Analysis on the Different Results of Seed Purity ldentification for the Same Hybrid Rice Variety Using Different SSR Primers

Qingcai ZHAN，Guoqi ZHU，Jingping CHEN，Yijie ZHAN，Dan XUE

1.Agricultural Biotechnology Research Center of Hunan Province，Changsha 410125，China；

2.Hunan Rice Research Institute，Changsha 410125，China；3.College of Applied Arts and Science of Beijing Union University，Beijing 100083，China

Analysis on the Different Results of Seed Purity ldentification for the Same Hybrid Rice Variety Using Different SSR Primers

Qingcai ZHAN1*，Guoqi ZHU2，Jingping CHEN1，Yijie ZHAN3，Dan XUE2

1.Agricultural Biotechnology Research Center of Hunan Province，Changsha 410125，China；

2.Hunan Rice Research Institute，Changsha 410125，China；3.College of Applied Arts and Science of Beijing Union University，Beijing 100083，China

Different results of seed purity identification for Gangyou 158，ⅡYou 808，Wuyou 308 and Tianfengyou 316 were obtained using different SSR primers in our early work.To find out the reasons，the four hybrid combinations were grown in field to identify their purity according to their phenotypic traits.Then，the results of field identification were compared with that of laboratory tests using different SSR primers.The comparison revealed that only sterile lines （female parent）were distinguished from true hybrids using the primers RM208，RM264，RM242 and RM164 for the purity identification of Gangyou 158，ⅡYou 808，Wuyou 308 and Tianfengyou 316，so the results were higher than that of field identification.In contrast，the primers RM341，RM297，RM21 and RM110 were able to distinguish not only the sterile plants but also the cross-pollinated ones from the true hybrids of Gangyou 158，ⅡYou 808，Wuyou 308 and Tianfengyou 316，and the results of purity identification using them were close that of field identification.In summary，several pairs of primers should be used for the purity identification of rice hybrids to distinguish all the off-type plants and thus improve the accuracy.

Hybrid rice；SSR primers；Seed purity；Different results

Hybrid rice has made tremendous contributions to the food security in China and even around the world.In both two-line and three-line hybrid rice breeding，highly pure hybrid seed is the basis of high yield and high quality. The sterility of two-line sterile lines is susceptible to environmental factors such as light and temperature.Abnormaltemperature atsome certain growth stages of two-line sterile lines may change their sterility，and then they may produce seeds by selfcrossing，and thus reducing the purity of true hybrids.Moreover，other offtype seeds may also be generated during the whole plant growth stage due to human factors.The purity of hybrid seeds should be detected before sale.Field identification is a direct and the most reliable method to determine hybrid purity at present.However，this method not only takes a long time period，but also is limited by seasons and regions.In contrast，SSR marker，most commonly used in laboratory for hybrid purity identification，is rapid，stable，repeatable and accurate. Since 2007，we have detected more than 20 000 samples from over 500 seed companies using SSR markers，and most of the test results were consistent with field identification.However，we also found that the identification results of some samples significantly differed when different SSR primers were used.Therefore，in this study，we compared the results of hybrid purity between laboratory tests and field identification，with an attempt to find out the reasons causing differences in results.

Materials and Methods

Materials

The hybrid rice combinations

Supported by Special Fund for the Screening and Breeding of Low-Cd-accumulating Crop Varieties.

*Corresponding author.E-mail:zhanqc@hotmail.com.

Received:September 15，2015 Accepted:November 15，2015Gangyou 158，Wuyou 308，ⅡYou 808 and Tianfengyou 316 were the experimental materials.According to the identification for a large number of samples，the difference in identification results for the seeds with purity higher than 96%using two pairs of primers should be less than 2%.However，the purity identification for the four combinations using two pairs of primers had a difference more than 15%.All the SSR primers used in this study were synthesized by Beijing AuGCT Biotechnology Co.，Ltd.

Methods

Seed purity identification with laboratory tests

The purity of every hybrid combination was identified using two pairs of SSR primers.Two repetitions were set，and 96 plants were included in each repetition.In detail，young leaf about 0.5 cm long was sampled from each plant，put in a 96-well plate，soaked with 150 μl of 80%acetone solution overnight，and then washed with ultrapure water to remove the remaining acetone.DNA was extracted with the method previously reported［1-4］with some modifications.Then，1 μl of the extracted DNA solution was used as the template for PCR amplification with the system and procedures described by Liu et al［2］.

