.

Table 1. The geographical distribution and climatic characteristic in 8 geographical colonies of Saccharum spontaneum L in China.

Colony

Number of samples

Geographical distribution and climatic characteristic samples

Yunnan (YN)

86

locate in the southwest of China, attrib to semi-tropic, plateau, moisture and monsoon climate

Sichuan(SC)

36

locate in the southwest of China, attrib to semi-tropic, plateau, moisture and monsoon climate

Guizhou(GZ)

10

locate in the southwest of China, attrib to semi-tropic, plateau, moisture and monsoon climate

Guangxi (GX)

13

locate in the south of China, attrib to tropic, moisture and monsoon climate

Guangdong(GD)

18

locate in the south of China, attrib to tropic, moisture and monsoon climate

Hainan(HN)

14

locate in the south of China, attrib to tropic, moisture and monsoon climate

Fujian(FJ)

14

locate in the southeast of China, attrib to semi-tropic, moisture and monsoon climate

Jiangxi(JX)

4

locate in the southeast of China, attrib to semi-tropic, moisture and monsoon climate

Methods

DNA was isolated from apical meristem and young leaf tissues using a modified method (Fan et al. 1999). PCR mixtures (10μL total) contained: 25ng DNA, 2.5mM dNTP, 0.2μM primers (products of Operon Technology Company),10 mM Tris-HCl (pH8.9), 50 mM KCl, 0.2mM BSA, 2.5mM MgCl2, and 1.0 unit of Taq polymerase (products of Takara Biotechnology Company). Reactions were overlaid with 20μL of mineral oil to prevent evaporation. Samples for enzymatic amplification were subjected to initial denaturation at 95C for 3 min follow by 40 cycles of 94C for 1min (denaturation), 36C for 1min (annealing), and 72C for 2min (extending) with a final extension at 72C for 5min. Fragments generated by amplification were separated according to size electrophoresis on 1.5% agarose gel in 1TAE, stained with ethidium bromide, and photographed by an EAGLE EYE imager (Williams et al. 1990; Wachiro et al. 1995; Chen et al. 2001).

Results

RAPD Analysis

Out of a total of 25 primers used, 20 (80%) generated polymorphic loci. A total of 266 bands were scored of which 145 (54.5%) were reproducibly polymorphic. The number of products generated by each primer varied from 7 to 15 with an average of 13. The size of the amplified fragments that were scored ranged from 0.2-2kb.

Genetic Diversity

The phenotypic frequencies detected with the 20 primers were calculated and used in estimating diversity (Ho) within population types (Table 2). The Jiangxi population exhibited the lowest within population variability (0.6937), but had the smallest population size, which affects Ho. The Yunnan population exhibited highest within population variability (1.7126).

Shannon’s index of phenotypic diversity was used to partition the diversity into within and between population components (Table 3). Primer OPI-08 detected the most within population variability, while primer OPA-19 detected the least.. An assessment of the proportion of diversity present within population Hpop/Hsp, compared with that between populations, (Hsp-Hpop)/Hsp, indicates that, on average, most of the diversity (62.95%) is detected between populations (38.15%-71.86%). 37.05% of the variation was detected within populations. However, the distribution of variability did vary slightly between and within populations with different primers.

Table 2. The genetic diversity index of 8 geographical colonies of Saccharum Spontaneum L. in China

