Sequencing And Analysis Of The Chloroplast Genomes Of Cytoplasmic Sterile Line And Its Maintainer Line In Soybean

Soybean plays a key role in providing oil and protein. It is one of major crop that no large-scale application of heterosis to improve yields. Based on the cytoplasmic male sterility (CMS) line, Jilin Acdemy of Agricultural Sciences has first implemented the "three lines" matching and developed Hybsoy-1 that bred and released in 2002, the first commercial hybrid soybean in the world. China is the world leader in application of hybrid soybean. But the genetic mechanism underling has not been fully understand. It is still in the initial stage, need for further study.As a peculiar organelle in plant cell, it performs important role in physiological processes such as photosynthesis. The chloroplast genome (cpDNA) often transfersgenetic materialand nuclear genomes. Therefore, when analyzing the quality of soybean nuclear male sterility molecular mechanism of interaction, in addition to considering the mitochondria, but also concerned about the chloroplast genome. However, it has not well established whether chloroplast DNA (cpDNA) play important role in male sterility. The current research on the impact of the chloroplast genome of the CMS is not complete, whether male sterility associated with soybean, nor the exact nature of the conclusions.In this study, RN type JLCMS9A and its maintainer JLCMS9Bwere performed with next-generation sequencing of chloroplast genome DNA. Based onthe results obtained polymorphism analysis and validate the use of molecular biology, and it is hoped to search for evidence that chloroplast DNA may play some roles in cytoplasmic male sterility.1. Chloroplast genomic DNA was isolated and purified using the enzymatic lysis buffer method from leaves of field-grown JLCMS9A and JLCMS9B plants. A male-fertile line (F) is performed sequencing of chloroplast genome. After removal of nuclear and mitochondrial genome sequences, we got 1,043,280 bp of JLCMS9A and 865,510 bp of JLCMS9B. Clean reads from each DNA library were then assembled separately using ABySS, SSPACE, and GapCloser assembly software. After annotation of regions by alignment against the PI 437654 soybean chloroplast reference genome, oligonucleotide primers were designed to amplify across problematic and remaining gap regions. The whole chloroplast genome sequence of JLCMS9A is 152,222 bp and JLCMS9Bis 152,215 bp. Genome annotations is performed using DOGMA software according to CpBase database, and the two chloroplast genome all consist of 96 mRNA genes,39 tRNA genes and 8 rRNA genes.2. According to alignment with PI 437654 soybean chloroplast reference genome, we found the chloroplast genome of soybean showed highly conservation. and after genome annotations is performed using DOGMA software according to CpBase database. the two chloroplast genome all consist of 96 mRNA genes,39 tRNA genes and 8 rRNA genes.43 SNPs and 3 InDels exist between JLCMS9A and JLCMS9B. of which 23 SNPs located in the gene coding region. On averagel SNP per 3.3kb, which is far below the frequency of that one SNP per 0.44kb in nuclear genome.3. Using PCR method we sequenced 12 male-sterile and 60 male-fertile materials of RN type which came from China, the United States and Japan, and represents majority of the world’s male-fertile materials.The results showed that the 23 SNPs which located in the gene coding region andthe 3SNPs whichlocated in the non-coding region exist in all RN type male cytoplasmic male-sterile and corresponding maintainer soybean lines.4. After PCR amplification of 4 SNPs and with subsequent digestion by restriction enzyme we candistinguish the RN typemale-sterile and other typemale-sterile soybean lines, and also we can distinguish the hybrid and maintainer lines. The four specific chloroplast marker used are listed here:(1) Mmarker RE 1, the PCR amplification length is 463 bp. After digested by Bpu10 Ⅰ, the PCR product of maintainer line kept the original length and the male-sterile line is digested into two bands, which are 255 bp and 208 bp respectively;(2) Marker RE2, the PCR amplification length is 489 bp. After digested by psi Ⅰ, the PCR product of maintainer line kept the original length and the male-sterile line is digested into two bands, which are 323 bp and 166 bp respectively;(3) Marker RE3, the PCR amplification length is 490 bp. After digested by psi Ⅰ, the PCR product of maintainer line kept the original length and the male-sterile line is digested into two bands, which are 288 bp and 192 bp respectively; (4) Marker RE4, the PCR amplification length is 399 bp. After digested by EcoR Ⅰ, the PCR product of maintainer line kept the original length and the male-sterile line is digested into two bands, which are 247 bp and 152 bp respectively;5. There are 23 SNPs between JLCMS9A and JLCMS9B,20 of which are also exist between JLCMS9B and PI 437654. The remainder 3 SNPs between JLCMS9A and JLCMS9B is specific to JLCMS9A and may related to male-sterile. These 3 SNPs located in the gene coding region within matK and ycfl, and then tested the expression level of these 2 genes in leaf and flower tissue of the two male-sterile and male-fertile lines by qPCR. The result showed that the 2 genes have the highest expression level in maintainer line and the lowest expression level in restoring line, while the male-sterile have the modest expression level. Because the expression level of these 2 genes in the male-sterileline fall in between the two male-fertile lines, we cann’t get any correlation with cytoplasmic male-sterile.Due to previous studies cytoplasmic male-sterile is highly related to the genome structure and function of mitochondria, but whether chloroplast may have also played some role in cytoplasmic male-sterile is unknown. In this study, the CMS line JLCMS9A and male-fertile line JLCMS9B were sequenced. and no cytoplasmic male-sterile related genes have been found. But it is found that some SNPs can be used to distinguish male-sterile and male-fertile lines.