Seed purity identification with field trials in Hainan

Each of the four hybrid combinations were planted in three plots，with 500 plants in each plot to identify their purity.The sterile plants and cross pollinated plants were calculated.In addition，100 plants of each combination were tag numbered，and the fertility and phenotypic traits of each of the plants were recorded，their leaves were sampled and identified in laboratory with two pairs of primers.

Results and Analysis

The difference in purity of hybrids identified in laboratory and field

As could be seen from Table 2，the purity of each hybrid combination identified usingtwo pairsofSSR primers was greatly different.The results of purity identification in laboratory using primers RM341，RM297，RM21 and RM110 for Gangyou 158，II You 808，Wuyou 308 and Tianfengyou 316 were close to the results in field identification.The purity obtained using primers RM208，RM264，RM242 and RM164 for Gangyou 158，II You 808，Wuyou 308 and Tianfengyou 316 were significantly higher than that obtained from field trials，and the difference was far beyond of the allowable limit of error.

Table 1 Sequences of SSR markers

Table 2 Comparison of the results of field experiment and laboratory test with different SSR markers %

Phenotypic analysis ofoff-type plants

The leaves of 100 plants of each hybrid were sampled from field and identified using SSR markers in laboratory.The results revealed that the results were similar to the first identification in laboratory for each hybrid combination.And the purity of the 100 samples identified according to phenotypic traits was also close to that of the 1 500 plants in field identification.

The phenotypic traits of the offtype plants identified by SSR primers were analyzed，and found that the offtype plants of Gangyou 158 by primer RM208 were sterile （female parent），accounting for 9%of total，like the samples No.2，9，16，30 and 45 in Fig. 1.The identification using primer RM341 proved that 68%of Gangyou 158 plants were true hybrid，and the off-type plants included all the sterile plants identified with primer RM208（like the samples No.2，9，16，30 and 45 in Fig.2）and the cross-pollinated plants（23%，like samples No.1，7，8，22，24，25，33，34，39，41 and 48 in Fig.2）.Similarly，forⅡYou 808，Wuyou 308 and Tianfengyou 316，only sterile （female parent）plants were identified as off-types using primers RM264，RM242 and RM164，so the results of the identification with RM264，RM242andRM164were higher than that in field identification. On the contrary，primers RM297，RM21 and RM110 identified not only the sterile （female parent）plants but also the cross-pollinated plants，so theresults of the identification with RM297，RM21 and RM110 were close to that of field identification.

不同引物鉴定同一杂交水稻种子样品纯度结果的差异及其原因分析

詹庆才1*，朱国奇2，陈静萍1，詹祎捷3，薛丹2
（1.湖南省农业生物技术研究中心，湖南长沙410125；2.湖南省水稻研究所，湖南长沙410125；3.北京联合大学应用文理学院，北京100083）

在对冈优158、Ⅱ优808、五优308及天丰优316的杂交种子样品进行室内纯度鉴定时，发现不同SSR引物鉴定的纯度结果存在明显差异。为了探讨其原因，将这4个组合的种子样品在海南进行田间种植鉴定，并按单株进行了分子与田间表型的对比分析。结果表明，冈优158、Ⅱ优808、五优308和天丰优316分别用引物 RM208、RM264、RM242和RM164鉴定种子纯度时，只能鉴定出不育系（母本）杂株，室内分子鉴定纯度的结果均高于相应的田间纯度鉴定结果；而引物RM341、RM297、RM21和RM110不仅能鉴定出不育系（母本）杂株，还能鉴定出串粉株，室内分子鉴定纯度与田间鉴定纯度相当。因此，室内纯度鉴定时应采用多对引物进行检测，以增加识别串粉株的几率。

杂交水稻；SSR引物；种子纯度鉴定；纯度差异

湖南省镉低积累农作物品种筛选与选育项目。

詹庆才（1962-），男，湖南桃江人，硕士，研究员，从事水稻分子育种和杂交稻种子质量检测技术研究，E-mail：zhanqc@hotmail. com。*通讯作者。

2015-09-15

修回日期 2015-11-15