Primer

YN

SC

GZ

GX

GD

HN

FJ

JX

OPA-07

2.0387

1.7673

1.4536

1.3849

1.2767

1.0885

1.1456

0.9764

OPA-19

2.0909

1.8421

1.7654

1.4308

1.5692

1.2048

1.0923

0.8732

OPB-14

1.4758

1.8122

0.9091

0.7422

0.7288

0.7059

0.4378

0.6496

OPD-01

2.1492

1.9678

1.6216

1.4358

1.2697

0.9739

0.7262

0.6519

OPF-01

1.3591

1.2943

1.0249

0.9739

0.7618

0.7618

0.8732

0.6348

OPF-05

2.2241

1.9769

2.0349

1.3851

1.0624

1.0348

0.9792

0.7567

OPF-12

1.8526

1.9769

1.7743

1.3155

1.2349

0.9849

0.8492

0.6599

OPH-01

1.5101

1.3844

1.0989

0.9432

1.1143

0.8938

0.7607

0.8349

Primer

YN

SC

GZ

GX

GD

HN

FJ

JX

OPH-19

1.8122

1.4278

1.3342

1.1893

0.9866

0.8654

0.7348

0.6549

OPI-08

1.4637

1.3859

1.2248

1.1049

1.0876

0.9703

0.8785

0.7786

OPJ-07

1.2917

1.0977

0.9771

0.8718

0.7185

0.6507

0.5873

0.4336

OPJ-09

1.7298

1.6348

1.3285

1.2853

1.0928

0.9385

0.6418

0.5186

OPJ-14

1.2875

1.4637

1.1524

1.0335

0.8785

0.8324

0.6545

0.5448

OPJ-18

1.8819

1.6405

1.4969

1.1721

0.9898

0.8671

0.7703

0.6479

OPK-18

1.9416

1.8803

1.6581

1.3272

1.2717

1.0068

0.8781

0.7589

OPL-17

1.9769

1.7734

1.5266

1.6534

1.2548

0.9849

0.8834

0.6599

OPM-04

1.6309

1.3154

1.4403

1.1191

1.0348

0.8779

0.5946

0.6343

OPM-07

1.7898

1.9416

1.6034

1.4432

1.0781

1.2249

0.9789

0.8744

OPN-02

1.1143

1.5533

0.9073

0.6471

0.8411

0.5489

0.5302

0.4812

OPN-11

1.6309

1.2951

1.4038

1.1438

0.9779

1.0433

0.9342

0.8495

Average

1.7126

1.6216

1.3868

1.1801

1.0615

0.9230

0.7965

0.6937


Table 3. Partitioning of the genetic diversity into within and between populations for 20 primers

Primer

Hsp

Hpop

Hpop/Hsp

(Hsp-Hpop)/Hsp

OPA-07

3.9898

1.3915

0.3487

0.6513

OPA-19

5.2715

1.4836

0.2814

0.7186

OPB-14

2.3744

0.9327

0.3928

0.6072

OPD-01

4.0458

1.3495

0.3335

0.6665

OPF-01

1.6233

0.9605

0.5917

0.4083

OPF-05

4.8902

1.4318

0.2928

0.7072

OPF-12

3.9773

1.3311

0.3347

0.6653

OPH-01

1.9903

1.0675

0.5364

0.4636

OPH-19

2.8188

1.1256

0.3993

0.6007

OPI-08

1.7977

1.1118

0.6184

0.3815

OPJ-07

1.7671

0.8285

0.4688

0.5312

OPJ-09

3.2252

1.1463

0.3554

0.6446

OPJ-14

2.2187

0.9809

0.4421

0.5579

OPJ-18

3.4079

1.1833

0.3472

0.6528

OPK-18

4.3257

1.3403

0.3098

0.6902

OPL-17

4.5241

1.3392

0.2961

0.7039

OPM-04

2.5573

1.0809

0.4227

0.5773

OPM-07

4.0844

1.3668

0.3346

0.6654

OPN-02

2.3378

0.8279

0.3541

0.6459

OPN-11

2.0307

1.1598

0.5711

0.4289

Average

3.1619

1.1719

0.3705

0.6295

Genetic Differentiation and Genetic Relationship

To examine the genetic differentiation and genetic relationship between the different populations, a genetic distance matrix based on the proportion of shared fragments (Nei 1978) was used to establish the level of relatedness between the different populations of S. spontaneum studied. Figure 1 shows a dendrogram generated by UPGMA cluster analysis, based on the estimates of genetic distances between populations.

Figure 1. A dendrogram generated by UPGMA cluster analysis, based on the estimates of genetic distances between populations.

Conclusion

This study demonstrated that S. spontaneum clones within the Yunnan population exhibited more extensive genetic variability, as well as more abundant genetic diversity and many original types of wild sugarcane. Yunnan province has the widest geographical distribution of S. spontaneum of all provinces in China. Moreover, according to our results, studies on sugarcane collection, maintenance, evaluation and exploitation of its genetic potential over many years, and related archeological information, we tentatively proposed that Yunnan province was the possible origin center of S. spontaneum in China, which then spread into Sichuan, Guizhou and Guangxi provinces. S. spontaneum in Guangxi further spread into Guangdong province, followed by spread into Fujiang and Jiangxi provinces northward and Hainan province southward.(Figure 2)

Figure 2. The possible origin center and diffusion paths of Saccharum Spontaneum L. in China were demonstrated by the dot and the arrows respectively.

References

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